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Functional analysis of zebrafish GDNF
Abstract
We have identified zebrafish orthologues of glial cell line-derived neurotrophic factor (GDNF) and the ligand-binding component of its receptor GFRα1. We examined the mRNA expression pattern of these genes in the developing spinal cord primary motor neurons (PMN), kidney, and enteric nervous systems (ENS) and have identified areas of correlated expression of the ligand and the receptor that suggest functional significance. Many aspects of zebrafish GDNF expression appear conserved with those reported in mouse, rat, and avian systems. In the zebrafish PMN, GFRα1 is only expressed in the CaP motor neuron while GDNF is expressed in the ventral somitic muscle that it innervates. To test the functional significance of this correlated expression pattern, we ectopically overexpressed GDNF in somitic muscle during the period of motor axon outgrowth and found specific perturbations in the pattern of CaP axon growth. We also depleted GDNF protein in zebrafish embryos using morpholino antisense oligos and found that GDNF protein is critical for the development of the zebrafish ENS but appears dispensable for the development of the kidney and PMN.
Supplementary resources (2)
Nucleotide Sequence
December 2000
Nucleotide Sequence
December 2000
... For over 20 years now, the zebrafish, Danio rerio, has been leveraged as a relevant model to understand vertebrate ENS development, form, and function [24,25]. Zebrafish ENS development is under the control of genetic circuits, such as Sox10 [26], Phox2b [27], and the Ret-GDNF pathway [23,[28][29][30], demonstrating the conservation of genetic pathways giving rise to the ENS, and thus the utility of this model for studying ENS development. In a nutshell, zebrafish offer many outstanding characteristics to study ENS development, including: 1. ...
... Following emigration from the neural tube, zebrafish vagal NCCs localize in ventrolateral domains posterior to the otic vesicles ( Figure 1A). By 36 hours post fertilization (hpf ), ENCCs then begin to migrate as two single chains medially toward and along the foregut entrance [28,31,32] ( Figure 1B). Zebrafish ENCCs express a defining combination of marker genes that encode for various transcription factors and receptors; including sox10, foxd3, hoxb5b, phox2bb, ret, and gfra1a/b [23,27,28,[33][34][35][36][37]. ...
... By 36 hours post fertilization (hpf ), ENCCs then begin to migrate as two single chains medially toward and along the foregut entrance [28,31,32] ( Figure 1B). Zebrafish ENCCs express a defining combination of marker genes that encode for various transcription factors and receptors; including sox10, foxd3, hoxb5b, phox2bb, ret, and gfra1a/b [23,27,28,[33][34][35][36][37]. Once resident along the gut, ENCC chains migrate caudally within gut mesenchyme until they reach their final destinations, with the wavefront (a.k.a. ...
The enteric nervous system (ENS) is a complex series of interconnected neurons and glia that reside within and along the entire length of the gastrointestinal tract. ENS functions are vital to gut homeostasis and digestion, including local control of peristalsis, water balance, and intestinal cell barrier function. How the ENS develops during embryological development is a topic of great concern, as defects in ENS development can result in various diseases, the most common being Hirschsprung disease, in which variable regions of the infant gut lack ENS, with the distal colon most affected. Deciphering how the ENS forms from its progenitor cells, enteric neural crest cells, is an active area of research across various animal models. The vertebrate animal model, zebrafish, has been increasingly leveraged to understand early ENS formation, and over the past 20 years has contributed to our knowledge of the genetic regulation that underlies enteric development. In this review, I summarize our knowledge regarding the genetic regulation of zebrafish enteric neuronal development, and based on the most current literature, present a gene regulatory network inferred to underlie its construction. I also provide perspectives on areas for future zebrafish ENS research.
... The zebrafish GDNF has an amino acid sequence identity of 49% with human GDNF (Shepherd, Beattie, & Raible, 2001) and GDNF expression is conserved in the enteric nervous systems of zebrafish and mice during development (Sánchez et al., 1996;Shepherd et al., 2001;Treanor et al., 1996). Overexpression of glial cell line-derived neurotrophic factor (gdnf) in zebrafish somitic muscles led to axon pathfinding defects in primary motor neurons, while transient knockdown of gdnf by morpholino (MO) antisense oligonucleotides caused a drastic reduction in the number of enteric neurons, suggesting functional correlations with the mouse (Shepherd et al., 2001). ...
... The zebrafish GDNF has an amino acid sequence identity of 49% with human GDNF (Shepherd, Beattie, & Raible, 2001) and GDNF expression is conserved in the enteric nervous systems of zebrafish and mice during development (Sánchez et al., 1996;Shepherd et al., 2001;Treanor et al., 1996). Overexpression of glial cell line-derived neurotrophic factor (gdnf) in zebrafish somitic muscles led to axon pathfinding defects in primary motor neurons, while transient knockdown of gdnf by morpholino (MO) antisense oligonucleotides caused a drastic reduction in the number of enteric neurons, suggesting functional correlations with the mouse (Shepherd et al., 2001). ...
... The zebrafish GDNF has an amino acid sequence identity of 49% with human GDNF (Shepherd, Beattie, & Raible, 2001) and GDNF expression is conserved in the enteric nervous systems of zebrafish and mice during development (Sánchez et al., 1996;Shepherd et al., 2001;Treanor et al., 1996). Overexpression of glial cell line-derived neurotrophic factor (gdnf) in zebrafish somitic muscles led to axon pathfinding defects in primary motor neurons, while transient knockdown of gdnf by morpholino (MO) antisense oligonucleotides caused a drastic reduction in the number of enteric neurons, suggesting functional correlations with the mouse (Shepherd et al., 2001). In addition, expression of gdnf and its receptor has been documented in adult zebrafish, including in the ventral diencephalon (vDC) which suggests that gdnf expression is not restricted to the developmental period (Lucini et al., 2008(Lucini et al., , 2011. ...
Glial cell line‐derived neurotrophic factor (GDNF) has been reported to enhance dopaminergic neuron survival and differentiation in vitro and in vivo, although those results are still being debated. Glial cell line‐derived neurotrophic factor (gdnf) is highly conserved in zebrafish and plays a role in enteric nervous system function. However, little is known about gdnf function in the teleost brain. Here, we employed clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 to impede gdnf function in the maintenance of dopaminergic neuron development. Genotyping of gdnf crispants revealed successful deletions of the coding region with various mutant band sizes and down‐regulation of gdnf transcripts at 1, 3 and 7 day(s) post fertilization. Notably, ~20% reduction in ventral diencephalic dopaminergic neuron numbers in clusters 8 and 13 was observed in the gdnf‐deficient crispants. In addition, gdnf depletion caused a modest reduction in dopaminergic neurogenesis as determined by 5‐ethynyl‐2'‐deoxyuridine pulse chase assay. These deleterious effects could be partly attributed to deregulation of dopaminergic neuron fate specification‐related transcription factors (otp,lmx1b,shha,and ngn1) in both crispants and established homozygous mutants with whole mount in‐situ hybridization (WISH) on gdnf mutants showing reduced otpb and lmx1b.1 expression in the ventral diencephalon. Interestingly, locomotor function of crispants was only impacted at 7 dpf, but not earlier. Lastly, as expected, gdnf deficiency heightened crispants vulnerability to 1‐methyl‐4‐phenylpyridinium toxic insult. Our results suggest conservation of teleost gdnf brain function with mammals and revealed the interactions between gdnf and transcription factors in dopaminergic neuron differentiation.
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... The BMP2b MO antisense oligonucleotide (Gene Tools) was designed to target the splicing of BMP2b mRNA based on the following sequence: 5 -CAAATCAGGCCTCACCTTCGTGATG-3 (Wise and Stock, 2010). The GDNF MO antisense oligonucleotide (Gene Tools) was designed to target the splicing of GDNF mRNA based on the following sequence: 5 -TGTCCCATAACTTCATTTTAGACT-3 (Shepherd et al., 2001). The standard control MO (cont MO: 5 -CCTCTTACCTCAGTTACAATTTATA-3 ) was used as control for MO injection. ...
... Most of the plasmids were from Luo lab. Digoxigenin-labeled riboprobes were synthesized from a linearized plasmid as follows: crestin (Rubinstein et al., 2000); phox2b (Elworthy et al., 2005); α-SMA (Georgijevic et al., 2007); GDNF (Shepherd et al., 2001). The probes for identifying zebrafish BMPRIa and BMPRIb were amplified from cDNA pools of 72hpf embryos using the appropriate sets of primers. ...
... In a previous study, GDNF was reported to be a key chemoattractant for ENS NCCs in vitro and to regulate ENS NCC proliferation (Gianino et al., 2003). Furthermore, knockdown of GDNF and its receptor disrupted ENS NCC migration (Shepherd et al., 2001). To identify whether the defects in ENS precursor migration in BMP2b MO-treated embryos were related to changes in GDNF expression, we first assessed GDNF expression in BMP2b MO-treated embryos and found that GDNF expression was greatly reduced at the posterior end of the intestine at 72 hpf in the BMP2b morphants (Figures 7A,c,d). ...
The enteric nervous system (ENS) is derived from neural crest cells (NCCs). Defects in ENS NCCs colonizing in the intestines lead to an absence of enteric ganglia in the colon and results in Hirschsprung’s disease (HSCR). Bone morphogenetic proteins (BMPs) play diverse roles in the proliferation, migration and survival of ENS NCCs; however, whether BMPs are involved in HSCR and the underlying mechanism remains largely unknown. In this study, we found that BMP2 expression is significantly decreased in HSCR patients. Further experiments demonstrated that BMP2 is involved in the regulation of NCC proliferation, migration and differentiation. In a detailed analysis of the role of BMP2 in HSCR development in vivo, we demonstrated that BMP2b regulates the proliferation, migration and differentiation of vagal NCCs in zebrafish and that BMP2b is required for intestinal smooth muscle development. In addition, we showed that BMP2b is involved in regulating the expression of glial cell line-derived neurotrophic factor (GDNF) in the intestine, which mediates the regulation of ENS development by BMP2b in zebrafish. These results highlight a central role of the BMP-GDNF cascade in intestinal patterning and ENS development. Our results further demonstrate the key role of BMP2 in the etiology of HSCR in vitro and in vivo.
... The role of GDNF in ENS development has been conserved evolutionarily. We previously showed that blocking zebrafish GDNF function through injection of antisense morpholino oligonucleotides disrupted ENS neuronal differentiation (Shepherd et al., 2001). ...
... We extended the partial sequences we had previously identified for the zebrafish gfra1a and gfra1b orthologs (Shepherd et al., 2001) by using RACE (rapid amplification of cDNA ends) (Frohman, 1993). RACE cDNA was isolated from 24 hours postfertilization (hpf) and 48 hpf embryos using a Smart RACE cDNA Amplification Kit (Clonetech). ...
... We isolated clones representing the complete ORF for zebrafish gfra1a, gfra1b and gfra2 (Fig. 1). We used 5′ and 3′ RACE and genomic RT-PCR to identify flanking coding regions to the partial cDNAs of gfra1a and gfra1b that we had previously reported (Shepherd et al., 2001). We also isolated a cDNA encoding a zebrafish gfra2 ortholog by screening an arrayed zebrafish EST library (Clark et al., 2001) with probes for the partial gfra1a and gfra1b cDNAs. ...
Components of the zebrafish GDNF receptor complex are expressed very early in the development of enteric nervous system precursors, and are already present as these cells begin to enter the gut and migrate caudally along its length. Both gfra1a and gfra1b as well as ret are expressed at this time, while gfra2 expression, the receptor component that binds the GDNF-related ligand neurturin, is not detected until the precursors have migrated along the gut. Gfra genes are also expressed in regions of the zebrafish brain and peripheral ganglia, expression domains conserved with other species. Enteric neurons are eliminated after injection with antisense morpholino oligonucleotides against ret or against both Gfra1 orthologs, but are not affected by antisense oligonucleotides against gfra2. Blocking GDNF signaling prevents migration of enteric neuron precursors, which remain positioned at the anterior end of the gut. Phenotypes induced by injection of antisense morpholinos against both Gfra orthologs can be rescued by introduction of mRNA for gfra1a or for gfra2, suggesting that GFRα1 and GFRα2 are functionally equivalent.
... In this study, we developed and validated a new zebrafish BAC transgenic line TgBAC(ret:EGFP)b1331 (referred to as ret:GFP from here forward) that expresses green fluorescent protein (GFP) under the ret promoter ( Figure 1A). In addition to GFP + ENS cells, other cells expressing GFP include cells in the developing pronephric ducts, enteroendocrine cells in the gut epithelium, neurons in the spinal cord and retina, and cells in the pharyngeal arches, in accordance with described ret expression in these cell types (Figure 1B,C, [8,15,[27][28][29]). In this paper, we focus on ret:GFP expression in the ENS. ...
... Antisense-RNA probe templates were obtained by NotI linearization of ret plasmid [8,27], and PCR amplification of GFP from genomic DNA isolated from finclips of TgBAC(ret:EGFP)b1331 carriers with primers containing T7 promoter sequence (T7: 5′-TAATACGACTCACTATAGGG-3′). Primer sequences were EGFP_ISH_F1: 5′-CAAGGGCGAGGAGCTGTT-3′, EGFP_ISH_R1: 5′-TAATACGACTCACTATAGGG CTCGTCCATGCCGAGAGT-3′. ...
The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10—another important ENS developmental regulator gene—GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.
... Shepherd et al. were the first to characterize gdnf in zebrafish. Whole-mount in situ hybridization showed that gdnf was expressed in the central nervous system (CNS) of zebrafish larvae as well as in the enteric neurons and pronephric ducts [22]. As reported for rodent Gdnf, zebrafish gdnf was shown to be critical in enteric nervous system development and peripheral axons of sensory neurons [22,23]. ...
... Whole-mount in situ hybridization showed that gdnf was expressed in the central nervous system (CNS) of zebrafish larvae as well as in the enteric neurons and pronephric ducts [22]. As reported for rodent Gdnf, zebrafish gdnf was shown to be critical in enteric nervous system development and peripheral axons of sensory neurons [22,23]. Moreover, neuroprotection against neuronal death induced by a mutated human Tau protein was seen in zebrafish overexpressing gdnf [24]. ...
