Fig 4 - uploaded by Stephen Carl Ekker
Content may be subject to copyright.
ponzr1 is required for correct kidney patterning by pax2a. (A)pax2a mRNA expression in anterior pronephric tubules (arrows) at 18S. (B)Expanded expression of pax2a at 18S (arrows) in ponzr1 morphants. (C)Wild-type expression of pax2a in the anterior pronephric tubules (arrows) at 24 hpf. (D)ponzr1 morphants show expanded/ectopic pax2a expression toward the midline at 24 hpf (arrow). (E)Wild-type pax2a at 21S (arrows). (F,G)ponzr1 morphants show a pax2a phenotype intermediate 18S and 24 hpf where the invagination (F) of pax2aexpressing cells and the start of the midline fusion (G) can be seen. (H)Wild-type pax2a at 3 dpf. (I)ponzr1 morphants show a maintenance of the ectopic midline expression of pax2a (arrow). (J)Ectopic pax2a phenotype is seen using two independent ponzr1 morpholinos but not in ponzr1 mismatch control. (K)Ectopic pax2a phenotype is rescued using ponzr1 mRNA (P<0.001) but not dsRed mRNA. (L-O)wt1a (L) and podocin (N) mark future podocytes with two distinct expression domains at 24 hpf in controls (arrows). A single midline wt1a (M) and podocin (O) expression domain is seen in ponzr1 morphants at 24 hpf. (P,Q)wt1a phenotype is seen with two ponzr1 morpholinos (P) and can be rescued using ponzr1 mRNA (Q, P<0.001). (R,S)cdh17 expression is detected in the pronephric ducts (R, arrows) and tubules at 48 hpf in controls but is expanded in the pronephric ducts (S, arrows) in ponzr1 morphants at 48 hpf. ***P<0.001
Source publication
The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecula...
Contexts in source publication
Context 1
... we assayed for altered expression of intermediate mesoderm markers during early pronephric development to determine the effect of ponzr1 knockdown on early kidney development. Between 4S and 12S, we noted no phenotype (supplementary material Fig. S2). At 18S, ponzr1 morphants exhibit expanded pax2a expression in the anterior pronephric tubules (Fig. 4B) compared with uninjected controls (Fig. 4A). Ectopic and expanded midline expression of pax2a was visible at 24 hpf in ponzr1 morphants (Fig. 4D) compared with controls ( Fig. 4C). At the 21S intermediate developmental time point, deformation of the anterior kidney is apparent in ponzr1 morphants when assayed using pax2a expression ...
Context 2
... mesoderm markers during early pronephric development to determine the effect of ponzr1 knockdown on early kidney development. Between 4S and 12S, we noted no phenotype (supplementary material Fig. S2). At 18S, ponzr1 morphants exhibit expanded pax2a expression in the anterior pronephric tubules (Fig. 4B) compared with uninjected controls (Fig. 4A). Ectopic and expanded midline expression of pax2a was visible at 24 hpf in ponzr1 morphants (Fig. 4D) compared with controls ( Fig. 4C). At the 21S intermediate developmental time point, deformation of the anterior kidney is apparent in ponzr1 morphants when assayed using pax2a expression (Fig. 4F,G, compared with 4E). The pax2a ...
Context 3
... on early kidney development. Between 4S and 12S, we noted no phenotype (supplementary material Fig. S2). At 18S, ponzr1 morphants exhibit expanded pax2a expression in the anterior pronephric tubules (Fig. 4B) compared with uninjected controls (Fig. 4A). Ectopic and expanded midline expression of pax2a was visible at 24 hpf in ponzr1 morphants (Fig. 4D) compared with controls ( Fig. 4C). At the 21S intermediate developmental time point, deformation of the anterior kidney is apparent in ponzr1 morphants when assayed using pax2a expression (Fig. 4F,G, compared with 4E). The pax2a midline fusion is apparent at 3 dpf in ponzr1 morphants (Fig. ...
