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No detectable Purkinje cell loss occurs in the cerebella of rescue animals. Two P30 C57BL/6J animals of each genotype (En1 / and En1 Hd/Hd ) were analyzed. There is no significant difference in Purkinje cell number between the two genotypes ( p 0.29; one-tailed Student's t test). The raw counts per 10 m section plotted as a function of distance from the midline is shown.
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The mouse homeodomain protein, Engrailed-1, is generally viewed as an essential player in the early establishment and maintenance of the midbrain/hindbrain region that gives rise to the cerebellum and midbrain. In keeping with this, engineered null mutations at this locus have been reported to lead to perinatal lethality accompanied by near-total a...
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... normal appearance of the cells of the cerebellar cortex is extended to the more quantitative features of the struc- ture as well. We estimated the Purkinje cell number in two C57BL/6 -En1 Hd/Hd animals and found no significant differences ( p 0.3; one-tailed Student's t test) compared with wild-type controls (Fig. 3). We did not apply any geometric or stereologic correction to our counts; thus, our analysis is not sufficiently accurate to rule out changes of 10%. Nonetheless, the counts offer clear semiquan- titative evidence that the C57BL/6J-En1 Hd/Hd animals have cere- bella that differ little from their wild-type counterparts. Thus, on the ...
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Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in brain development and neuronal migration. Cdk5 is abundant in postmitotic, terminally differentiated neurons. The ability of Cdk5 to phosphorylate substrates is dependent on activation by its neuronal-specific activators p35 and p39. There exist striking differences in the phenotypic severity...
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... Both the absence and enhancement of Wnt/βcatenin signaling in GCPs led to abnormal cerebellar gross structures and reduced cerebellar size, although they caused different changes at the cellular level: the absence of signaling facilitated GCP proliferation, leading to an accumulation of abnormally matured GCs near the pial surface, whereas increasing signaling inhibited GCP proliferation, leading to a reduction of GCs. Homeobox transcription factors, Engrailed 1 (En1) and 2 (En2), have also been implicated in cerebellar folium formation (Bilovocky et al., 2003;Sudarov and Joyner, 2007;Cheng et al., 2010). However, based on a study that used En1/2 conditional knockout mice either in the RL or the ventricular zone (VZ), the latter of which gives rise to cerebellar γ-aminobutyric acid (GABA)-ergic neurons, their expression in GCs appears to contribute more to cerebellar enlargement than to the regulation of foliation (Orvis et al., 2012). ...
The well-organized cerebellar structures and neuronal networks are likely crucial for their functions in motor coordination, motor learning, cognition, and emotion. Such cerebellar structures and neuronal networks are formed during developmental periods through orchestrated mechanisms, which include not only cell-autonomous programs but also interactions between the same or different types of neurons. Cerebellar granule cells (GCs) are the most numerous neurons in the brain and are generated through intensive cell division of GC precursors (GCPs) during postnatal developmental periods. While GCs go through their own developmental processes of proliferation, differentiation, migration, and maturation, they also play a crucial role in cerebellar development. One of the best-characterized contributions is the enlargement and foliation of the cerebellum through massive proliferation of GCPs. In addition to this contribution, studies have shown that immature GCs and GCPs regulate multiple factors in the developing cerebellum, such as the development of other types of cerebellar neurons or the establishment of afferent innervations. These studies have often found impairments of cerebellar development in animals lacking expression of certain molecules in GCs, suggesting that the regulations are mediated by molecules that are secreted from or present in GCs. Given the growing recognition of GCs as regulators of cerebellar development, this review will summarize our current understanding of cerebellar development regulated by GCs and molecules in GCs, based on accumulated studies and recent findings, and will discuss their potential further contributions.
... 177 Moreover, inbred mice cannot mimic the diversity of humans. 178 Another limitation of CRISPR-Cas9mediated genome editing in rodents is the precise knowledge of the reproductive cycle of rodents, e.g., time of fertilization and time to recover the fertilized eggs. Therefore, it is important to consider these limitations and obtain in-depth knowledge of the animal model used in preclinical studies before entering into clinical use. ...