Glial cell line-derived neurotrophic factor (GDNF) was initially described as important for dopaminergic neuronal survival and is involved in many other essential functions in the central nervous system. Characterization of GDNF phenotype in mammals is well described; however, studies in non-mammalian vertebrate models are scarce. Here, we characterized the anatomical distribution of gdnf-expressing cells in adult zebrafish brain by means of combined in situ hybridization (ISH) and immunohistochemistry. Our results revealed that gdnf was widely dispersed in the brain. gdnf transcripts were co-localized with radial glial cells along the ventricular area of the telencephalon and in the hypothalamus. Interestingly, Sox2 positive cells expressed gdnf in the neuronal layer but not in the ventricular zone of the telencephalon. A subset of GABAergic precursor cells labeled with dlx6a-1.4kbdlx5a/6a: green fluorescence protein (GFP) in the pallium, parvocellular preoptic nucleus, and the anterior and dorsal zones of the periventricular hypothalamus also showed expression with gdnf mRNA. In addition, gdnf signals were detected in subsets of dopaminergic neurons, including those in the ventral diencephalon, similar to what is seen in mammalian brain. Our work extends our knowledge of gdnf action sites and suggests a potential role for gdnf in adult brain neurogenesis and regeneration.
... Zebrafish has two GFRα1 orthologs and morpholino-mediated antisense knockdown of the two orthologs (GFRα1a and GFRα1b) results in complete loss of ENS neurons and their precursors (Shepherd et al., 2004). Similar to knockdown of RET and GFRα1, knockdown of GDNF also results in complete loss of zebrafish ENS neurons and their precursors (Shepherd et al., 2001). Two other GDNF family members, Neurturin and Artemin, are reported, by immunoreactivity, to be present in zebrafish, although their function in zebrafish ENS development is unknown (Lucini et al., 2005(Lucini et al., , 2004. ...
... A morning's work of injecting antisense morpholino oligos (MOs), targeted against a gene of interest, into onecell stage embryos can generate hundreds of putative knock-down embryos for analysis (Bill et al., 2009). This strategy was used to study the roles of gdnf, gfrα1, ret, phox2b, and demonstrated that gene function of each of these genes was required for normal ENS development (Elworthy et al., 2005;Heanue and Pachnis, 2008;Shepherd et al., 2001Shepherd et al., , 2004. More recently, genome editing approaches, such as zinc finger nucleases, TALEN and now CRISPR/ Cas9, are enabling the efficient and rapid generation of targeted mutations in genes of interest (Peng et al., 2014), and are likely to play a major role in future studies. ...
Our current understanding of the developmental biology of the enteric nervous system (ENS) and the genesis of ENS diseases is founded almost entirely on studies using model systems. Although genetic studies in the mouse have been at the forefront of this field over the last 20 years or so, historically it was the easy accessibility of the chick embryo for experimental manipulations that allowed the first descriptions of the neural crest origins of the ENS in the 1950 s. More recently, studies in the chick and other non-mammalian model systems, notably zebrafish, have continued to advance our understanding of the basic biology of ENS development, with each animal model providing unique experimental advantages. Here we review the basic biology of ENS development in chick and zebrafish, highlighting conserved and unique features, and emphasising novel contributions to our general understanding of ENS development due to technical or biological features.
... Interestingly, no deficits were observed in GFR␣2 mutants (Garces et al., 2000). In zebrafish, GFR␣1 is expressed in a specific primary motor neuron, CaP, while GDNF is expressed specifically in the muscle that it innervates (Shepherd et al., 2001). Ectopic expression of GDNF results in perturbations in growth by the CaP axon. ...
... Intriguingly, despite the similarities in inductive interactions in formation of the pro, meso, and metanephric kidneys, in mice GDNF/GFR␣1/c-ret signaling is essential only for development of the metanephros. Consistent with this, no deficit in pronephric kidney development is observed following GDNF depletion in zebrafish using anti-sense morpholino-oligonucleotides (Shepherd et al., 2001). In contrast, GDNF signaling through GFR␣1 has been shown to be essential for normal migration of pronephric duct cell precursors in axolotl (Drawbridge et al., 2000). ...
... In addition, the different activity patterns may lead to optimal release of retrograde factors from innervated muscles. In this regard, CaPs, but not MiPs, express the glial cell line-derived neurotrophic factor (GDNF) receptor GFRa1 (Shepherd et al. 2001). Further, ventral, but not dorsal axial, muscle expresses GDNF (Shepherd et al. 2001). ...
... In this regard, CaPs, but not MiPs, express the glial cell line-derived neurotrophic factor (GDNF) receptor GFRa1 (Shepherd et al. 2001). Further, ventral, but not dorsal axial, muscle expresses GDNF (Shepherd et al. 2001). ...
Different muscle targets and transcription factor expression patterns reveal the presence of motor neuron subtypes. However, it is not known whether these subtypes also differ with respect to electrical membrane properties. To address this question, we studied primary motor neurons (PMNs) in the spinal cord of zebrafish embryos. PMN genesis occurs during gastrulation and gives rise to a heterogeneous set of motor neurons that differ with respect to transcription factor expression, muscle targets, and soma location within each spinal cord segment. The unique subtype-specific soma locations and axonal trajectories of two PMNs-MiP (middle) and CaP (caudal)-allowed their identification in situ as early as 17 h postfertilization (hpf), prior to axon genesis. Between 17 and 48 hpf, CaPs and MiPs displayed subtype-specific electrical membrane properties. Voltage-dependent inward and outward currents differed significantly between MiPs and CaPs. Moreover, by 48 hpf, CaPs and MiPs displayed subtype-specific firing behaviors. Our results demonstrate that motor neurons that differ with respect to muscle targets and transcription factor expression acquire subtype-specific electrical membrane properties. Moreover, the differences are evident prior to axon genesis and persist to the latest stage studied, 2 days postfertilization.
... During development, gdnf expression is well conserved in the zebrafish brain and enteric nervous system (Reichenbach et al., 2008;Shepherd, Beattie, & Raible, 2001). gdnf transcripts are widespread in the whole larvae brain, but no specific expression pattern was observed (Reichenbach et al., 2008). ...
Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.
... In this study, we developed and validated a new zebrafish BAC transgenic line TgBAC(ret:EGFP) b1331 (referred to as ret:GFP from here forward) that expresses green fluorescent protein (GFP) under the ret promoter representing ret expression in the ENS from development through adulthood. In addition to GFP + ENS cells, other cells expressing GFP include cells in the developing pronephric ducts, enteroendocrine cells in the gut epithelium, neurons in the spinal cord and retina, and cells in the pharyngeal arches, in accordance with described ret expression in these cell types [7,15,[27][28][29]. In this paper, we focus on ret:GFP expression in the ENS. ...
The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g. Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in female and male. In homozygous mutants of ret and sox10 – another important ENS developmental regulator gene – GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.
... 46 Similarly, components of the GDNF/Ret signaling pathway are expressed in the subset of vagal NCCs that give rise to the ENS after the onset of migration towards the gut and play a crucial role in precursor migration. 47,223 In zebrafish, ret is expressed as two isoforms, ret9 and ret51, and the Ret9 isoform is sufficient for colonization of the gut by enteric neurons. 48 Moreover, when gfra1a/gfra1b or ret are knocked down, enteric NCCs are still capable of expressing phox2b and entering the anterior gut, indicating that their initial specification is not affected. ...
Our understanding of the neural crest, a key vertebrate innovation, is built upon studies of multiple model organisms. Early research on neural crest cells (NCCs) was dominated by analyses of accessible amphibian and avian embryos, with mouse genetics providing complementary insights in more recent years. The zebrafish model is a relative newcomer to the field, yet it offers unparalleled advantages for the study of NCCs. Specifically, zebrafish provide powerful genetic and transgenic tools, coupled with rapidly developing transparent embryos that are ideal for high‐resolution real‐time imaging of the dynamic process of neural crest development. While the broad principles of neural crest development are largely conserved across vertebrate species, there are critical differences in anatomy, morphogenesis, and genetics that must be considered before information from one model is extrapolated to another. Here, our goal is to provide the reader with a helpful primer specific to neural crest development in the zebrafish model. We focus largely on the earliest events—specification, delamination, and migration—discussing what is known about zebrafish NCC development and how it differs from NCC development in non‐teleost species, as well as highlighting current gaps in knowledge.
... Amphioxus and tunicates, chordates without neural crest cells, have also ENs (41, 42) whose cell lineages are unclear, raising a possible evolutionary scenario in which endodermderived ENs may be a conserved characteristic of deuterostomes. Although all reported enteric neurons are derived from neural crest cells in mammals and birds (43)(44)(45), zebrafish morphants, in which neural crest cell-essential genes such as GDNF or Phox2b were attenuated, had significantly decreased ENs, but the few neurons that remained (46,47) suggests some vertebrates have nonneural crest cell ENs. ...
Significance
In vertebrate digestive tracts, pyloric sphincters play an important role in controlling the passage of food from stomach to intestine. A major regulator of sphincter relaxation is nitric oxide (NO). However, it is unknown when and how this important means of control was acquired, in part because we lack information on how nonvertebrate deuterostome sphincters are regulated. Here, we show that a NO-dependent regulatory system is present in the pyloric sphincters of sea urchin larvae. Our data suggest that NO-dependent regulation of the pyloric sphincter was present in deuterostome stem groups, and the common deuterostome ancestor had endodermally derived cells that regulated gut function.
... Furthermore, visualization with broad neural crest gene markers like crestin, sox10, foxd3, tfap2a, and enteric progenitor markers phox2bb, ret and gfra1a/b label vagal neural crest en route to the gut ( Barrallo-Gimeno et al., 2004;Knight et al., 2003;Luo et al., 2001;Shepherd et al., 2004;Stewart et al., 2006). These studies show that zebrafish vagal neural crest cells migrate medially from post-otic vagal domains toward the foregut entrance as two chains and migrate caudally along the left and right sides of the gut tube to fully colonize the hindgut by the third day of development (Dutton et al., 2001;Elworthy et al., 2005;Kelsh and Eisen, 2000;Olden et al., 2008;Shepherd and Eisen, 2011;Shepherd et al., 2001;Uribe and Bronner, 2015). It is not yet known if the post-otic vagal population of neural crest cells gives rise to tissues other than the ENS in zebrafish. ...
Arising within the neural tube between the cranial and trunk regions of the body axis, the vagal neural crest shares interesting similarities in its migratory routes and derivatives with other neural crest populations. However, the vagal neural crest is also unique in its ability to contribute to diverse organs including the heart and enteric nervous system. This review highlights the migratory routes of the vagal neural crest and compares them across multiple vertebrates. We also summarize recent advances in understanding vagal neural crest ontogeny and discuss the contribution of this important neural crest population to the cardiovascular system and endoderm-derived organs, including the thymus, lungs and pancreas.
... The zebrafish (Danio rerio) has been used as a model organism for studying RET, in which it was shown that the function of RET and its bipartite ligand (defined as two GDNF and two GFRα1 molecules) is conserved in enteric nervous system development (Shepherd et al., 2004, Shepherd et al., 2001, Marcos-Gutierrez et al., 1997). ...
The RET receptor tyrosine kinase (RTK) is crucial for embryonic and adult development of multiple organs, tissues and neurons. Gain-of-function mutations in the RET gene are found in human cancer, while loss-of-function mutations are associated with congenital anomalies of the kidney and urinary tract (CAGUT) and Hirschsprung's Disease (HSCR). Previous work has identified that some HSCR RET mutations result in a bottleneck in RET folding and a subsequent loss of RET export. This thesis presents work examining the characteristics of wild type (WT) and HSCR RET maturation, export and signalling in stably transfected mammalian cell lines. High throughput siRNA screening was used to identify components involved in WT and HSCR RET maturation and export; preliminary validation has implicated Endoplasmic Reticulum associated degradation (ERAD), autophagy and the N-glycosylation pathway. RET is also a validated cancer target, as a driver of cancers including multiple endocrine neoplasia (MEN) 2A and B. While there are several FDA-approved RET inhibitors available, their lack of specificity and potency has resulted in high levels of off-target toxicity and low life expectancy extensions. As such, a new generation of more optimal inhibitors is required. This thesis presents the investigation of the molecular basis of RET kinase inhibition, through the elucidation of the RET kinase domain (KD) structure bound to several ATP-competitive chemical inhibitors that are known to inhibit RET in vitro. Preliminary development of an updated RET pharmacophore is described, defining key residue interactions and combining observations with biochemical and thermal stability data.
... GDNF-GFRα-1 complexes recruit the tyrosine kinase transmembrane protein to execute differentiation of DA-ergic neurons in brain (Naughton et al., 2006;Durbec et al., 1996). The expression of GFRα-1in certain areas of the brain where neurons responsive to GDNF family members reside have been analyzed in mammals (Quartu et al., 2007;Matsuo et al., 2000) and less studied in lower vertebrates including teleosts (Shepherd et al., 2001;Lucini et al., 2010;Lucini et al., 2011). Teleosts serve as an excellent animal model to study neurodegeneration partially due to the abundance of neuronal precursor cells such as glial cells (Zupanc and Clint, 2003). ...
Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor that preferentially binds to GDNF family receptor α-1 (GFRα-1) by regulating dopaminergic (DA-ergic) neurons in brain. Present study aimed to evaluate the significance of GFRα-1 expression during early brain development in catfish. Initially, the full-length cDNA of GFRα-1 was cloned from adult brain which showed high homology with other vertebrate counterparts. Quantitative PCR analysis of tissue distribution revealed ubiquitous expression of GFRα-1 in the tissues analyzed with high levels in female brain and ovary. Significant higher expression was evident in brain at 75 and 100 days post hatch females than the respective age-match males. High expression of GFRα-1 was evident in brain during the spawning phase in comparison to other reproductive phases. Localization of GFRα-1 revealed its presence in preoptic area-hypothalamus which correlated well with the expression profile in discrete areas of brain in adult catfish. To investigate further, transient silencing of GFRα-1 through siRNA resulted in lower expression levels of GFRα-1, which further downregulated the expression of certain brain-specific genes. Expression level of GFRα-1 in brain was significantly diminished upon treatments with the 1-methyl-1,2,3,6-tetrahydropyridine causing neurodegeneration which further correlated with catecholamines, L-3,4-dihydroxyphenylalanine, DA and norepinephrine levels. Taken together, GFRα-1 may be associated to DA-ergic neuron in brain either directly or indirectly and it plausibly have a role in gonadotropin-releasing hormone and gonadotropin (GnRH-GTH) axis, at least by targeting DA-ergic activity, partially.