Context 4
... 4S and 12S, we noted no phenotype (supplementary material Fig. S2). At 18S, ponzr1 morphants exhibit expanded pax2a expression in the anterior pronephric tubules (Fig. 4B) compared with uninjected controls (Fig. 4A). Ectopic and expanded midline expression of pax2a was visible at 24 hpf in ponzr1 morphants (Fig. 4D) compared with controls ( Fig. 4C). At the 21S intermediate developmental time point, deformation of the anterior kidney is apparent in ponzr1 morphants when assayed using pax2a expression (Fig. 4F,G, compared with 4E). The pax2a midline fusion is apparent at 3 dpf in ponzr1 morphants (Fig. ...
Context 5
... (Fig. 4B) compared with uninjected controls (Fig. 4A). Ectopic and expanded midline expression of pax2a was visible at 24 hpf in ponzr1 morphants (Fig. 4D) compared with controls ( Fig. 4C). At the 21S intermediate developmental time point, deformation of the anterior kidney is apparent in ponzr1 morphants when assayed using pax2a expression (Fig. 4F,G, compared with 4E). The pax2a midline fusion is apparent at 3 dpf in ponzr1 morphants (Fig. ...
Context 6
... of pax2a was visible at 24 hpf in ponzr1 morphants (Fig. 4D) compared with controls ( Fig. 4C). At the 21S intermediate developmental time point, deformation of the anterior kidney is apparent in ponzr1 morphants when assayed using pax2a expression (Fig. 4F,G, compared with 4E). The pax2a midline fusion is apparent at 3 dpf in ponzr1 morphants (Fig. ...
Context 7
... markers of the future podocytes of the glomerulus, wt1a and podocin, are normally expressed in two distinct domains at 24 hpf (Fig. 4L,N). However, ponzr1 morphants exhibit ectopic wt1a and podocin expression in a single midline position (Fig. 4M,O). To ensure the phenotype was specific to ponzr1 loss of function, we tested for the pax2a and wt1a 24 hpf phenotypes with a second ponzr1 morpholino as well as a mismatch control (Fig. 4J,P) and found both morpholinos were ...
Context 8
... markers of the future podocytes of the glomerulus, wt1a and podocin, are normally expressed in two distinct domains at 24 hpf (Fig. 4L,N). However, ponzr1 morphants exhibit ectopic wt1a and podocin expression in a single midline position (Fig. 4M,O). To ensure the phenotype was specific to ponzr1 loss of function, we tested for the pax2a and wt1a 24 hpf phenotypes with a second ponzr1 morpholino as well as a mismatch control (Fig. 4J,P) and found both morpholinos were significantly different from wild type and the mismatch control. Additionally, we noted a statistically ...
Context 9
... expressed in two distinct domains at 24 hpf (Fig. 4L,N). However, ponzr1 morphants exhibit ectopic wt1a and podocin expression in a single midline position (Fig. 4M,O). To ensure the phenotype was specific to ponzr1 loss of function, we tested for the pax2a and wt1a 24 hpf phenotypes with a second ponzr1 morpholino as well as a mismatch control (Fig. 4J,P) and found both morpholinos were significantly different from wild type and the mismatch control. Additionally, we noted a statistically significant rescue of the phenotype by co- injection of ponzr1 mRNA (Fig. 4K,Q). We tested ponzr1 MO knockdown efficiency using an artificial ponzr1 5 UTR/green fluorescent protein (GFP) synthetic mRNA ...
Context 10
... loss of function, we tested for the pax2a and wt1a 24 hpf phenotypes with a second ponzr1 morpholino as well as a mismatch control (Fig. 4J,P) and found both morpholinos were significantly different from wild type and the mismatch control. Additionally, we noted a statistically significant rescue of the phenotype by co- injection of ponzr1 mRNA (Fig. 4K,Q). We tested ponzr1 MO knockdown efficiency using an artificial ponzr1 5 UTR/green fluorescent protein (GFP) synthetic mRNA injected into zebrafish embryos [based on Chen et al. (Chen et al., 2004)]. Briefly, GFP intensity was quantified by fluorescent imaging. Each experiment was standardized with wild-type embryos as 0% (supplementary ...