The concept of genome alteration in both plant and animal kingdom has come a long way and has transformed medicinal research in the modern era. The predetermined gene expression may be modulated (i.e., upregulated or downregulated) as a result of the specific genetic change. CRISPR/Cas9 is a hopeful gene editing technique that can modify DNA sequences, correcting the genes for gain-of-function or loss-of-functions mutation to treat genetic illnesses. Although other gene editing techniques are also known, CRISPR/Cas9 is the most efficient and precise technique to gene correction. In the last decade, CRISPR/Cas9 has entered under clinical trials and very soon it might enter into clinics. In this chapter we discuss the origins of CRISPR-Cas9 systems and their biomedical applications.
... Furthermore, En2 is involved in the tectal laminar formation, the rostral part of the optic tectum showing more advanced lamination than the caudal one, as a potential regulator of neuronal cell migration (Omi et al., 2014). In summary, these findings strongly suggest that En2 expression is controlled by environmental influences, governing the rostrocaudal polarity of the optic tectum (Friedman and O'Leary, 1996;Logan et al., 1996;Shigetani et al., 1997;Bilovocky et al., 2003; reviewed by Omi and Nakamura, 2015). ...
A crucial event during the development of the central nervous system (CNS) is the early subdivision of the neural tube along its anterior-to-posterior axis to form neuromeres, morphogenetic units separated by transversal constrictions and programed for particular genetic cascades. The narrower portions observed in the developing neural tube are responsible for relevant cellular and molecular processes, such as clonal restrictions, expression of specific regulatory genes, and differential fate specification, as well as inductive activities. In this developmental context, the gradual formation of the midbrain-hindbrain (MH) constriction has been an excellent model to study the specification of two major subdivisions of the CNS containing the mesencephalic and isthmo-cerebellar primordia. This MH boundary is coincident with the common Otx2-(midbrain)/Gbx2-(hindbrain) expressing border. The early interactions between these two pre-specified areas confer positional identities and induce the generation of specific diffusible morphogenes at this interface, in particular FGF8 and WNT1. These signaling pathways are responsible for the gradual histogenetic specifications and cellular identity acquisitions with in the MH domain. This review is focused on the cellular and molecular mechanisms involved in the specification of the midbrain/hindbrain territory and the formation of the isthmic organizer. Emphasis will be placed on the chick/quail chimeric experiments leading to the acquisition of the first fate mapping and experimental data to, in this way, better understand pioneering morphological studies and innovative gain/loss-of-function analysis.
... Both EN1 and Hedgehog signaling play crucial roles in developmental processes. EN1 is essential in the early establishment and maintenance of the midbrain/hindbrain [44]; interestingly, SHH signaling together with Wnt signaling also plays essential roles in the patterning and development of the midbrain/hindbrain [7]. In Drosophila, interactions between Engrailed (en) and Hedgehog signaling pathways revealed that the Hedgehog pathway is transcriptionally activated by the Engrailed protein [45]. ...
Glioblastoma is an aggressive cancer of the nervous system that accounts for the majority of brain cancer-related deaths. Through cross-species transcriptome studies, we found that Engrailed 1 (EN1) is highly expressed in serum-free cultured glioma cells as well as glioma tissues, and increased expression level predicts a worse prognosis. EN1 controls glioma cell proliferation, colony formation, migration, and tumorigenic capacity in vivo. It also influences sensitivity of glioma cells to γ-ray irradiation by regulating intracellular ROS levels. Mechanistically, EN1 influences Hedgehog signaling by regulating the level of Gli1 as well as primary cilia length and the primary cilia transport-related protein TULP3. In conclusion, we demonstrate that EN1 acts as an oncogenic regulator that contributes to glioblastoma pathogenesis and could serve as a diagnostic/prognostic marker and therapeutic target for glioblastoma.
... En1tm1Alj/+ animals were back-crossed to the C57BL6/J line generating En1+/+ (WT), En1tm1Alj/+ (Het) and viable En1tm1Alj/tm1Alj (KO) offspring, and genotyped using specific primers [16,19,43]. Nkx2.9LacZ/LacZ and Nkx2.9LacZ/+ animals were back crossed in C57BL/6 mice and genotyped using specific primers, as described previously [22]. ...
Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.