... Furthermore, visualization with broad neural crest gene markers like crestin, sox10, foxd3, tfap2a, and enteric progenitor markers phox2bb, ret and gfra1a/b label vagal neural crest en route to the gut ( Barrallo-Gimeno et al., 2004;Knight et al., 2003;Luo et al., 2001;Shepherd et al., 2004;Stewart et al., 2006). These studies show that zebrafish vagal neural crest cells migrate medially from post-otic vagal domains toward the foregut entrance as two chains and migrate caudally along the left and right sides of the gut tube to fully colonize the hindgut by the third day of development (Dutton et al., 2001;Elworthy et al., 2005;Kelsh and Eisen, 2000;Olden et al., 2008;Shepherd and Eisen, 2011;Shepherd et al., 2001;Uribe and Bronner, 2015). It is not yet known if the post-otic vagal population of neural crest cells gives rise to tissues other than the ENS in zebrafish. ...
... GDNF mainly binds to GFRA1, but may also interact with GFRA2 (Airaksinen and Saarma, 2002). In zebrafish, as in other vertebrates, Gdnf signaling through Ret plays a very important role during ENS development (Shepherd et al., 2001;Shepherd et al., 2004;Heanue and Pachnis, 2008;Heanue et al., 2016a). During early stages of ENS development, ret, and the two gene duplicates gfra1a and gfra1b are expressed in EPCs after they have started to migrate towards the gut [Fig. ...
The enteric nervous system (ENS) is the largest part of the peripheral nervous system and is entirely neural crest derived. It provides the intrinsic innervation of the gut controlling different aspects of gut function, such as motility. In this review, we will discuss key points of zebrafish ENS development, genes and signaling pathways regulating ENS development as well as contributions of the zebrafish model system to better understand ENS disorders. During their migration, enteric progenitor cells display a gradient of developmental states based on their proliferative and migratory characteristics and show spatio-temporal heterogeneity based on gene expression patterns. Many genes and signaling pathways that regulate the migration and proliferation of EPCs have been identified, but later stages of ENS development, especially steps of neuronal and glial differentiation remain poorly understood. In recent years, zebrafish have become increasingly important to test candidate genes for ENS disorders, e.g. from genome-wide association studies, to identify environmental influences on ENS development e.g. through large-scale drug screens, and to investigate the role the gut microbiota play in ENS development and disease. With its unique advantages as a model organism, zebrafish will continue to contribute to a better understanding of ENS development, function and disease. This article is protected by copyright. All rights reserved.
... The MOs application reduces gene expression via steric blocking of pre-mRNA splicing or translation (Galderisi et al. 1999;Summerston 1999;Morcos 2007). The MOs have been employed for studying the role of genes and disrupting their expression during fish embryonic development (Ekker & Larson 2001;Shepherd et al. 2001;Boonanuntanasarn et al. 2002). Although many researchers have used MOs to assess gene function in fish, few studies have applied this approach to specifically suppress genes involved in embryonic PGCs development. ...
Interest in reproductively sterile fish in aquaculture has prompted research into their production. Several methods are available for inducing sterility and optimizing its application in the global fishery industry. Sterilization can potentially be accomplished through irradiation, surgery, or chemical and hormonal treatment. Alternative approaches include triploidization, hybridization, and generation of new lines via advanced biotechnological techniques. Triploids of many commercially important species have been studied extensively and have been produced on a large scale for many years. Novel approaches, including disruption of gonadotropin releasing hormone signalling and genetic ablation of germ cells, have been developed that are effective in producing infertile fish but have the disadvantage of not being 100% reliable or are impractical
for large-scale aquaculture. We review currently used technologies and recent advances in induction of sterility in fish, especially those intended for use in germ cell transplantation. Knowledge of the implications of these approaches remains incomplete, imposing considerable limitations.
... 36) Ciliary neurotrophic factor (CNTF) also promotes neurite outgrowth of fish RGCs. Although other neurotrophic factors such as NT-6, 37) NT-7, 38) brain derived neurotrophic factor (BDNF) 39) and glial cell line derived neurotrophic factor (GDNF) 40) have been identified in fish, the functional role of these factors in ON regeneration is not yet known. Two low molecular weight factors (axogenesis factor 1 and 2; AF-1 and AF-2) are isolated from the goldfish ON. 41) AF-1 is a neurite outgrowth promoting factor with potential for priming treatment of ON crush. ...
... Here, we further investigated whether certain NTs can prevent neuronal death induced by overexpression of wildtype human and zebrafish tau proteins. The C-terminal ends of BDNF [59] and GDNF [60] were tagged with HA peptide for easy detection of protein production. We expressed BDNF-HA or GDNF-HA under the control of a glia-specific glial fibrillary acidic protein (GFAP) gene promoter [40] to examine whether these proteins are able to promote the survival of neuronal cells expressing human and zebrafish tau-GFP proteins. ...
Background:
The axonal tau protein is a tubulin-binding protein, which plays important roles in the formation and stability of the microtubule. Mutations in the tau gene are associated with familial forms of frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Paired helical filaments of tau and extracellular plaques containing beta-amyloid are found in the brain of Alzheimer's disease (AD) patients.
Results:
Transgenic models, including those of zebrafish, have been employed to elucidate the mechanisms by which tau protein causes neurodegeneration. In this study, a transient expression system was established to express GFP fusion proteins of zebrafish and human tau under the control of a neuron-specific HuC promoter. Approximately ten neuronal cells expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording, in order to evaluate the neurotoxicity induced by tau-GFP proteins. Expression of tau-GFP was observed to cause high levels of neuronal death. However, multiple signaling factors, such as Bcl2-L1, Nrf2, and GDNF, were found to effectively protect neuronal cells expressing tau-GFP from death. Treatment with chemical compounds that exert anti-oxidative or neurotrophic effects also resulted in a similar protective effect and maintained human tau-GFP protein in a phosphorylated state, as detected by antibodies pT212 and AT8.
Conclusions:
The novel finding of this study is that we established an expression system expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording to evaluate the neurotoxicity induced by tau-GFP proteins. This system may serve as an efficient in vivo imaging platform for the discovery of novel drugs against tauopathy.
... To examine whether enteric neural crest localization was altered after loss of Meis3, we analyzed serial transverse sections from the postotic and foregut levels at 36 hpf. In control embryos, at the postotic level, sox10:GFP + neural crest cells localized laterally streams from the left and right postotic vagal regions (Kelsh and Eisen, 2000;Dutton et al., 2001;Shepherd et al., 2001;Olden et al., 2008). Once they reach their final destinations, enteric neural crest cells differentiate into neuronal subtypes or glia, subsequently forming interconnected ganglia within the myenteric and submucosal plexuses that run within the mucosa and smooth muscle layers in zebrafish (Olsson et al., 2008;Shepherd and Eisen, 2011). ...
During development, vagal neural crest cells fated to contribute to the enteric nervous system migrate ventrally away from the neural tube toward and along the primitive gut. The molecular mechanisms that regulate their early migration en route to and entry into the gut remain elusive. Here, we show that the transcription factor meis3 is expressed along vagal neural crest pathways. Meis3 loss of function results in a reduction in migration efficiency, cell number and the mitotic activity of neural crest cells in the vicinity of the gut, while having no effect on neural crest or gut specification. Later, during enteric nervous system differentiation, Meis3 depleted embryos exhibit colonic aganglionosis, a disorder in which the hindgut is devoid of neurons. Accordingly, the expression of Shh pathway components, previously shown to have a role in the etiology of Hirschsprung's disease, was misregulated within the gut following loss of Meis3. Taken together, these findings support a model in which Meis3 is required for neural crest proliferation, migration into and colonization of the gut such that its loss leads to severe defects in enteric nervous system development.
... GDNF. 52 Reduction of gdnf or ret1 activity has no effect on primordium migration, but significantly reduces the extension of the lateral line nerve. 53 After initial leader axons have extended along with the primordium, other axons follow, with both leaders and followers showing distinct somatotopy. ...
The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprised of clusters of mechanosensory hair cells called neuromasts. The lateral line is initially established by a migratory group of cells, called a primordium, that deposits neuromasts at stereotyped locations along the surface of the fish. Wnt, FGF , and Notch signaling are all important regulators of various aspects of lateral line development, from primordium migration to hair cell specification. As zebrafish age, the organization of the lateral line becomes more complex in order to accommodate the fish's increased size. This expansion is regulated by many of the same factors involved in the initial development. Furthermore, unlike mammalian hair cells, lateral line hair cells have the capacity to regenerate after damage. New hair cells arise from the proliferation and differentiation of surrounding support cells, and the molecular and cellular pathways regulating this are beginning to be elucidated. All in all, the zebrafish lateral line has proven to be an excellent model in which to study a diverse array of processes, including collective cell migration, cell polarity, cell fate, and regeneration. WIREs Dev Biol 2015, 4:1–16. doi: 10.1002/wdev.160
This article is categorized under: Nervous System Development > Vertebrates: Regional Development
Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration
... Major factors that control the specification, differentiation or proliferation of these cells in zebrafish include endodermderived cues (Pietsch et al. 2006), such as Hh, mesoderm factors (Reichenbach et al. 2008), and GDNF. Blocking GDNF function or the GDNF receptor c-Ret and co-receptors GFL-alpha through injection of antisense morpholinos impaired both enteric precursors differentiation and migration (Shepherd et al. 2001;Shepherd et al. 2004;Heanue and Pachnis 2008). In addition, several transcription factors (Phox2b, Sox10) have been implicated (Elworthy et al. 2005 AU :2 ), and ongoing forward genetic screens promise to uncover yet-unknown enteric determinants (Kuhlmann and Eisen 2007 AU :3 ). ...
This chapter discusses the development of neural functions, basic neurophysiology, and the control of behavior in zebrafish. The first step in the development of the nervous system is the specification of the neural plate by a process called “neural induction.” It takes place at a very early developmental stage, preceding or concomitant to gastrulation. Like in other vertebrates, neural plate specification within the zebrafish embryo integrates antagonistic activities from different signaling pathways, such as meso- and endoderm inducers, neural inducers, and ventralizing versus dorsalizing agents. The commitment of neuroepithelial progenitors towards neuronal or glial differentiation is initiated concomitantly to neural plate formation and neurulation. The general organization of the zebrafish central nervous system (CNS) is very similar to that of other vertebrates. Along the antero-posterior axis, the central nervous system is divided into four parts. The spinal cord is the most caudal part of the CNS, protected by the bony column of vertebrae. It is continued anteriorly by the rhombencephalon, the most caudal part of the encephalon, within the skull. The forebrain is the most rostral and largest area of the brain, separated from the rhombencephalon by the intervening mesencephalon. These four main regions of the CNS are subdivided into morphological and functional areas. In particular, the forebrain or prosencephalon, comprises the caudally located diencephalon and the anterior “secondary prosencephalon.” The latter gives rise ventrally to the hypothalamus and dorsally to the telencephalon.
... These features make MOs insensitive to nucleases, yet allow MOs to speci fi cally and stably bind to its complementary nucleotide polymer, particularly RNA sequences, with high af fi nity to block access of cell components, resulting in inhibition of translation ( 2 ) and splicing ( 3 ) of mRNA or maturation of microRNA (miRNA) (4)(5)(6) . They were fi rst introduced for developmental biology studies in Xenopus by Janet Heasman and colleagues in 2000 ( 7 ) and since then have been used in variety of organisms, including sea urchin ( 8 ) , Ciona savignyi ( 9 ) , Xenopus laevis (10)(11)(12) , Xenopus tropicalis ( 13 ) , zebra fi sh (14)(15)(16) , chick ( 17 ) , and mouse ( 18 ) . In Xenopus , the effectiveness of MOs in early tadpole stages has been well documented using transgenic animals expressing GFP ( 13 ) . ...
Here we present a protocol, which allows loss-of-function studies in Xenopus embryos using antisense morpholino oligonucleotides (MOs). Gene knockdown studies provide a critical method for assessing gene function in vitro and in vivo. Such studies are currently performed in Xenopus using primarily one of the two main methods: (1) overexpression of dominant negative constructs or (2) inhibition of gene function by using MOs targeting either the initiation of translation or mRNA splicing. While a dominant negative approach is very effective, it often suffers from specificity. Given that MOs target very specific nucleotide sequences in the target RNA, it suffers considerably less from issues of specificity. The most convenient method for introducing MOs into embryos is through microinjection, which is a simple procedure. Therefore, a reverse genetics approach in Xenopus using MOs is an extremely powerful tool to study gene function, particularly when taking advantage of available sequence data in the post-genomic era. Furthermore, given the well-established fate map in Xenopus, it is also very easy to generate mosaic knockdown embryos, where the gene of interest is affected in defined regions of the embryo. Finally it should be noted that MOs can also be used to block miRNA function and processing, so that it provides a convenient method to not only perform gene knockdown studies on protein coding genes, but also noncoding genes. The protocol we describe here is for both Xenopus laevis and Xenopus tropicalis.
... However, in both phyla, there is expression of homologues of mammalian NGF [257][258][259][260], BDNF [261], and NT-3, together with Trk-A/-B/-C, and p75 NTR (e.g., Xenopus xp75 NTF a/x p75 NTF b) [262], and additional neurotrophin family members (e.g. NT6 [263] and NT-7 [264], GDNF [265], CNTF [153,266], and FGF-2 [267]. Novel axogenic factors are also present which may account for the successful CNS axon regeneration in fish and amphibia, including AF-1/2 [116,268] which regulates axon regeneration through a purine-sensitive pathway [266]. ...
Normal visual function may be compromised by optic nerve (ON) injury and a range
of inherited and acquired retinal degenerative diseases, which affect retinal ganglion cells
(RGC), photoreceptors and/or retinal pigment epithelium. Since the visual system is part
of the central nervous system (CNS), recovery from penetrant traumatic injury is
incomplete because lost neurons are not replaced and severed axons do not regenerate.