Context 11
... three markers pax2a, wt1a and podocin label the anterior kidney, specifically the developing glomerulus. Next, we examined the pronephric tubules and ducts in ponzr1 MO knockdown embryos. cdh17 staining in 48 hpf morphants demonstrated a widening of expression in the anterior pronephric duct (Fig. 4S) compared with controls (Fig. 4R). In the Tg(atp1a1a.4:GFP) ( Liu et al., 2007) (supplementary material Fig. S3) and Tg(enpep:GFP) (Seiler and Pack, 2011) (supplementary material Fig. S4) lines, no obvious phenotypes were noted at 28 hpf (A-H) and 48 hpf ...
Context 12
... three markers pax2a, wt1a and podocin label the anterior kidney, specifically the developing glomerulus. Next, we examined the pronephric tubules and ducts in ponzr1 MO knockdown embryos. cdh17 staining in 48 hpf morphants demonstrated a widening of expression in the anterior pronephric duct (Fig. 4S) compared with controls (Fig. 4R). In the Tg(atp1a1a.4:GFP) ( Liu et al., 2007) (supplementary material Fig. S3) and Tg(enpep:GFP) (Seiler and Pack, 2011) (supplementary material Fig. S4) lines, no obvious phenotypes were noted at 28 hpf (A-H) and 48 hpf ...
Context 13
... tubules and ducts in ponzr1 MO knockdown embryos. cdh17 staining in 48 hpf morphants demonstrated a widening of expression in the anterior pronephric duct (Fig. 4S) compared with controls (Fig. 4R). In the Tg(atp1a1a.4:GFP) ( Liu et al., 2007) (supplementary material Fig. S3) and Tg(enpep:GFP) (Seiler and Pack, 2011) (supplementary material Fig. S4) lines, no obvious phenotypes were noted at 28 hpf (A-H) and 48 hpf ...
Context 14
... embryos (Fig. 8E). Injections of ponzr1-VP16 mRNA also resulted in a reduction in pax2a expression in the anterior pronephric tubules (Fig. 8G,J) compared with controls ( Fig. 8F,I). By contrast, expression of the ponzr1-EnR mRNA resulted in enhanced pax2a expression (Fig. 8H,K), resembling the change of pax2a noted in ponzr1 morphant embryos (Fig. 4B). However, we did not observe a cdh17 phenotype in ponzr1-EnR injected embryos (Fig. 8E). The pax2a phenotypes seen in the ponzr1-VP16 and ponzr1-EnR injections are significantly different from wild type (Fig. 8L). Together, these data strongly suggest that ponzr1 normally functions as a transcription factor or co-factor during kidney ...
Context 15
... data presented here implicate ponzr1 as a downstream modulator of anterior pax2a expression in zebrafish kidney specification (Fig. 4). pax2a is able to specify pronephric tubule and duct fates; however, pax2a alone is insufficient to specify the glomerulus. We demonstrate that the zebrafish embryo deploys ponzr1 to provide an additional layer of complexity beyond the regulatory gene pax2a, thus facilitating the formation of a glomerular pronephric kidney (Fig. 5). ...
Similar publications
Transcription factors of the achaete-scute and atonal bHLH proneural gene family play important roles in neuronal differentiation. They are also involved in neuronal subtype specification through collaboration with homeodomain (HD) transcription factors. However, concerted regulation of these genes and in turn progenitor fate toward distinct lineag...
Citations
... In plants, most studies showed that plac8 homologs regulate cell proliferation negatively and control the weight and size of fruits in tomato, maize, etc. (Guo et al., 2010;Guo and Simmons, 2011;Libault et al., 2010). On the other hand, diverse functions of plac8 in animals, including regulation of cell differentiation, proliferation, apoptosis and migration, have been reported (Bedell et al., 2012;Li et al., 2006;Mao et al., 2019). Here, we revealed that plac8-A is involved in induced hyperproliferation of aPSCs via a decrease of its expression level after both amputation and feeding. ...