... During cerebellar development, it has been suggested that genetic modifiers can alter the impact of genes that are essential for morphogenesis (Sillitoe & Joyner, 2007). For instance, based on the genetic background and the related modifiers of those alleles, loss of engrailed1 (En1) can have polar opposite effects, to the extremes that on the C57BL/6 background, En1 null mice have a cerebellum, whereas on the 129/S1 background strain the entire cerebellum is eliminated if En1 is deleted (Bilovocky et al. 2003;Murcia et al. 2007). Therefore, why do not all carriers with dystonia-causing mutations develop twisting and postural abnormalities or tremor? ...
Key points
Loss‐of‐function mutations in the Thap1 gene cause partially penetrant dystonia type 6 (DYT6).
Some non‐manifesting DYT6 mutation carriers have tremor and abnormal cerebello‐thalamo‐cortical signalling.
We show that Thap1 heterozygote mice have action tremor, a reduction in cerebellar neuron number, and abnormal electrophysiological signals in the remaining neurons.
These results underscore the importance of Thap1 levels for cerebellar function.
These results uncover how cerebellar abnormalities contribute to different dystonia‐associated motor symptoms.
Abstract
Loss‐of‐function mutations in the Thanatos‐associated domain‐containing apoptosis‐associated protein 1 (THAP1) gene cause partially penetrant autosomal dominant dystonia type 6 (DYT6). However, the neural abnormalities that promote the resultant motor dysfunctions remain elusive. Studies in humans show that some non‐manifesting DYT6 carriers have altered cerebello‐thalamo‐cortical function with subtle but reproducible tremor. Here, we uncover that Thap1 heterozygote mice have action tremor that rises above normal baseline values even though they do not exhibit overt dystonia‐like twisting behaviour. At the neural circuit level, we show using in vivo recordings in awake Thap1+/− mice that Purkinje cells have abnormal firing patterns and that cerebellar nuclei neurons, which connect the cerebellum to the thalamus, fire at a lower frequency. Although the Thap1+/− mice have fewer Purkinje cells and cerebellar nuclei neurons, the number of long‐range excitatory outflow projection neurons is unaltered. The preservation of interregional connectivity suggests that abnormal neural function rather than neuron loss instigates the network dysfunction and the tremor in Thap1+/− mice. Accordingly, we report an inverse correlation between the average firing rate of cerebellar nuclei neurons and tremor power. Our data show that cerebellar circuitry is vulnerable to Thap1 mutations and that cerebellar dysfunction may be a primary cause of tremor in non‐manifesting DYT6 carriers and a trigger for the abnormal postures in manifesting patients.
... The genetic background greatly influences the phenotypes of transgenic mice (Kelly et al. 1998;Magara et al. 1999;Bilovocky et al. 2003;Lloret et al. 2006;Kovacs and Pearce 2015) and is also known to affect the gut microbiota diversity and composition (Laukens et al. 2016). Comparing the gut microbiota of Cln1 R151X mice on two different genetic backgrounds, a mixed 129S6/SvEv × C57BL/6J and pure C57BL/6J, revealed several background-specific changes. ...
Accumulated evidence indicates that the gut microbiota affects brain function and may be altered in neurological diseases. In this study, we analyzed the gut microbiota in Cln1R151X and Cln2R207X mice, models of the childhood neurodegenerative disorders, infantile CLN1 and late infantile CLN2 Batten diseases. Significant alterations were found in the overall gut microbiota composition and also at the individual taxonomic ranks as compared to wild-type mice. The disease-specific alterations in the gut microbiota of Cln1R151X and Cln2R207X mice may contribute to the disease phenotypes observed in these mouse models. We also compared the gut microbiota composition of three wild-type mouse strains frequently used in transgenic studies: 129S6/SvEv, C57BL/6J and mixed 129S6/SvEv × C57BL/6J. Our results show that the gut microbiota of 129S6/SvEv and C57BL/6J mice differs remarkably, which likely contributes to the known, pronounced differences in behavior and disease susceptibility between these two wild-type mouse strains.
... 177 Moreover, inbred mice cannot mimic the diversity of humans. 178 Another limitation of CRISPR-Cas9mediated genome editing in rodents is the precise knowledge of the reproductive cycle of rodents, e.g., time of fertilization and time to recover the fertilized eggs. Therefore, it is important to consider these limitations and obtain in-depth knowledge of the animal model used in preclinical studies before entering into clinical use. ...