The failure of axon regeneration is attributed to a lack of stimulation by neurotrophic
factors (NTF) which ‘prime’ neurons to growth readiness and promote axon
regeneration, the presence of CNS myelin and extracellular matrix-associated axon
growth inhibitors in the neuropil and wound, and a failure of neurons to survive. CNS
myelin-derived axon growth inhibitory ligands bind to the non-signalling NgR receptor
expressed on axon growth cones. NgR complexes with Lingo-1 and a signalling coreceptor,
either p75NTR, or TROY. The latter mediate inhibition either through a Rho-A
mediated signalling cascade that culminates in changed actin dynamics and growth cone
collapse, or by activation of the epidermal growth factor receptor (EGFR) through an
unknown signalling pathway that converges downstream of RhoA. Other CNS axon
growth inhibitors include semaphorins, ephrins and chondroitin sulphate proteoglycans
(CSPG), and their signalling pathways also converge on and activate Rho-A. Using the
ON as a model of CNS injury, we and others have made advances in understanding the
relative contribution of axon growth stimulators versus inhibitors to the regenerative
outcome of axotomised CNS neurons. For example, we have shown that regulated
intramembraneous proteolysis (RIP) is induced by NTF and cleaves p75NTR into intra-and extracellular domain fragments, blocks Rho activation and attenuates EGFR
phosphorylation, thereby blocking inhibitory signalling after CNS myelin inhibitory
ligand binding to NgR. Furthermore, robust axon regeneration arrests the development of
the scar matrix that normally forms after CNS injury at the lesion site, by release of
matrix metalloproteases (MMPs) and suppression of their inhibitors (TIMPs) from glia
into the environment of the regenerating axon. Others have also reported different
strategies to promote RGC survival and axon regeneration, including lens injury,
suppression of RhoGTP, addition of cAMP and inhibition of EGFR phosphorylation, all
of which can promote RGC axon regeneration to varying degrees and have dispelled the
myth that CNS axons are inherently incapable of regeneration. All these studies support
the contention that CNS axon regeneration occurs when: (1) a ‘growth activated state’ is
induced in neurons; (2) axons are blinded to putative inhibitory ligands; and (3) neuron
survival and axon regeneration is promoted. This chapter will highlight current research
on therapeutic approaches, such as gene therapy and RNA interference aimed at
promoting these three priorities.
... 36) Ciliary neurotrophic factor (CNTF) also promotes neurite outgrowth of fish RGCs. Although other neurotrophic factors such as NT-6, 37) NT-7, 38) brain derived neurotrophic factor (BDNF) 39) and glial cell line derived neurotrophic factor (GDNF) 40) have been identified in fish, the functional role of these factors in ON regeneration is not yet known. Two low molecular weight factors (axogenesis factor 1 and 2; AF-1 and AF-2) are isolated from the goldfish ON. 41) AF-1 is a neurite outgrowth promoting factor with potential for priming treatment of ON crush. ...
IntroductionThe Time Course of Optic Nerve Regeneration in Goldfish Morphological Changes in RGCs and Their Axon Terminals During Optic Nerve RegenerationA Computer Image Processing System to Quantify Goldfish BehaviorTime-Specific Molecular Expressions During Optic Nerve Regeneration in Goldfish Purpurin: A Retinol-Binding Protein in the Retina During the Early Stage of Optic Nerve RegenerationMolecular Involvement of Na, K-ATPase in the Retina During Axonal Regeneration in GoldfishRetinal Transglutaminase (TGR) During Axonal Regeneration in GoldfishUp-Regulation of a Molecule in the Optic Tectum During the Late Stage of Optic Nerve RegenerationApplications of Fish-Derived Regeneration-Associated Molecules to Promote Regrowth of Mammalian Optic AxonsSummary Morphological Changes in RGCs and Their Axon Terminals During Optic Nerve RegenerationA Computer Image Processing System to Quantify Goldfish Behavior Purpurin: A Retinol-Binding Protein in the Retina During the Early Stage of Optic Nerve RegenerationMolecular Involvement of Na, K-ATPase in the Retina During Axonal Regeneration in GoldfishRetinal Transglutaminase (TGR) During Axonal Regeneration in GoldfishUp-Regulation of a Molecule in the Optic Tectum During the Late Stage of Optic Nerve Regeneration
... These modifications make morpholino antisense oligonucleotides resistant to enzymatic degradation and nontoxic while permitting hybridization to target transcripts and the selective blocking of the initiation of translation (Summerton, 1999). Morpholino antisense oligonucleotides have been used successfully in zebrafish Shepherd et al., 2001), Xenopus (Heasman et al., 2000), sea urchin (Howard et al., 2001), and chicken (Kos et al., 2001) embryos to investigate the roles of specific gene products in developmental events. By using the method of Kos et al. (2001) we have introduced tenascin-C morpholino antisense oligonucleotides into avian neural crest cell precursors in ovo by microinjection into the lumen of the early neural tube and subsequently driving the oligonucleotides into the neuroepithelium by square-pulse electroporation. ...
A key feature of vertebrate development is the formation of the neural crest. In the trunk, neural crest cells delaminate from the neural tube shortly after the fusion of the neural folds and migrate ventrally along specific pathways to form the neurons and glia of the peripheral nervous system. As neural crest cells leave the neural tube during the initial stages of their migration, they express the extracellular matrix glycoprotein tenascin-C, which is also found in the stroma of many tumors. We have studied the possible role for tenascin-C during neural crest morphogenesis in vivo by microinjecting tenascin-C morpholino antisense oligonucleotides into the lumen of the avian neural tube in ovo and electroporating the morpholino antisense oligonucleotides into the precursors of the neural crest. After 24 hr, tenascin-C immunostaining is reduced around the dorsal neural tube in the experimental microinjected embryos (12 of 13) but not in embryos microinjected with control morpholino antisense oligonucleotides (n = 3) or subjected to electroporation only (n = 2). In each of the 12 tenascin-C knockdown embryos neural crest cells are seen ectopically in the lumen of the neural tube and in the neuroepithelium; cells that do leave the neural tube after the microinjection fail to disperse laterally from the surface of the neural tube into the somites. The observation that neural crest cells must express tenascin-C to migrate normally is consistent with a role for this glycoprotein in contributing to the invasive behavior of neural crest cells. © 2001 Wiley-Liss, Inc.
... Whether this gene functions later in ENS development has not been addressed. Similar to knockdown of RET and Gfrα1, knockdown of GDNF also results in complete loss of zebrafish ENS neurons and their precursors (Shepherd et al., 2001). Two other GDNF family members, Neurturin and Artemin, are reported to be present in zebrafish by immunoreactivity, although whether they function in zebrafish ENS development is unknown (Lucini et al., 2005;Lucini et al., 2004). ...
The enteric nervous system (ENS) is composed of neurons and glia that modulate many aspects of intestinal function. The ability to use both forward and reverse genetic approaches and to visualize development in living embryos and larvae has made zebrafish an attractive model in which to study mechanisms underlying ENS development. In this chapter, we review the recent work describing the development and organization of the zebrafish ENS and how this relates to intestinal motility. We also discuss the cellular, molecular, and genetic mechanisms that have been revealed by these studies and how they are providing new insights into human ENS diseases.
... To determine the effects of Gdnf on ENS development, we modulated its activity in avian embryos using retrovirus-mediated gene overexpression and retroviral vector-based gene silencing. While the essential role of Gdnf signaling in ENS development has been established in humans (Amiel and Lyonnet, 2001), rodents (Moore et al., 1996;Pichel et al., 1996;Sanchez et al., 1996), and zebrafish (Shepherd et al., 2001), most of these studies used cultured neural crest-derived cells devoid of the mesenchymal environment known to be important during ENS development (Chalazonitis et al., 1998;Hearn et al., 1998;Heuckeroth et al., 1998;Taraviras et al., 1999;Worley et al., 2000;Young et al., 2001;Natarajan et al., 2002;Ngan et al., 2008). Our approach allowed us to modulate Gdnf activity in vivo, specifically in the presumptive mesoderm of the distal gut, which has major clinical importance in neurointestinal diseases, such as Hirschsprung's disease and intestinal neuronal dysplasia. ...
Glial-derived neurotrophic factor (Gdnf) is required for morphogenesis of the enteric nervous system (ENS) and it has been shown to regulate proliferation, differentiation, and survival of cultured enteric neural crest-derived cells (ENCCs). The goal of this study was to investigate its in vivo role in the colon, the site most commonly affected by intestinal neuropathies such as Hirschsprung's disease. Gdnf activity was modulated in ovo in the distal gut of avian embryos using targeted retrovirus-mediated gene overexpression and retroviral vector-based gene silencing. We find that Gdnf has a pleiotropic effect on colonic ENCCs, promoting proliferation, inducing neuronal differentiation, and acting as a chemoattractant. Down-regulating Gdnf similarly induces premature neuronal differentiation, but also inhibits ENCC proliferation, leading to distal colorectal aganglionosis with severe proximal hypoganglionosis. These results indicate an important role for Gdnf signaling in colonic ENS formation and emphasize the critical balance between proliferation and differentiation in the developing ENS.
... Quail NCCs were grafted into chick embryos prior to the onset of migration and this led to the presence of quail neurons and glia in the gut of the grafted chick embryos supporting the neural crest origin of the ENS (Le Douarin & Teillet, 1973). Cell lineage studies revealed a similar origin for the ENS in mammals and zebrafish (Serbedzija et al., 1991;Shepherd et al., 2001). Even though NCCs are produced from the entire length of the neural axis, only certain tightly defined regions of the neural crest give rise to the ENS, more specifically the vagal and sacral regions . ...
The enteric nervous system (ENS) in vertebrates is derived from neural crest cells which emerge during embryogenesis from the hindbrain and, following stereotypical migratory pathways, colonize the entire gastrointestinal tract. Assembly of enteric ganglia and formation of functional neuronal circuits throughout the gut depends on the highly regulated differentiation of enteric neural crest stem cells (eNCSCs) into a plethora of neuronal subtypes and glia. The identification of eNCSCs and the lineages they generate is fundamental to understand ENS organogenesis. However, the study of the properties of eNCSCs has been hindered by the lack of specific markers and genetic tools to efficiently identify and follow these cells in vivo.
Although previous in vitro studies have suggested that Sox10-expressing cells of the mammalian gut generate both enteric neurons and glia, the differentiation potential of these Sox10+ cells in vivo is currently unclear. Here, we have developed a genetic marking system which allows us to identify Sox10+ cells and follow their fate in vivo. Using this system we demonstrate that Sox10+ cells of the gut generate both enteric neurons and glia in vivo, thus representing multilineage ENS progenitors.
To examine whether the neurogenic potential of Sox10+ eNCSCs is temporally regulated over the course of gut organogenesis, we generated additional transgenic mouse lines expressing a tamoxifen-inducible Cre recombinase (iCreERT2) under the control of the Sox10 locus (Sox10iCreERT2). Activation of iCreERT2 in Sox10iCreERT2 transgenic mice at specific developmental stages and analysis of enteric ganglia from adult animals showed that the pool of Sox10+ cells progressively lose their neurogenic potential.
These findings raise the question of the origin of multilineage ENS progenitors isolated from cultures of post-neurogenic gut. By combining genetic fate mapping in mice, cultures of enteric ganglia and an ENS injury model, we demonstrate that glial cells in the adult ENS retain neurogenic potential which can be activated both in vitro and in vivo, in response to injury. The signals that lead Sox10+ progenitor cells to become either neurons or glial cells remain unclear. We hypothesized that the receptor tyrosine kinase RET may be part of the molecular fate switch between the two lineages being able to divert differentiation of eNCSCs away from the glial lineage and towards the neuronal fate. Here, we describe a genetic strategy to attain persistent expression of RET in vivo, in a temporally and spatially controlled manner. Such a strategy will allow us to assess the role of RET in ENS differentiation during development. Taken together, our data provide a framework for exploring the molecular mechanisms that control enteric neurogenesis in vivo and identify glial cells as a potential target for cell replacement therapies in cases associated with congenital absence or acquired loss of enteric neurons.
... In both phyla, there is expression of homologues of mammalian NGF (Turner et al., 1980;Turner et al., 1982;Yip & Grafstein, 1982), BDNF (Nilsson et al., 1998;Duprey-Diaz et al., 2002), and NT-3, together with Trk-A/-B/-C, and p75 NTR (e.g., Xenopus xp75 NTF a/x p75 NTF b) (Hutson & Bothwell, 2001;Tamura et al., 2004), and additional neurotrophin family members, e.g. NT-6 and NT-7 (Hallbook et al., 1991;Gotz et al., 1994;Lewin & Barde, 1996;Caminos et al., 1999), together with GDNF (Shepherd et al., 2001), CNTF (Jo et al., 1999;Petrausch et al., 2000), and FGF-2 (Soto et al., 2003). Novel axogenic factors are also present including AF-1/2 (Schwalb et al., 1995;Schwalb et al., 1996;Yin et al., 2003) which may regulate axon regeneration (Sivron & Schwartz, 1994a;Sivron & Schwartz, 1994b) through a purine-sensitive pathway (Petrausch et al., 2000;Irwin et al., 2006). ...
This review will describe the unique advantages that are offered by the visual system of mammals and other vertebrates for studying the regenerative responses of the central nervous system (CNS) to injury, and recent insights provided by such studies. In the mouse and rat visual system a variety of experimental paradigms promote survival of retinal ganglion cells (RGC) and optic nerve regeneration, probably through stimulation by neurotrophic factors (NTF) either directly, or indirectly through retinal astrocyte/Müller cell intermediary activation. NTF induce disinhibition of axon growth through regulated intramembranous proteolysis of p75NTR, and the inactivation of RhoA and EGFR signalling. The concomitant release of metalloproteinases (MMP) and plasminogen activators from RGC axons, and tissue inhibitors of metalloproteinases from optic nerve glia repress scarring and thereby reduce titres of scar-derived inhibitory ligands expressed in the wound. MMP also degrade myelin-derived inhibitory ligands along regenerating axon trajectories after regulated release from glia at the growing front of regenerating RGC axons. Optic nerve transection induces apoptosis of RGC which is blocked by anti-apoptotic regimes and thus, in combination with blockers of axon-growth inhibitory signalling and promoters of axon growth may be a therapeutic formula for promoting sustained axon regeneration. All these findings in the visual system are translatable to the CNS as a whole and thus strategies that successfully promote visual axon regeneration will be equally effective elsewhere in the CNS. Future developments likely to advance the field of regenerative research include a greater understanding of phylogenetic differences in the response of the CNS to injury, the role of NTF, cAMP, EGFR, glia/neuron interactions in disinhibiting and promoting axon growth, the control of neuron death, and effective drug delivery.