The regenerative ability of planarians relies on their adult pluripotent stem cell population. Although all stem cells express a piwi homolog, recently it has become possible to classify the piwi+ stem cell population into specialized subpopulations according to the expression of genes related to differentiation. However, piwi+ stem cells behave practically as a homogeneous population after amputation, during which stem cells show accelerated proliferation, named “induced hyper-proliferation”. Here we show that Djplac8-A was expressed in almost all of the stem cells, and that a decrease of the Djplac8-A expression level led to induced hyper-proliferation uniformly in a broad stem cell subpopulation after amputation. This reduction of Djplac8-A expression was caused by activated JNK signaling after amputation. Pharmacological inhibition of JNK signaling caused failure to induce hyper-proliferation and resulted in regenerative defects. Such defects were abrogated by simultaneous knockdown of Djplac8-A expression. Thus, JNK-dependent suppression of Djplac8-A expression is indispensable for stem cell dynamics involved in regeneration. These findings suggest that Djplac8-A acts as a molecular switch of piwi+ stem cells for entry into the regenerative state after amputation.
... Additionally, 13 genes were differentially expressed in both pairs of sympatric crater lake cichlids (Fig. IV.2C). An interesting candidate was ponzr1, which is involved in the development of pharyngeal arches in zebrafish (Bedell et al. 2012). However, no single gene was found to be differentially expressed in all contrasts. ...
... Reyon et al. (2012) designed the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a fast and economical tool for the large-scale assembly of TALENs. Targeted zebrafish genome editing, which represented a bottleneck, has also benefited from an advanced TALENs approach (Bedell et al., 2012). ...
Gene, genetic inheritance, genome, or even CRISPR (clustered regularly interspaced short palindromic repeats) are well-known names, but they have an amazing historical and scientific development that converges in the current tools for genome editing. This is the result of the knowledge accumulated along one and a half century on genetics, microbiology, chemistry, biochemistry, biotechnology, omics tools, and technological developments. Nowadays, synthetic biology represents a consolidated discipline that allows scientists to transform and redesign biological systems in a controlled manner. It provides solutions for pharmaceutical, materials, or energy fields. As a result, the genome editing can be applied either to the de novo synthesis of an organism or to the modification of an existing one. The present chapter presents the timeline of the findings, developments, and technological improvements of synthetic biology aimed to reach new products, increase productions, as well as the development of genomic chassis from scratch by means of genome design, assembly, and editing.
... Tg(enpep:GFP) larvae label both pronephric tubules and ducts as well as podocytelike cells of the glomeruli in early development [227]. This line has been used to identify and characterize the genes associated with pronephric development, kidney diseases and cellular dynamics during adult zebrafish kidney regeneration [228][229][230][231]. ...
Since its debut in the biomedical research fields in 1981, zebrafish have been used as a vertebrate model organism in more than 40,000 biomedical research studies. Especially useful are zebrafish lines expressing fluorescent proteins in a molecule, intracellular organelle, cell or tissue specific manner because they allow the visualization and tracking of molecules, intracellular organelles, cells or tissues of interest in real time and in vivo. In this review, we summarize representative transgenic fluorescent zebrafish lines that have revolutionized biomedical research on signal transduction, the craniofacial skeletal system, the hematopoietic system, the nervous system, the urogenital system, the digestive system and intracellular organelles.
... PLAC8 expression in embryo, placenta and/or different reproductive tissues has been reported in several species, including humans [15,16], mice [23,42], zebrafish [51,52], tigers [52,53], rabbits [54,55], cows [17e20], and buffaloes [56]. However, little is known about PLAC8 expression in equine placenta which has a diffuse, microvillous epitheliochorial morphology distinct from species described to date [57]. ...
... PLAC8 expression in embryo, placenta and/or different reproductive tissues has been reported in several species, including humans [15,16], mice [23,42], zebrafish [51,52], tigers [52,53], rabbits [54,55], cows [17e20], and buffaloes [56]. However, little is known about PLAC8 expression in equine placenta which has a diffuse, microvillous epitheliochorial morphology distinct from species described to date [57]. ...