At present, the idea of genome modification has revolutionized the modern therapeutic research era. Genome modification studies have travelled a long way from gene modifications in primary cells to genetic modifications in animal. The targeted genetic modification may result in the modulation (i.e. either upregulation or downregulation) of the predefined gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease 9 (CRISPR/Cas9) is a promising genome editing tool that has therapeutic potential against incurable genetic disorders by modifying their DNA sequences. In comparison with other genome editing techniques, CRISPR/Cas9 is simple, efficient and very specific. This enabled CRISPR/Cas9 genome editing technology to enter into the clinical trials against cancer. Besides therapeutic potential, CRISPR/Cas9 tool can also be applied to generate genetically inhibited animal models for drug discovery and development. This comprehensive review paper discussed the origin of CRISPR/Cas9 systems and its therapeutic potential against various genetic disorders includes cancer, allergy, immunological disorders, duchenne muscular dystrophy, cardiovascular disorders, neurological disorders, liver-related disorders, cystic fibrosis, blood-related disorders, eye-related disorders and viral infection. Finally, we talk about the different challenges, safety concerns and the strategies that can be applied to overcome the obstacles during CRISPR/Cas9-mediated therapeutic approach.
... The earliest studies on En1-ablated mice reported perinatal lethality, due to cerebellar ablation [32], however our group and others reported that this phenomenon may be circumvented by back-crossing the original 129/Sv line to a C57BL/6J background [13,33]. To determine the influence of the mouse-model background in these double En1KO;Pitx3GFP/GFP animals, we first examined the cytoarchitecture of the mid-and hindbrain area (using a DAPI staining). ...
... This phenotype is identical to the En1-mutation in the 129/Sv mouse background [32]. Both Pitx3 gfp/gfp animals and En1 tm1Alj/+ animals were backcrossed onto a C57BL6/J background, however it takes several generations of back-crossing to completely neutralize the 129/Sv mouse background, and generate viable En1KO animals [13,30,33]. We thus hypothesize that the perinatal lethality is most likely the consequence of the presence of the 129/Sv mouse background. ...
Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral midbrain. These neurons form the substantia nigra (SNc) and the ventral tegmental area (VTA). Two transcription factors that play important roles in the process of terminal differentiation and subset-specification of mdDA neurons, are paired-like homeodomain transcription factor 3 (Pitx3), and homeobox transcription factor Engrailed 1 (En1). We previously investigated the single Pitx3KO and En1KO and observed important changes in the survival of mdDA neurons of the SNc and VTA as well as altered expression of pivotal rostral- and caudal-markers, Ahd2 and Cck, respectively. To refine our understanding of the regional-specific relationships between En1 and Pitx3 and their (combined) role in the programming mdDA neurons on the rostral-to-caudal axis, we created double En1tm1Alj/tm1Alj;Pitx3gfp/gfp (En1KO; Pitx3GFP/GFP) animals. Here we report, that in absence of En1 and Pitx3, only a limited number of mdDA neurons are present at E14.5. These mdDA neurons have a rudimentary dopaminergic cell fate, as they express Nurr1, Pbx3 and Otx2 but have lost their rostral or caudal subset identity. Furthermore, we report that the expression of Cck depends on En1 expression, while (in contrast) both Pitx3 and En1 are involved in the initiation of Ahd2 expression. Thus we reveal in this manuscript that regulated levels of Pitx3 and En1 control the size and rostral/caudal-identity of the mdDA neuronal population. © 2017 Kouwenhoven et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... Later it became apparent that part of the hindbrain phenotype together with the perinatal lethality was due to dosage differences of En2 caused by genome background differences in the used mutant strains. After several backcrosses to C57BL6J as a background strain it became possible to study adult En1 mutants which did not have a hindbrain defect (Bilovocky et al., 2003). Initial analyzes of these En1 mutants indicated that specific part of the SNc and VTA regions were affected by En1 ablation . ...
Dopamine neurons of the substantia nigra compacta (SNc) and ventral tegmental area (VTA) are critical components of the neuronal machinery to control emotion and movement in mammals. The slow and gradual death of these neurons as seen in Parkinson's disease has triggered a large investment in research toward unraveling the molecular determinants that are used to generate these neurons and to get an insight in their apparent selective vulnerability. Here, I set out to summarize the current view on the molecular distinctions that exist within this mesodiencephalic dopamine (mdDA) system and elaborate on the molecular programming that is responsible for creating such diversity.