The enteric nervous system (ENS) is the intrinsic nervous system of the intestine and regulates important gut functions, including motility, nutrient uptake, and immune response. The development of the ENS begins during early organogenesis and continues to develop once feeding begins, with ongoing plasticity in adulthood. There has been increasing recognition that the intestinal microbiota and ENS interact during critical periods, with implications for normal development and potentially disease pathogenesis. In this review, we will focus on insights from mouse and zebrafish model systems to compare and contrast how each model can serve in elucidating the bidirectional communication between the ENS and the microbiome. At the end of this review, we further outline implications for human disease and highlight research innovations that can lead the field forward.
The Enteric Nervous System (ENS) is a large network of enteric neurons and glia that regulates various processes in the gastrointestinal tract including motility, local blood flow, mucosal transport and secretion. The ENS is derived from stem cells coming from the neural crest that migrate into and along the primitive gut. Defects in ENS establishment cause enteric neuropathies, including Hirschsprung disease (HSCR), which is characterized by an absence of enteric neural crest cells in the distal part of the colon. In this review, we discuss the use of zebrafish as a model organism to study the development of the ENS. The accessibility of the rapidly developing gut in zebrafish embryos and larvae, enables in vivo visualization of ENS development, peristalsis and gut transit. These properties make the zebrafish a highly suitable model to bring new insights into ENS development, as well as in HSCR pathogenesis. Zebrafish have already proven fruitful in studying ENS functionality and in the validation of novel HSCR risk genes. With the rapid advancements in gene editing techniques and their unique properties, research using zebrafish as a disease model, will further increase our understanding on the genetics underlying HSCR, as well as possible treatment options for this disease.
Rett syndrome is a severe neurodevelopmental disorder. It is caused by a mutation in methyl-CpG binding protein 2 (MecP2), a transcriptional regulator that recruits protein complexes involved in histone modification and chromatin remodeling. However, the role of Mecp2 in Rett syndrome remains unclear. In this study, we investigated the function of Mecp2 in neuronal development using zebrafish embryos. Mecp2 expression was detected ubiquitously in the central nervous system and muscles at 28 hours post fertilization (hpf). We injected an antisense morpholino oligonucleotide (AMO) to induce Mecp2 knockdown phenotype. In mecp2 morphants (embryos with Mecp2 knockdown by AMO) at 28 and 72 hpf, we found an increase in abnormal axonal branches of caudal primary motor neurons and a decrease in motor activity. In mecp2 morphants at 24 hpf, we observed an increase in the expression of an mecp2 downstream candidate gene, brain derived neurotrophic factor (bdnf). In mecp2 morphants at 72 hpf, the presynaptic area stained by an anti-SV2 antibody was increased at the neuromuscular junction (NMJ). Interestingly, the size of SV2-positive presynaptic area at the NMJ was also increased following bdnf mRNA injection, while it was normalized in a double knockdown of mecp2 and bdnf. These results imply that Mecp2 is an important functional regulator of bdnf gene expression during neural circuit formation in zebrafish embryo. This article is protected by copyright. All rights reserved.
The RET (REarranged during Transfection) gene encodes the tyrosine kinase membrane receptor (RET) for glial cell line-derived neurotrophic factor (GDNF) family ligands (GFL). The human RET gene is located on the long arm of chromosome 10 (10q11.2). RET protein contains an N-terminal glycosylated extracellular portion, with four cadherin-like and one cysteine-rich domain, a central hydrophobic transmembrane segment, and a cytosolic domain that has the tyrosine kinase (TK) activity. RET activation is achieved through the formation of a ternary complex with GFLs and glycosylphosphatidylinositol (GPI)-anchored co-receptors of the GDNF receptor-α (GFRα) family. Upon binding to GFL-GFRα, RET protein undergoes dimerization and activation. RET loss-of-function mutations are found in developmental disorders such as Hirschsprung’s disease, characterized by the congenital absence of the enteric innervation, and congenital anomalies of the kidney or lower urinary tract. Gain-of-function mutations in RET are the driver events of the hereditary cancer syndromes named multiple endocrine neoplasia (MEN) type 2A and 2B. Gene rearrangements fusing the tyrosine kinase domain of RET with the N-terminal portion of heterologous proteins lead to the formation of chimeric oncoproteins endowed with constitutive catalytic activity in papillary thyroid carcinoma and other human malignancies. Finally, altered RET expression has been linked to several additional human neoplasms, including breast carcinoma.
This chapter focuses on those molecules, which in a variety of biological systems, have been shown to have conserved distributions and/or functions in the vertebrate kidney forms. The chapter focuses on the pronephric studies that have been carried out and attempts to draw parallels in structure, developmental expression patterns, and function among the four developmental models for kidney development—mouse, chick, zebrafish, and Xenopus. Many of the genes that have a role in kidney organogenesis have been identified by targeted mutagenesis studies. A brief review of these results is included in the chapter to allow developmental comparisons to be made. The chapter discusses the roles of transcription factors and signaling molecules in pronephric development followed by some other molecular players in pronephric development in amphibia and fish. There are three ways in which major advances are likely to be made in understanding the molecular control of pronephros development and differentiation. The first is the continued search for new genes that are expressed early in the developing pronephros at the time of specification. The second is the development of morpholino antisense technology that can be used to specifically inhibit the translation of mRNAs in early stages of development. The final approach is to fully exploit transgenics and targeted expression.
Glial cell-line derived neurotrophic factor (GDNF) and the GFRα co-receptors play a role in the developing enteric nervous system. The co-receptors elicit their action by binding receptor tyrosine kinase RET.
This immunohistochemical study reports the presence of GDNF and its specific co-receptor GFRα1 in the cat gastrointestinal apparatus during development, from stage 9 to 22. At stage 9 and 11, immunoreactivity (IR) to GDNF was observed in the cells of mesenchyme of the anterior gut. From stage 14 to 22, GDNF IR was detected in nervous plexuses; moreover, GDNF and GFRα1 IR appeared localized in gastrointestinal endocrine cells. The presence of GDNF in the enteric nervous system and in the endocrine cells suggests an involvement of this neurotrophic factor in the gastrointestinal development. Moreover, the presence of the co-receptor GFRα1 in endocrine cells and its absence in the enteric nervous system seems to indicate a different mode of transduction of GDNF signal. GFRα2 and GFRα3 co-receptors were not detected.
In most fish species investigated, the gastrointestinal tract is a highly innervated organ. Like in other vertebrates, the autonomic nerves control a variety of gut functions, from breakdown and transport of food to blood flow, osmoregulation and barrier functions. The autonomic innervation includes both local intrinsic nerves (the enteric nervous system) and extrinsic sympathetic and parasympathetic nerves. The autonomic nerves take part in involuntary reflex signaling. Hence, sensory extrinsic neurons, although strictly speaking not autonomic, are often considered in the same context as the autonomic motor neurons. They respond to various stimuli, many of which can be related to the presence of food in the gastrointestinal tract, and make up elaborate reflex pathways. The final effectors include smooth muscle cells in the gut wall and blood vessels, as well as glandular cells. They may also include other cell types that are involved in the signaling pathways like endocrine cells, interstitial cells of Cajal (ICCs) and glial cells. In this chapter, the layout of the enteric innervation in fish is presented, focusing on transmitter distribution and effect on gut motility. Furthermore, areas where knowledge of fish is still substantially lacking, like functional cell types and reflexes, are discussed.
The chapter summarizes current knowledge on the neuronal and endocrine control of gut functions such as motility, secretion and absorption in the fish gut. Most knowledge is on elasmobranch and teleost species, but what little is known from the other groups is included when relevant. The anatomy of the gut innervation and the endocrine system of the gut is outlined. Many studies have concerned the identity and distribution of neurotransmitters and gut hormones, and this is summarized in one section. Most functional studies concern different aspects of motility control, and this comprises a major part of the chapter, but the more scarce knowledge on control of gut circulation, secretion, water and ion transport, and absorptive processes are also included. Recent studies dealing with the development of the fish gut nervous and endocrine control systems are reported. Comparisons are made between fish species and groups, and, when relevant, with vertebrates in general.
Glial fibrillary acidic protein (GFAP) is a commonly used marker to identify enteric glia in the mammalian gut. Little is however known about enteric glia in other vertebrates. The aim of the present study was to examine the distribution of GFAP immunoreactivity in adult and developing fish. In adult shorthorn sculpin (Myoxocephalus scorpius) and zebrafish (Danio rerio), GFAP immunoreactivity was seen in the myenteric plexus in all regions of the gut. Co-staining for the neuronal markers Hu C/D and acetylated tubulin showed that GFAP immunoreactivity was not associated with nerves. GFAP immunoreactivity was predominantly seen in processes with few glial cell bodies being demonstrated in adult fish. GFAP immunoreactivity was also found in the gut in larval zebrafish from 3 days post-fertilisation, i.e. at approximately the same time that differentiated enteric nerve cells first occur. Immunoreactivity was most prominent in areas with no or a low density of Hu-immunoreactive nerve cell bodies, indicating that the developing glia follows a different pattern from that of enteric neurons. The results suggest that GFAP can be used as a marker for enteric glia in fish, as in birds and mammals. The distribution of GFAP immunoreactivity implies that enteric glia are widespread in the fish gastrointestinal tract. Glia and neurons diverge early during development of the gastrointestinal tract.
In this review we summarize current understanding of the development of autonomic neurons in vertebrates. The mechanisms controlling the development of sympathetic and enteric neurons have been studied in considerable detail in laboratory mammals, chick and zebrafish, and there are also limited data about the development of sympathetic and enteric neurons in amphibians. Little is known about the development of parasympathetic neurons apart from the ciliary ganglion in chicks. Although there are considerable gaps in our knowledge, some of the mechanisms controlling sympathetic and enteric neuron development appear to be conserved between mammals, avians and zebrafish. For example, some of the transcriptional regulators involved in the development of sympathetic neurons are conserved between mammals, avians and zebrafish, and the requirement for Ret signalling in the development of enteric neurons is conserved between mammals (including humans), avians and zebrafish. However, there are also differences between species in the migratory pathways followed by sympathetic and enteric neuron precursors and in the requirements for some signalling pathways.
Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2002. Includes bibliographical references (p. 165-178). Antisense oligonucleotides have the potential to selectively inhibit the expression of any gene with a known sequence. Antisense-based therapies are under development for the treatment of infectious diseases as well as complex genetic disorders. Although there have been some remarkable successes, realizing this potential is proving difficult because of problems with oligonucleotide stability, specificity, affinity, and delivery. Each of these limitations has been addressed experimentally through the use of chemically-modified oligonucleotides and oligonucleotide conjugates, with much success in enhancing oligonucleotide efficacy. These early studies have shown that selection of target site, once considered a trivial problem, is critical to the success of antisense strategies. It has become clear that the efficacy of antisense oligonucleotides is a strong function of the structure of the target mRNA. Though single-stranded, RNA molecules are typically folded into complex three-dimensional structures, formed primarily by intramolecular Watson-Crick base-pairing. If an oligonucleotide is complementary to a sequence embedded in the three dimensional structure, the oligonucleotide may not be able to bind to its target site and exert its therapeutic effect. Because the majority of the structure of RNA molecules is due to Watson-Crick base-pairing, relatively accurate predictions of these folding interactions can be made from algorithms that locate the structure with the most favorable free energy of folding. (cont.) Taking advantage of the predictability of RNA structures, this thesis addresses the problem of antisense target site selection, first from a theoretical and subsequently an experimental standpoint. A thermodynamic model to predict the binding affinity of oligonucleotides for their target mRNA is described and validated using multiple in vitro and cell-culture based experimental data sets. Subsequently, direct experimental comparisons with theoretical predictions are made on the well-characterized rabbit-[beta]-globin (RBG) mRNA, using a novel, centrifugal, binding affinity assay. The importance of the hybridization kinetics is also explored, as is the role of association kinetics in defining the rate of cleavage by the enzyme ribonuclease H (RNase H). Finally, the applicability of the model in identifying biologically active oligonucleotides is demonstrated. by S. Patrick Walton. Sc.D.
The enteric nervous system is the largest subdivision of the peripheral nervous system that plays a critical role in digestive functions. Despite considerable progress over the last 15 years in understanding the molecular and cellular mechanisms that control the development of the enteric nervous system, several questions remain unanswered. The present review will focus on recent progress on understanding the development of the mammalian enteric nervous system and highlight interesting directions of future research.
Glial cell line-derived neurotrophic factor (GDNF) has a neuroprotective role in Purkinje cells of cerebellum, promoting the survival and the differentiation of these cells. Its signalling is mediated by a receptorial complex GFRalpha1/RET. In the brain of adult zebrafish (Danio rerio) we previously investigated GDNF expression and localization, but no data exist regarding GFRalpha1 and RET presence. Thus, the present study was designed to clarify the morphological relation between GDNF and its receptorial complex GFRalpha1/RET immunoreactivity in the cerebellum of adult zebrafish. The expression of gdnf, GFRalpha1 and ret genes was demonstrated in adult zebrafish cerebellum by a standard RT-PCR. The distribution of GDNF and its receptorial complex GFRalpha1/RET was examined by single and double immunocytochemical stainings. In the valvula and corpus cerebelli GDNF, GFRalpha1 and RET immunoreactivity was seen co-localized in Purkinje cells, identified morphologically and by using an antiserum against a specific marker for these cells, aldolase C enzyme. In the vestibulolateralis lobe, Purkinje neurons were lacking in both the eminentiae granulares and medial caudal lobe. These results demonstrated the expression of the GDNF receptorial complex in adult zebrafish cerebellum and suggest an autocrine mode of action of GDNF in Purkinje cells.
The enteric nervous system (ENS) is the largest and most complicated subdivision of the peripheral nervous system. Its action is necessary to regulate many of the functions of the gastrointestinal tract including its motility. Whilst the ENS has been studied extensively by developmental biologists, neuroscientists and physiologists for several decades it has only been since the early 1990s that the molecular and genetic basis of ENS development has begun to emerge. Central to this understanding has been the use of genetic model organisms. In this article, we will discuss recent advances that have been achieved using both mouse and zebrafish model genetic systems that have led to new insights into ENS development and the genetic basis of Hirschsprung's disease.