Placenta-specific 8 (PLAC8) is one of the placenta-regulatory genes which is highly conserved among eutherian mammals. However, little is known about its expression in equine placenta (chorioallantois; CA and endometrium; EN) during normal and abnormal pregnancy. Therefore, the current study was designed to 1) elucidate the expression of PLAC8 in equine embryonic membranes during the preimplantation period, 2) characterize the expression profile of PLAC8 in equine CA (45d, 4mo, 6mo, 10 mo, 11 mo and postpartum) and EN (14d, 4mo, 6mo, 10 mo, and 11 mo) obtained from pregnant mares (n = 4/timepoint), as well as, d14 non-pregnant EN (n = 4), and 3) investigate the expression profile of PLAC8 in ascending placentitis (n = 5) and in nocardioform placentitis (n = 6) in comparison to normal CA. In the preimplantation period, PLAC8 mRNA was not abundant in the trophectoderm of d8 equine embryo and d14 conceptus, while it was abundant later in d 30, 31, 34, and 45 chorion. In normal pregnancy, PLAC8 mRNA expression in CA at 45 d gradually decline to reach nadir at 6mo before gradually increasing to its peak at 11mo and postpartum CA. The mRNA expression of PLAC8 was significantly upregulated in CA from mares with ascending and nocardioform placentitis compared to control mares. Immunohistochemistry revealed that PLAC8 is localized in equine chorionic epithelium and immune cells. Our results revealed that PLAC8 expression in equine chorion is dynamic during pregnancy and is regulated in an implantation-dependent manner. Moreover, PLAC8 is implicated in the immune response in CA during equine ascending placentitis and nocardioform placentitis.
... This cluster also expresses ponzr1 and slc9a3.2 which are expressed not only in the intestine but also the pronephric duct, tubules, and nephrons (83,84). This raises the possibility that this cluster may include cells from the pronephric duct or tubule. ...
Bile salt synthesis, secretion into the intestinal lumen, and resorption in the ileum occurs in all vertebrate classes. In mammals, bile salt composition is determined by host and microbial enzymes, affecting signaling through the bile salt-binding transcription factor Farnesoid X receptor (Fxr). However, these processes in other vertebrate classes remain poorly understood. We show that key components of hepatic bile salt synthesis and ileal transport pathways are conserved and under control of Fxr in zebrafish. Zebrafish bile salts consist primarily of a C27 bile alcohol and a C24 bile acid which undergo multiple microbial modifications including bile acid deconjugation that augments Fxr activity. Using single-cell RNA sequencing, we provide a cellular atlas of the zebrafish intestinal epithelium and uncover roles for Fxr in transcriptional and differentiation programs in ileal and other cell types. These results establish zebrafish as a non-mammalian vertebrate model for studying bile salt metabolism and Fxr signaling.
... Transcriptional factor irx3b, evi1, and pou3f3a/pou3f3b are required for the development of distal segments of pronephros [6]. The hnf1ba and hnf1bb are required for proper nephron patterning by regulating the expression of other genes, like pax2a establishes boundary of podocyte and the neck by directly inhibiting wt mediated podocyte formation [11]. Knock-down of hnf1ba and hnf1bb showed defects in the formation of proximal and distal segments of pronephros, indicating their role in nephron segmentation [12,13]. ...
Background:
The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function.
Results:
efhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development.
Conclusion:
In this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development.
... The DV axis specifies the separation of paraxial mesoderm, intermediate mesoderm, and lateral plate mesoderm. In zebrafish and Xenopus studies, misexpression of early kidney markers, such as lhx1 or pax2, promotes ectopic kidney formation but only in paraxial mesoderm and not in lateral plate mesoderm (Bedell et al. 2012;Carroll and Vize 1999). Additionally, perturbation of anterior somite formation in Xenopus embryos precludes pronephrogenesis (Seufert et al. 1999). ...
... In mice, Wt1 is a negative regulator of Pax2 (Ryan et al. 1995), and a similar epistatic relationship between wt1a and pax2a may be present in zebrafish as the anterior pax2a expression domain initially overlaps with wt1a in P/PECs, but then restricts to the neck segment at 24 hpf (O'Brien et al. 2011). In addition, pax2a overexpression reduces wt1a expression and promotes formation of an aglomerular pronephros (Bedell et al. 2012). These data suggest that pax2a and wt1a are negative regulators of each other. ...