The enteric nervous system follows a similar overall arrangement in all vertebrate groups. In fish, the majority of nerve cell bodies are found in the myenteric plexus, innervating muscles, blood vessels and glands. In this review, I describe similarities and differences in size, shape and transmitter content in enteric neurons in different fish species and also in comparison with other vertebrates, foremost mammals. The use of different histological and immunochemical methods is reviewed in a historical perspective including advantages and disadvantages of different methods. Lately, zebrafish have become an important model species for developmental studies of the nervous system, including the enteric nervous system, and this is briefly discussed. Finally, examples of how the enteric nervous system controls gut activity in fish is presented, focussing on the effect on gastrointestinal motility.
The rapid embryonic development and high fecundity of zebrafish contribute to the great advantages of this model for the study of developmental genetics. Transient disruption of the normal function of a gene during development can be achieved by microinjecting mRNA, DNA or short chemically stabilized anti-sense oligomers, called morpholinos (MOs), into early zebrafish embryos. The ensuing develop ment of the microinjected embryos is observed over the following hours and days to analyze the impact of the microinjected products on embryogenesis. Compared to stable reverse genetic approaches (sta ble transgenesis, targeted mutants recovered by TILLING), these transient reverse genetic approaches are vastly quicker, relatively affordable, and require little animal facility space. Common applications of these methodologies allow analysis of gain-of-function (gene overexpression or dominant active), loss-of-function (gene knock down or dominant negative), mosaic analysis, lineage-restricted studies and cell tracing experiments. The use of these transient approaches for the manipulation of gene expression has improved our understanding of many key developmental pathways including both the Wnt/beta-catenin and Wnt/PCP pathways, as covered in some detail in Chapter 17 of this book. This chapter describes the most common and versatile approaches: gain of function and loss of function using DNA and mRNA injections and loss of function using MOs.
Development of the enteric nervous system is critical for normal functioning of the digestive system. In vertebrates, enteric precursors originate from the neural crest and migrate into the digestive system. Enteric neurons enable the digestive system to sense and respond to local conditions without the need for central nervous system input. Here we describe major steps in differentiation of the zebrafish enteric nervous system. During migration and neural differentiation of enteric precursors, we identify regions of the enteric nervous system in different phases of differentiation. Early in migration, a small group of anterior enteric neurons are first to form. This is followed by an anterior to posterior wave of enteric neural differentiation later in the migratory phase. Enteric precursors continue proliferating and differentiating into the third day of embryogenesis. nNOS neurons form early while serotonin neurons form late toward the end of enteric neural differentiation. Numbers of enteric neurons increase gradually except during periods of circular and longitudinal intestinal smooth muscle differentiation.
The zebrafish pronephric kidney provides a simplified model of nephron development and epithelial cell differentiation which is amenable to genetic analysis. The pronephros consists of two nephrons with fused glomeruli and paired pronephric tubules and ducts. Nephron formation occurs after the differentiation of the pronephric duct with both the glomeruli and tubules being derived from a nephron primordium. Fluorescent dextran injection experiments demonstrate that vascularization of the zebrafish pronephros and the onset of glomerular filtration occurs between 40 and 48 hpf. We isolated fifteen recessive mutations that affect development of the pronephros. All have visible cysts in place of the pronephric tubule at 2–2.5 days of development. Mutants were grouped in three classes: (1) a group of twelve mutants with defects in body axis curvature and manifesting the most rapid and severe cyst formation involving the glomerulus, tubule and duct, (2) the fleer mutation with distended glomerular capillary loops and cystic tubules, and (3) the mutation pao pao tang with a normal glomerulus and cysts limited to the pronephric tubules. double bubble was analyzed as a representative of mutations that perturb the entire length of the pronephros and body axis curvature. Cyst formation begins in the glomerulus at 40 hpf at the time when glomerular filtration is established suggesting a defect associated with the onset of pronephric function. Basolateral membrane protein targeting in the pronephric duct epithelial cells is also severely affected, suggesting a failure in terminal epithelial cell differentiation and alterations in electrolyte transport. These studies reveal the similarity of normal pronephric development to kidney organogenesis in all vertebrates and allow for a genetic dissection of genes needed to establish the earliest renal function.
The members of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors (GDNF, neurturin, persephin, and artemin) are able to promote in vivo and in vitro survival of different neuronal populations, including spinal cord motoneurons. These factors signal via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a member of the GDNF family receptor α (GRFα) family of glycosylphosphatidylinositol-linked coreceptors. Activation of the receptor induces Ret phosphorylation that leads the survival-promoting effects. Ret phosphorylation causes the activation of several intracellular pathways, but the biological effects caused by the activation of each of these pathways are still unknown. In the present work, we describe the ability of the GDNF family members to promote chicken motoneuron survival in culture. We show the presence of Ret and GFRα-1, GFRα-2, and GFRα-4 in chicken motoneurons using in situ hybridization and reverse transcription-PCR techniques. By Western blot analysis and kinase assays, we demonstrate the ability of these factors to induce the phosphatidylinositol 3 kinase (PI 3-kinase) and the extracellular regulated kinase (ERK)–mitogen-activated protein (MAP) kinase pathways activation. To characterize the involvement of these pathways in the survival effect, we used the PI 3-kinase inhibitor LY 294002 and the MAP kinase and ERK kinase (MEK) inhibitor PD 98059. We demonstrate that LY 294002, but not PD 98059, prevents GDNF-, neurturin-, and persephin-induced motoneuron survival, suggesting that PI 3-kinase intracellular pathway is responsible in mediating the neurotrophic effect.
The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFR alpha-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFR alpha-1 or GFR alpha-2, a novel receptor related to GFR alpha-1. Both GFR alpha-1 and GFR alpha-2 mediate neurturin-induced Ret phosphorylation. GDNF can also bind to either GFR alpha-1 or GFR alpha-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFR alpha-1 bind GDNF more efficiently than neurturin, while cells expressing GFR alpha-2 bind neurturin preferentially. Crosslinking and Ret activation data also suggest that while there is cross-talk, GFR alpha-1 is the primary receptor for GDNF and GFR alpha-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFR alpha receptor family. This putative receptor, designated GFR alpha-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFR alpha-1 and GFR alpha-2. Analysis of the tissue distribution of GFR alpha-1, GFR alpha-2, GFR alpha-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFR alpha s and Ret are expressed in many of the same tissues, suggesting that GFR alpha s mediate the action of GDNF family ligands in vivo.
Neurturin (NTN) is a neurotrophic factor that shares homology with glial cell line-derived neurotrophic factor (GDNF). Recently,
a receptor complex has been identified for GDNF that includes the Ret tyrosine kinase receptor and a glycosylphosphatidylinositol-linked
protein termed “GDNFRα.” However, differences in the phenotype of Ret and GDNF knockout animals suggest that Ret has at least
one additional ligand. In this report, we demonstrate that NTN induces Ret phosphorylation in primary cultures of rat superior
cervical ganglion (SCG) neurons. NTN also caused Ret phosphorylation in fibroblasts that were transfected stably with Ret
and GDNFRα but not in cells expressing Ret alone. A glycosylphosphatidylinositol-linked protein also was important for NTN
and GDNF signaling in SCG neurons; phosphatidylinositol-specific phospholipase C treatment of SCG cultures reduced the ability
of NTN to phosphorylate Ret and the ability of NTN or GDNF to activate the mitogen-activated protein kinase pathway. NTN and
GDNF also caused sustained activation of Ret and the mitogen-activated protein kinase pathway in SCG neurons. Finally, both
NTN and GDNF activated the phosphatidylinositol 3-kinase pathway in SCG neurons, which may be important for the ability of
NTN and GDNF to promote neuronal survival. These data indicate that NTN is a physiologically relevant ligand for the Ret receptor
and suggest that NTN may have a critical role in the development of many neuronal populations.
In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.
We describe a new primer design strategy for PCR amplification of unknown targets that are related to multiply-aligned protein
sequences. Each primer consists of a short 3′ degenerate core region and a longer 5′ consensus clamp region. Only 3–4 highly
conserved amino acid residues are necessary for design of the core, which is stabilized by the clamp during annealing to template
molecules. During later rounds of amplification, the non-degenerate clamp permits stable annealing to product molecules. We
demonstrate the practical utility of this hybrid primer method by detection of diverse reverse transcriptaselike genes in
a human genome, and by detection of C5 DNA methyltransferase homologs in various plant DNAs. In each case, amplified products were sufficiently pure to be cloned
without gel fractionation. This COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy has been implemented
as a computer program that is accessible over the World Wide Web (http://blocks.fhcrc.org/codehop.html) and is directly linked from the BlockMaker multiple sequence alignment site for hybrid primer prediction beginning with
a set of related protein sequences.
Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.
Many peptide hormones are produced from larger precursors by endoproteolysis at pairs of basic amino acids (e.g. Lys-Arg and Arg-Arg) within the regulated secretory pathway in endocrine cells. However, many other secretory and membrane proteins appear to be produced from precursors through cleavage at multiple, rather than paired, basic residues within the constitutive secretory pathway in non-endocrine cells. By surveying various precursors processed constitutively, we noticed that most of them have the consensus sequence, Arg-X-Lys/Arg-Arg (RXK/RR), at the cleavage site. When expressed in endocrine and non-endocrine cells, a precursor with the RXKR sequence was cleaved in both types of cells, whereas that with the Lys-Arg pair was cleaved only in the endocrine cells. When the RXKR precursor was coexpressed with furin and PC3, both of which are mammalian homologues of the yeast precursor-processing endoprotease Kex2, in non-endocrine cells, enhancement of the precursor cleavage by furin but not by PC3 was observed. By contrast, when the Lys-Arg precursor was coexpressed with the two mammalian proteases in endocrine cells with no endogenous processing activity at dibasic sites, it was cleaved only by PC3. These results indicate that the basic pair and the RXK/RR sequence are the signals for precursor cleavages catalyzed by PC3 within the regulated secretory pathway and by furin within the constitutive pathway, respectively.
Every trunk hemisegment of the zebrafish is innervated by 3 identified primary motoneurons whose development can be observed directly in living embryos. In this paper, we describe another identified neuron that is part of this system. Unlike the other primary motoneurons which are present in all trunk hemisegments, this cell is present in slightly less than half of the trunk hemisegments. Additionally, this cell has at least 2 different fates: it may become a primary motoneuron and arborize in an exclusive muscle territory, or it may die during embryonic development. We have named this cell VaP, for variable primary. We show that the presence of VaP does not affect the early development of the other primary motoneurons in the same hemisegment. Moreover, we show that ablation of both VaP and caudal primary does not alter pathfinding by another identified primary motoneuron.
To learn how neural segments are structured in a simple vertebrate, we have characterized the embryonic zebrafish hindbrain with a library of monoclonal antibodies. Two regions repeat in an alternating pattern along a series of seven segments. One, the neuromere centers, contains the first basal plate neurons to develop and the first neuropil. The other region, surrounding the segment boundaries, contains the first neurons to develop in the alar plate. The projection patterns of these neurons differ: those in the segment centers have descending axons, while those in the border regions form ventral commissures. A row of glial fiber bundles forms a curtain-like structure between each center and border region. Specific features of the individual hindbrain segments in the series arise within this general framework. We suggest that a cryptic simplicity underlies the eventual complex structure that develops from this region of the CNS.
cDNA encoding the alpha-subunit of the (Na+ + K+)-ATPase was cloned from a chicken kidney cDNA library and the nucleotide sequence determined. The deduced amino acid sequence showed 92% sequence homology with the alpha-subunit of the sheep kidney (Na+ + K+)-ATPase, and high cross-species homologies were found among nucleotide sequences both in the 5'- and 3'-untranslated regions of the "kidney-type" alpha-subunit mRNAs. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk- cells. Expression of the avian alpha-sub-unit could be activated by culture of the cells in 10 mM butyrate. Cells expressing avian alpha-subunits displayed high-affinity ouabain binding (KD = 2.6 +/- 0.7 x 10(-7) M) and ouabain-sensitive 86Rb+ uptake, characteristic of avian cells.
A new method for identifying secretory signal sequences and for predicting the site of cleavage between a signal sequence
and the mature exported protein is described. The predictive accuracy is estimated to be around 75–80% for both prokaryotic
and eukaryotic proteins.
Glial cell line-derived neurotrophic factor (GDNF) promotes survival of midbrain dopaminergic neurons and motoneurons. Expression of GDNF mRNA in cerebellum raises the possibility that cells within this structure might also respond to GDNF. To examine potential trophic activities of GDNF, dissociated cultures of gestational day 18 rat cerebellum were grown for < or = 21 days in the presence of factor. GDNF increased Purkinje cell number without affecting the overall number of neurons or glial cells. A maximal response (50% above control) was elicited with GDNF at 1 pg/ml. Effects of GDNF on Purkinje cell differentiation were examined by scoring the morphologic maturation of cells in treated and control cultures. GDNF increased the proportion of Purkinje cells that displayed relatively mature morphologies, characterized by dendritic thickening and the development of spines and filopodial extensions. Morphologic maturation of the overall neuronal population was unaffected. In sum, our data indicate that GDNF is a potent survival and differentiation factor for Purkinje cells, the efferent neurons of cerebellar cortex. Together with its other actions, these findings raise the possibility that GDNF might be a critical trophic factor at multiple loci in neuronal circuits that control motor function.
Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor-beta (TGF-beta), originally isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons, and more recently shown to be a potent neurotrophic factor for motorneurons. The biological activities and distribution of this molecule outside the central nervous system are presently unknown. We report here on the mRNA expression, biological activities and initial receptor binding characterization of GDNF and a shorter spliced variant termed GDNF beta in different organs and peripheral neurons of the developing rat. Both GDNF mRNA forms were found to be most highly expressed in developing skin, whisker pad, kidney, stomach and testis. Lower expression was also detected in developing skeletal muscle, ovary, lung, and adrenal gland. Developing spinal cord, superior cervical ganglion (SCG) and dorsal root ganglion (DRG) also expressed low levels of GDNF mRNA. Two days after nerve transection, GDNF mRNA levels increased dramatically in the sciatic nerve. Overall, GDNF mRNA expression was significantly higher in peripheral organs than in neuronal tissues. Expression of either GDNF mRNA isoform in insect cells resulted in the production of indistinguishable mature GDNF polypeptides. Purified recombinant GDNF promoted neurite outgrowth and survival of embryonic chick sympathetic neurons. GDNF produced robust bundle-like, fasciculated outgrowth from chick sympathetic ganglion explants. Although GDNF displayed only low activity on survival of newborn rat SCG neurons, this protein was found to increase the expression of vasoactive intestinal peptide and preprotachykinin-A mRNAs in cultured SCG neurons. GDNF also promoted survival of about half of the neurons in embryonic chick nodose ganglion and a small subpopulation of embryonic sensory neurons in chick dorsal root and rat trigeminal ganglia. Embryonic chick sympathetic neurons expressed receptors for GDNF with Kd 1-5 x 10(-9) M, as measured by saturation and displacement binding assays. Our findings indicate GDNF is a new neurotrophic factor for developing peripheral neurons and suggest possible non-neuronal roles for GDNF in the developing reproductive system.