... pax2a has also been shown to participate in a negative feedback loop by inducing the expression of plac8 onzin related protein 1 (ponzr1), a poorly studied gene that acts to antagonize pax2a expression. Morpholino-mediated knockdown of ponzr1 causes persistent expression of pax2a in P/PECs, resulting in the downregulation of wt1a and the formation of an aglomerular pronephros (Bedell et al. 2012). ...
The pronephros is the first kidney type to form in vertebrate embryos. The first step of pronephrogenesis in the zebrafish is the formation of the intermediate mesoderm during gastrulation, which occurs in response to secreted morphogens such as BMPs and Nodals. Patterning of the intermediate mesoderm into proximal and distal cell fates is induced by retinoic acid signaling with downstream transcription factors including wt1a, pax2a, pax8, hnf1b, sim1a, mecom, and irx3b. In the anterior intermediate mesoderm, progenitors of the glomerular blood filter migrate and fuse at the midline and recruit a blood supply. More posteriorly localized tubule progenitors undergo epithelialization and fuse with the cloaca. The Notch signaling pathway regulates the formation of multi-ciliated cells in the tubules and these cells help propel the filtrate to the cloaca. The lumenal sheer stress caused by flow down the tubule activates anterior collective migration of the proximal tubules and induces stretching and proliferation of the more distal segments. Ultimately these processes create a simple two-nephron kidney that is capable of reabsorbing and secreting solutes and expelling excess water—processes that are critical to the homeostasis of the body fluids. The zebrafish pronephric kidney provides a simple, yet powerful, model system to better understand the conserved molecular and cellular progresses that drive nephron formation, structure, and function.
... Expression of Pax2 in mouse podocytes affects the integrity of the glomerulus and leads to scarring (glomerulosclerosis) in transgenic models (Wagner et al. 2006). Similarly, in zebrafish embryos deficient in ponzr, a pax2a negative-feedback regulator, ectopic expression of pax2a in podocytes is associated with failed glomerulogenesis (Bedell et al. 2012). These results suggest that expression of Pax2 is incompatible with podocyte cell fate, possibly by antagonizing the function/expression of Wt1. ...
... In mouse embryos deficient in Pax2 and Pax8 the intermediate mesoderm fails to initiate kidney formation and the nephric duct does not form (Bouchard et al. 2002). Similarly, in zebrafish embryos doubly deficient in pax2a and pax8, the intermediate mesoderm does not activate expression of a number of early acting renal genes, including hnf1b, mecom, plac8 and lhx1a (Bedell et al. 2012;Naylor et al. 2013;Swanhart et al. 2010). Together, these data are consistent with Pax2/8 acting at the top of a gene network that controls the specification of tubule fate from the intermediate mesoderm ( Fig. 7.1). ...
The nephron is the conserved functional unit of vertebrate kidneys and is composed of a glomerular blood filter attached to a segmented tubule. The gene regulatory networks governing nephron formation during embryonic development are poorly understood and are challenging to study in complex kidney types such as the mammalian adult (metanephric) kidney. By contrast, the zebrafish embryonic (pronephric) kidney
offers a number of advantages including its linearly arranged, simple two-nephron structure, and ease of genetic manipulation. As the genes involved in nephrogenesis are largely conserved, the zebrafish model can provide valuable insights into the core gene networks involved in mammalian nephron formation, with relevance to birth defects and disease. In this chapter we review the structure and function of the zebrafish pronephric nephron and summarize our current understanding of the gene regulatory networks and signaling pathways that control the formation of glomerular and tubule cell types.
... There are also evidences that pax2a directly inhibits podocyte formation by antagonizing wt1 activity and establishing a boundary between the podocyte and the neck. Reciprocally, the gene ponzr1 modulates negatively pax2a to restrict its activity within the podocyte territory [45,46]. As in Xenopus, irx3b plays an essential role for DE segment patterning modulating the boundary between proximal and distal segments [47]. ...