We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups. We observed similarities but also unique features of the expression patterns of the zebrafish dlx genes. Among the three genes, dlx3 alone is expressed during gastrulation. Shortly after gastrulation, cells in the ventral forebrain rudiment express dlx2 and dlx4, but not dlx3, and hindbrain neural crest cells express only dlx2. Presumptive precursor cells of the olfactory placodes express dlx3 and dlx4 but not dlx2. Transcripts of dlx3 and dlx4 are present in overlapping subsets of cells in the auditory vesicle and in cells of the median fin fold, whereas dlx2 is never expressed in the auditory vesicle and only at low levels in localized regions of the median fin fold. Cells of the visceral arches and their primordia express all three dlx genes, but with different developmental time courses. We suggest that combinatorial expression of the dlx genes is part of a homeobox gene code specifying pattern formation or cell fate determination in the forebrain, in peripheral structures of the head, and in the fins.
Receptor tyrosine kinases (RTKs) are cell-surface molecules that transduce signals for cell growth and differentiation. The RTK encoded by the c-ret proto-oncogene is rearranged and constitutively activated in a large proportion of thyroid papillary carcinomas, and germ-line point mutations in c-ret seem to be responsible for the dominantly inherited cancer syndromes multiple endocrine neoplasia (MEN) types 2A and B. The gene is expressed in the developing central and peripheral nervous systems (sensory, autonomic and enteric ganglia) and the excretory system (Wolffian duct and ureteric bud epithelium) of mice, indicating that it may play a role in normal development. Here we show that mice homozygous for a targeted mutation in c-ret develop to term, but die soon after birth, showing renal agenesis or severe dysgenesis, and lacking enteric neurons throughout the digestive tract. Ret is thus an essential component of a signalling pathway required for renal organogenesis and enteric neurogenesis.
Mesoderm formation is critical for the establishment of the animal body plan and in Drosophila requires the snail gene. This report concerns the cloning and expression pattern of the structurally similar gene snail1 from zebrafish. In situ hybridization shows that the quantity of snail1 RNA increases at the margin of the blastoderm in cells that involute during gastrulation. As gastrulation begins, snail1 RNA disappears from the dorsal axial mesoderm and becomes restricted to the paraxial mesoderm and the tail bud. snail1 RNA increases in cells that define the posterior border of each somite and then disappears when somitic cells differentiate. Later in development, expression appears in cephalic neural crest derivatives. Many snail1-expressing cells were missing from mutant spadetail embryos and the quantity of snail1 RNA was greatly reduced in mutant no tail embryos. The work presented here suggests that snail1 is involved in morphogenetic events during gastrulation, somitogenesis and development of the cephalic neural crest, and that no tail may act as a positive regulator of snail1.
The c-ret proto-oncogene encodes a receptor tyrosine kinase whose normal function has yet to be determined. To begin to investigate the potential role of this gene in vertebrate development, we have isolated cDNA clones representing the murine c-ret gene, and have analyzed the pattern of expression during mouse embryogenesis, using northern blotting, in situ hybridization to histological sections and whole-mount hybridization histochemistry. c-ret transcripts were detected beginning at day 8.5 of embryogenesis, and were observed in a number of cell lineages in the developing peripheral and central nervous systems, as well as in the excretory system. In the cranial region at day 8.5-9.5, c-ret mRNA was restricted to a population of neural crest cells migrating from rhombomere 4 and forming the anlage of the facioacoustic ganglion, as well as to a closely associated domain of surface ectoderm and pharyngeal endoderm. At later stages (10.5-14.5 days), c-ret mRNA was observed in all cranial ganglia. In the peripheral nervous system of the trunk, c-ret was expressed in the autonomic ganglia and in subsets of cells in the dorsal root ganglia. In the enteric nervous system, c-ret was expressed in the presumptive enteric neuroblasts of the vagal crest (day 9.0-11.5), and in the myenteric ganglia of the gut (day 13.5-14.5). c-ret mRNA was observed in several regions of the central nervous system, including the undifferentiated neuroepithelial cells of the ventral neural tube (8.5 days), the motor neurons in the spinal cord and the hindbrain (10.5-14.5 days), the embryonic neuroretina (day 13.5) and the layers of the postnatal retina containing ganglion, amacrine and horizontal cells. Outside the nervous system, c-ret was expressed in the nephric (Wolffian) duct at day 8.5-10.5, the ureteric bud epithelium (but not the surrounding metanephric mesenchyme) at day 11.0-11.5, and the growing tips of the renal collecting ducts (but not the previously formed, subcortical portions of the collecting ducts, or the mesenchyme-derived renal vesicles) at day 13.5-17.5. Our results suggest that the c-ret gene may encode the receptor for a factor involved in the proliferation, migration, differentiation or survival of a variety of neuronal cell lineages, as well as in inductive interactions during organogenesis of the kidney.
Programmed cell death is a prominent feature of normal animal development. During neurogenesis, naturally occurring cell death is a mechanism to eliminate neurons that fail to make appropriate connections. To prevent accidental cell death, mechanisms that trigger programmed cell death, as well as the genetic components of the cell death program, are tightly controlled.
In a large-scale mutagenesis screen for embryonic lethal mutations in zebrafish Danio rerio we have found 481 mutations with a neural degeneration phenotype. Here, we present 50 mutations that fall into two classes (termed spacehead and fala-like) that are characterized by two main features: first, they appear to affect cell survival primarily within the neuroectodermal lineages during somitogenesis, and second, they show an altered brain morphology at or before 28 hours of development. Evidence for the specificity of cell death within the central nervous system comes from visual inspection of dying cells and analysis of DNA fragmentation, a process associated with apoptotic cell death. In mutants, the level of dying cells is significantly increased in brain and spinal cord. Furthermore, at the end of somitogenesis, the cell count of radial glia and trigeminal neurons is reduced in some mutants of the spacehead class.
A variety of neurodegenerative disorders in mouse and humans have been associated with abnormal levels of programmed cell death within the central nervous system. The mutations presented here might provide a genetic framework to aid in the understanding of the etiology of degenerative and physiological disorders within the CNS and the activation of inappropriate programmed cell death.
Overexpression of glial cell line–derived neurotrophic factor (GDNF) by muscle greatly increased the number of motor axons innervating neuromuscular junctions in neonatal mice. The extent of hyperinnervation correlated with the amount of GDNF expressed in four transgenic lines. Overexpression of GDNF by glia and overexpression of neurotrophin-3 and neurotrophin-4 in muscle did not cause hyperinnervation. Thus, increased amounts of GDNF in postsynaptic target cells can regulate the number of innervating axons.
A new family of neuronal survival factors comprised of glial cell line-derived neurotrophic factor (GDNF) and neurturin has
recently been described (Kotzbauer, P. T., Lampe, P. A., Heuckeroth, R. O., Golden, J. P., Creedon, D. J., Johnson, E. M.,
Jr., and Milbrandt, J. (1997)Nature 384, 467–470). These molecules, which are related to transforming growth factor-β, are important in embryogenesis and in the
survival of distinct neuronal populations. These molecules signal through a novel receptor system that includes the Ret receptor
tyrosine kinase, a ligand (i.e. GDNF or neurturin), and an accessory glycosyl-phosphatidylinositol-linked molecule that is responsible for high affinity
binding of the ligand. Two accessory molecules denoted GDNF family receptor 1 and 2 (GFRα-1 and GFRα-2) have been described
that function in GDNF and neurturin signaling complexes. We have identified a novel co-receptor belonging to this family based
on similarity to GFRα-1, which we have named GFRα-3. GFRα-3 displays 33% amino acid identity with GFRα-1 and 36% identity
with GFRα-2. Despite the similarity of GFRα-3 to GFRα-1 and GFRα-2, it is unable to activate Ret in conjunction with GDNF,
suggesting that there are likely additional undiscovered ligands and/or Ret-like receptors to be identified. GFRα-3 is anchored
to the cell membrane by a phosphatidylinositol-specific phospholipase C-resistant glycosyl-phosphatidylinositol linkage. GFRα-3
is highly expressed by embryonic day 11 but is not appreciably expressed in the adult mouse.In situ hybridization analyses demonstrate that GFRα-3 is located in dorsal root ganglia and the superior cervical sympathetic ganglion.
Comparison of the expression patterns of GFRα-3 and Ret suggests that these molecules could form a receptor pair and interact
with GDNF family members to play unique roles in development.
Glial cell line–derived neurotrophic factor (GDNF) signals through a receptor complex composed of the Ret tyrosine kinase and a glycosylphosphatidylinositol- (GPI-) anchored cell surface coreceptor, either GDNF family receptor α1 (GFRα1) or GFRα2. To investigate the usage of these coreceptors for GDNF signaling in vivo, gene targeting was used to produce mice lacking the GFRα1 coreceptor. GFRα1-deficient mice demonstrate absence of enteric neurons and agenesis of the kidney, characteristics that are reminiscent of both GDNF- and Ret-deficient mice. Midbrain dopaminergic and motor neurons in GFRα1 null mice were normal. Minimal or no neuronal losses were observed in a number of peripheral ganglia examined, including the superior cervical and nodose, which are severely affected in both Ret- and GDNF-deficient mice. These results suggest that while stringent physiologic pairing exists between GFRα1 and GDNF in renal and enteric nervous system development, significant cross-talk between GDNF and other GFRα coreceptors must occur in other neuronal populations.
Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF) are the first two members of the GDNF family (GF) of neurotrophic factors. These two proteins are potent survival factors for several populations of central and peripheral neurons in mature and developing rodents. The receptor for these factors is a multicomponent complex that includes the RET (rearranged during transfection) tyrosine kinase receptor and one of two glycosyl phosphatidylinositol (GPI)-linked ligand-binding components called GDNF family receptor alphas (GFRα-1 and GFRα-2). We have used in situ hybridization to study the mRNA expression of NTN, GDNF, RET, GFRα-1, and GFRα-2 in the central nervous system (CNS) of adult mice. GF receptors are expressed in several areas in which neuronal populations known to respond to NTN and GDNF are located, including the ventral horn of the spinal cord and the compacta region of the substantia nigra. In addition, we have demonstrated receptor expression in other areas of the brain including the thalamus and hypothalamus. Neurons in these areas express GF receptors, and therefore, may respond to NTN or GDNF. NTN and GDNF are expressed in targets of neurons that express GF receptors. The pattern of GF factor and receptor expression in the adult brain suggests a role for these factors in maintaining neuronal circuits in the mature CNS. J. Comp. Neurol. 398:139–150, 1998. © 1998 Wiley-Liss, Inc.
The localization of glial cell line-derived neurotrophic factor (GDNF) mRNA was studied by in situhybridization in rat from embryonic (E) day E10 to E15. At E10, GDNF mRNA is found in the urogenital field and the cranial part of the gut. At E11, the most abundant expression of GDNF mRNA is seen in the epithelial cells of the second, third and fourth pharyngeal pouches, the third and fourth pharyngeal arches and pharynx. Also mesenchymal cells of the gut and mesonephric tubules contain GDNF mRNA. At E13, expression is observed in the mesenchymal cell layers of the oesophagus, intestine and stomach, the mesenchymal cells around the condensing cartilages and metanephric kidney mesenchyme. Also, the epithelia of Rathke's pouch and pharynx are intensely labelled. High expression of GDNF mRNA continues at El5 in kidney, gastrointestinal tract and cartilage. At that stage, GDNF mRNA is seen also in whisker pad and skeletal muscles. The distribution of GDNF mRNA in embryonic rat suggests important roles for GDNF in the early differentiation of the kidney tubules, the innervation of the gastrointestinal tract and the differentiation process of the cartilage and muscle. Our results indicate novel functions for GDNF outside the nervous system.
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor-# family and has potent neurotrophic effects on several classes of neurons including dopamine neurons and motoneurons. Here, we have used in situ hybridization to describe the development of the cellular expression of GDNF mRNA pre- and postnatally. Consistent with dopaminotrophic activity, GDNF mRNA is expressed in the developing basal ganglia and the olfactory tubercle. It is also found in a thalamic nucleus, in neurons of the substantia innominata, in the developing Purkinje neurons and the developing locus coeruleus area, and in trigeminal brainstem nuclei. In the spinal cord, neuronal expression is found in Clarke's column. GDNF mRNA is also expressed in the dorsal horns during development. Additional GDNF mRNA expression in the head region includes the carotid body, the retina, the vibrissae, the inner ear, the ear canal, and epithelium in the nasal cavity. Prominent expression is also found in the developing teeth. The widespread expression of GDNF in developing skeletal muscle is consistent with trophic activity on !-motoneurons. The smooth muscle layers of the gastrointestinal tract are also strongly positive. A very strong signal is found in the outer mesenchyme of the developing metanephric kidney. We conclude that GDNF mRNA is expressed in many different cellular systems inside and outside the central nervous system during development, suggesting multiple functions of GDNF in the developing organism.
We have cloned a partial cDNA of chicken glial cell line-derived neurotrophic factor (GDNF) and systematically examined its expression pattern as well as that of GDNF-binding components (GDNF family receptor alpha-1 and 2: GFRα-1 and 2) and a common signal transduction receptor (c-ret protooncogene: RET) during very early developmental stages. In addition, we also examined the expression pattern of an apparent avian-specific binding component, GFRα-4. The cloned chicken cDNA for GDNF had approximately 80% homology to mammalian counterparts. The expression of GDNF mRNA occurred in many spatially and temporally discrete regions such as the intermediate mesoderm, the floor plate of the spinal cord, pharyngeal endoderm contacting the epibranchial placodes, distal ganglia of cranial nerves, subpopulations of mesenchyme cells in the craniofacial region, and in the mesodermal wall of the digestive tract. Both a GDNF receptor signal transduction component (RET) and a binding component (GFRα-1 or GFRα-2) were independently expressed in nearby interacting tissues such as the somites, peripheral and central nervous system, and mesenchyme cells in the craniofacial region. These observations suggest that possible combinations of novel unidentified receptors acting with RET or with GFRαs may mediate GDNF-derived signals and indicate that GDNF or other family members may have previously unidentified actions in early organogenesis in the chick embryo.
The role of cellular interactions in determining the fates of two identified motoneurons in the embryonic zebrafish was investigated by transplanting individual motoneurons from labeled donor embryos to unlabeled hosts. The results suggest that although these cells normally adopt different fates, they form an equivalence group in which one fate is primary and the other is secondary. Both cells are able to adopt the primary fate. A cell that has adopted the secondary fate can be induced to switch to the primary fate by ablating the cell that has adopted the primary fate, even many hours after axogenesis. Although interactions between the two cells appear to regulate which cell adopts the secondary fate, these interactions seem to be independent of neuromuscular activity.
Developing motoneurons in zebrafish embryos follow a stereotyped sequence of axonal outgrowth and accurately project their axons to cell-specific target muscles. During axonal pathfinding, an identified motoneuron pioneers the peripheral motor pathway. Growth cones of later motoneurons interact with the pioneer via contact, coupling, and axonal fasciculation. In spite of these interactions, ablation of the pioneer motoneuron does not affect the ability of other identified motoneurons to select the pathways that lead to appropriate target muscles. We conclude that interactions between these cells during pathfinding are not required for accurate pathway selection.
The mammalian permanent kidney consists of three cell lineages of different origin: the epithelial cells of the ureter bud, the mesenchymal cells of the nephric blastema and the endothelial cells of the capillaries. Organogenesis is governed by a cascade of morphogenetic interactions between these cell populations, a reciprocal epithelial-mesenchymal interaction between the branching ureter and the metanephric mesenchyme, homotypic interactions between cells of the tubular anlagen, stimulation of angiogenesis by the differentiating blastema and a mesenchymal--endothelial interaction guiding the migration of the capillary endothelial cells. While the biology of these interactive events is well known, as described in this overview, the molecular mechanisms are less well mapped out.
We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1-2 h) of the DNA precursor analog bromodeoxyuridine (BrdU) in conjunction with MAb 16A11, we observed Hu+/BrdU+ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu+ cells were ambiguously BrdU+ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation.
Protein blocks consist of multiply aligned sequence segments that correspond to the most highly conserved regions of protein families. Typically, a set of related proteins has more than one region in common and their relationship can be represented as a series of ungapped blocks separated by unaligned regions. Blockmaker is an automated system available by electronic mail ([email protected]
/* */) and the World Wide Web (http://www.blocks.fhere.org4) that finds blocks in a group of related protein sequences submitted by the user. It adapts and extends existing algorithms to make them useful to biologists looking for conserved regions in a group of related proteins sequences. Two sets of blocks are returned, one in which candidate blocks are detected using the MOTIF algorithm and the other using a Gibbs sampler algorithm that has been adapted for full automation. This use of two block-finding methods based on completely different principles provides a 'reality check', whereby a block detected by both methods is considered to be correct. Resulting blocks can be displayed using the information-based 'sequence logo' method, adapted to incorporate sequence weights, which provides an intuitive visual description of both the residue and the conservation information at each position. Blocks generated by this system are useful in diverse applications, such as searching databases and designing degenerate PCR primers. As an example, blocks made from amino acid sequences related to Caenorhabditis elegans Tcl transposase were used to search GenBank, revealing that several fish and amphibian genomic sequences harbor previously unreported Tcl homologs.
Human recombinant glial cell line-derived neurotrophic factor (GDNF) was tested for its ability to stimulate fiber formation and neuron survival in primary cultures of peripheral ganglia dissected from the chicken embryo. GDNF, first characterized by its actions on central nervous system (CNS) neurons, had a marked stimulatory effect on fiber outgrowth in sympathetic and ciliary ganglia. Weaker responses were evoked in sensory spinal and nodose ganglia and in the ganglion of Remak. In addition, survival of neurons from the sympathetic and ciliary ganglia was stimulated by GDNF at 50 ng/ml. The effects were not mimicked by the distant but related protein transforming growth factor beta 1 (TGF beta 1). The profile of neurons stimulated by GDNF is also distinct from the patterns of stimulation shown by nerve growth factor (NGF), stimulating strongly sympathetic but not ciliary ganglia, and ciliary neurotrophic factor (CNTF), stimulating mainly the ciliary ganglion. Moreover, using in situ hybridization histochemistry, GDNF was demonstrated to be present in the pineal gland in the newborn rat, a target organ for sympathetic innervation. The present results suggest that GDNF is likely to act upon receptors present in several autonomic and sensory neuronal populations. GDNF may serve to support fiber outgrowth and cell survival in peripheral ganglia, adding yet one more trophic factor to the list of specific proteins controlling development and maintenance of the peripheral nervous system.
The c-ret proto-oncogene encodes a receptor tyrosine kinase that plays important roles in human disease and in normal mammalian development. Mutations in the human RET gene are associated with multiple endocrine neoplasia syndromes and Hirschsprung's disease in humans, while targeted mutagenesis of murine c-ret resulted in severe developmental abnormalities affecting the excretory and peripheral nervous systems. To examine the evolutionary conservation of the ret protein sequence and its developmental expression pattern, we isolated and sequenced cDNA clones of chicken c-ret and examined its expression in chick embryos and adult tissues. The cytoplasmic domains of chicken and human ret were relatively well conserved (91% similar), but the extracellular domains were more divergent (68% similar), although the conservation of cysteine residues in this region suggests a conserved secondary structure. As in mouse and human, chicken c-ret encodes two protein isoforms. The number and sizes of the transcripts were similar to those in human and mouse cells, and during chick embryogenesis, c-ret mRNA was observed in many of the same sites as in the mouse, including the Wolffian duct and ureteric bud, the enteric, dorsal root, sympathetic and facioacoustic ganglia, and the ventral spinal cord. Evolutionary differences in expression were observed in the trigeminal ganglion, the ventral roots of the spinal cord, the mesenchymal cells of the branchial arches and the adult testes. The results are discussed with regard to the role of the ret receptor in normal development and disease.
For survival, embryonic motoneurons in vertebrates depend on as yet undefined neurotrophic factors present in the limb bud.
Members of the neurotrophin family are currently the best candidates for such neurotrophic factors, but inactivation of their
receptor genes leads to only partial loss of motoneurons, which suggests that other factors are involved. Glial cell line-derived
neurotrophic factor (GDNF), originally identified as a trophic factor specific for dopaminergic neurons, was found to be 75-fold
more potent than the neurotrophins in supporting the survival of purified embryonic rat motoneurons in culture. GDNF messenger
RNA was found in the immediate vicinity of motoneurons during the period of cell death in development. In vivo, GDNF rescues
and prevents the atrophy of facial motoneurons that have been deprived of target-derived survival factors by axotomy. GDNF
may therefore be a physiological trophic factor for spinal motoneurons. Its potency and specificity in vitro and in vivo also
make it a good candidate for treatment of motoneuron disease.
A potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons was purified and cloned. Glial cell line-derived
neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer that is a distantly related member of the transforming
growth factor-beta superfamily. In embryonic midbrain cultures, recombinant human GDNF promoted the survival and morphological
differentiation of dopaminergic neurons and increased their high-affinity dopamine uptake. These effects were relatively specific;
GDNF did not increase total neuron or astrocyte numbers nor did it increase transmitter uptake by gamma-aminobutyric-containing
and serotonergic neurons. GDNF may have utility in the treatment of Parkinson's disease, which is marked by progressive degeneration
of midbrain dopaminergic neurons.
Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for central and peripheral neurons, and is essential for the development of kidneys and the enteric nervous system. Despite the potential clinical and physiological importance of GDNF, its mechanism of action is unknown. Here we show that physiological responses to GDNF require the presence of a novel glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrate that GDNF promotes the formation of a physical complex between GDNFR-alpha and the orphan tyrosin kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF uses a multi-subunit receptor system in which GDNFR-alpha and Ret function as the ligand-binding and signalling components, respectively.
Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for embryonic midbrain dopaminergic, spinal motor, cranial sensory, sympathetic, and hindbrain noradrenergic neurons, and is available to these cells in vivo. It is therefore considered a physiological trophic factor and a potential therapeutic agent for Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Here we show that at postnatal day 0 (P0), GDNF-deficient mice have deficits in dorsal root ganglion, sympathetic and nodose neurons, but not in hindbrain noradrenergic or midbrain dopaminergic neurons. These mice completely lack the enteric nervous system (ENS), ureters and kidneys. Thus GDNF is important for the development and/or survival of enteric, sympathetic and sensory neurons and the renal system, but is not essential for catecholaminergic neurons in the central nervous system (CNS).
Glial-lial-cell-line-derived neurotrophic factor (GDNF) has been isolated as neurotrophic factor for midbrain dopaminergic neurons. Because of its neurotrophic activity on a wide range of neuronal populations in vitro and in vivo, GDNF is being considered as a potential therapeutic agent for neuronal disorders. During mammalian development, it is expressed not only in the nervous system, but also very prominently in the metanephric kidney and the gastrointestinal tract, suggesting possible functions during organogenesis. We have investigated the role of GDNF during development by generating a null mutation in the murine GDNF locus, and found that mutant mice show kidney agenesis or dysgenesis and defective enteric innervation. We demonstrate that GDNF induces ureter bud formation and branching during metanephros development, and is essential for proper innervation of the gastrointestinal tract.
Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for dopaminergic neurons and motor neurons in culture. It also protects these neurons from degeneration in vitro, and improves symptoms like Parkinson's disease induced pharmacologically in rodents and monkeys. Thus GDNF might have beneficial effects in the treatment of Parkinson's disease and amyotrophic lateral sclerosis. To examine the physiological role of GDNF in the development of the mammalian nervous system, we have generated mice defective in GDNF expression by using homologous recombination in embryonic stem cells to delete each of its two coding exons. GDNF-null mice, regardless of their targeted mutation, display complete renal agencies owing to lack of induction of the ureteric bud, an early step in kidney development. These mice also have no enteric neurons, which probably explains the observed pyloric stenosis and dilation of their duodenum. However, ablation of the GDNF gene does not affect the differentiation and survival of dopaminergic neurons, at least during embryonic development.
We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.
We describe the cloning of the mouse glial cell line-derived neurotrophic factor (GDNF) gene and its expression during embryogenesis. GDNF is a distant member of the superfamily of TGF-beta related genes that was originally identified on the basis of its striking neurotrophic activity. GDNF is expressed in a highly dynamic pattern in the anterior neuroectoderm during early stages of neurogenesis between E7.5 and E10.5. Beginning at E10.5 GDNF is also expressed in several organs that develop through inductive epithelial-mesenchymal interactions. In those organs, GDNF expression is strictly confined to mesenchymal tissues and is not found in epithelia. Our results suggest multiple roles for GDNF during early stages of neuronal development and in epithelial-mesenchymal interactions.
The locus coeruleus (LC), the main noradrenergic center in the brain, participates in many neural functions, as diverse as memory and motor output, and is severely affected in several neurodegenerative disorders of the CNS. GDNF, a neurotrophic factor initially identified as dopaminotrophic, was found to be expressed in several targets of central noradrenergic neurons in the adult rat brain. Grafting of genetically engineered fibroblasts expressing high levels of GDNF prevented > 80% of the 6-hydroxydopamine-induced degeneration of noradrenergic neurons in the LC in vivo. Moreover, GDNF induced a fasciculated sprouting and increased by 2.5-fold both tyrosine hydroxylase levels and the soma size of lesioned LC neurons. These findings reveal a novel and potent neurotrophic activity of GDNF that may have therapeutic applications in neurodegenerative disorders affecting central noradrenergic neurons, such as Alzheimer's, Parkinson's, and Huntington's diseases.
Glial cell line-derived neurotrophic factor (GDNF) is known to support the survival of motoneurons in vitro and in vivo, as well as subpopulations of sensory neurons in vitro. To clarify the mechanisms by which GDNF supports these neurons, we examined the patterns of GDNF mRNA expression in relation to motor and sensory axons during early stages of mouse development. Between embryonic days (E) 10 and 12, a time when motor and sensory axons are entering the periphery, GDNF mRNA is expressed at high levels in a restricted region in proximal limb buds where axons converge and enter the limb. At later ages (E14-16), GDNF mRNA was detected in non-neuronal cells along peripheral nerve, in dermis, and in some muscles. To characterize cells that express GDNF in the proximal limb, GDNF expression in the forelimb was compared to expression patterns of two markers of muscle, Pax 3 and myogenin, as well as with the pan neurotrophin receptor (p75) which is expressed by Schwann cell precursors. We show that expression of GDNF in the proximal limb bud at E11-12 does not correlate with markers of muscle or Schwann cell precursors, which supports the idea that GDNF is expressed by mesenchymal cells in this region. Our results suggest that GDNF expression in proximal limb buds may function as a transient survival factor, particularly for motor neurons, before they reach their final targets. GDNF expression in muscle and dermis at later stages suggests that GDNF may have additional functions as motor and sensory neurons mature.
The normal development of the vertebrate nervous system entails the death of 30-70% of the neurons originally generated in most neuronal populations. This naturally occurring cell death is regulated by specific neurotrophic factors that promote neuronal survival and which are produced in limiting quantities by target cells, glial cells and neurons. These factors are also of potential utility as therapeutic agents for neurodegenerative diseases. Here we describe the purification and cloning of a new neurotrophic factor, identified on the basis of its ability to support the survival of sympathetic neurons in culture. This factor, neurturin, is structurally related to glial-cell-line-derived neurotrophic factor (GDNF). These factors can each activate the MAP kinase signalling pathway in cultured sympathetic neurons and support the survival of sympathetic neurons, as well as of sensory neurons of the nodose and dorsal root ganglia. Thus, neurturin and GDNF together now define a new family of neurotrophic factors.