Le rein du Mammifère est un organe essentiel dont le rôle est de maintenir l’homéostasie et de purifier le sang en éliminant les déchets issus du métabolisme. Le développement du rein définitif, débute par l’émergence du bourgeon urétéral (BU) au niveau de l’extrémité postérieure du canal néphrique (CN). Le BU subit ensuite un processus complexe d’arborisations stéréotypées aboutissant à la formation du système de canaux collecteurs (CC) et de l’uretère. A chaque nouvelle arborisation, l’invasion de l’épithélium induit la condensation du mésenchyme adjacent pour former des agrégats pré-tubulaires, précurseurs des unités de filtration du rein, les néphrons. Ainsi, les deux structures tubulaires épithéliales rénales que constituent les CCs et les néphrons se développent de façon coordonnée par des mécanismes différents finement régulés. Le facteur de transcription HNF1B joue un rôle essentiel dans la morphogenèse d’organes complexes tels, que le rein, le pancréas ou le foie. Chez l’Homme, les mutations hétérozygotes de HNF1B entraînent l’apparition du syndrome « Renal Cysts And Diabetes » (RCAD), caractérisé par un diabète précoce, une hypoplasie pancréatique ainsi que des défauts de morphogenèse des tubules rénaux et du tractus génital. Notre laboratoire a montré que HNF1B est requis durant le développement précoce du rein, pour l’émergence du BU et l’initiation de la néphrogenèse chez la souris. Hnf1b s’exprime aussi plus tardivement lors de la formation du néphron et dans l’ensemble des CCs, suggérant ainsi un rôle plus tardif et spécifique dans ces structures. Étant donné les interactions réciproques existant entre le mésenchyme métanéphrique et le BU, et pour discriminer les fonctions spécifiques de Hnf1b dans chacun de ces compartiments, nous avons inactivé ce gène dans ces deux populations de cellules grâce à une stratégie d’invalidation conditionnelle chez la Souris. L’invalidation conditionnelle de Hnf1b dans les progéniteurs des néphrons avec la lignée Wnt4-EGFPCre entraîne une létalité précoce des mutants associée à une hypoplasie rénale. Les mutants présentent une arborisation normale mais ne développent aucun néphron. Ce défaut est associé à une diminution de l’expression de composants de la voie Notch (Lfng, Dll1, Jag1) et des facteurs Irx1/2 qui sont respectivement des régulateurs potentiels de la spécification des segments proximal et intermédiaire. De plus, HNF1B est recruté in vivo au niveau des séquences régulatrices de ces gènes. La surexpression d’une forme dominant-négatif de HNF1B dans des embryons de Xénope provoque la diminution de l’expression de marqueurs spécifiques des segments proximal et intermédiaire du pronéphros montrant ainsi que la fonction de HNF1B semble conservée au cours de l’évolution des Vertébrés. L’inactivation de Hnf1b dans le CN et ses dérivés, avec la lignée HoxB7-Cre, entraîne de multiples anomalies urogénitales, en partie dues à une activité mosaïque de la Cre-recombinase. L’analyse de l’architecture des branches du BU montre que le nombre d’extrémités ainsi que leur longueur sont massivement réduits chez les mutants. En outre, la géométrie générale de l’organe apparaît désordonnée. En associant l’invalidation mosaïque de Hnf1b avec une lignée portant un rapporteur fluorescent membranaire EGFP ou Tomato selon que les cellules sont recombinantes ou non, nous avons pu suivre par imagerie en temps réel le comportement des cellules lors de l’arborisation du BU. Les cellules mutantes apparaissent exclues des extrémités des branches ce qui suggère que HNF1B pourrait y agir de façon autonome-cellulaire pour promouvoir l’arborisation. De plus, la polarité des cellules des CCs semble perdue chez les mutants et le système collecteur ne peut se différencier correctement. On observe alors l’apparition de dilatations, voire même de kystes, dans les CCs, à la fois in vivo et in vitro [...]
