Estrogen related receptor beta (Esrrb) is an orphan nuclear receptor that is required for self-renewal and pluripotency in mouse embryonic stem (ES) cells. However, in the early post-implantation mouse embryo, Esrrb is specifically expressed in the extraembryonic ectoderm (ExE) and plays a crucial role in trophoblast development. Previous studies showed that Esrrb is also required to maintain trophoblast stem (TS) cells, the in vitro stem cell model of the early trophoblast lineage. In order to identify regulatory targets of Esrrb in vivo, we performed microarray analysis of Esrrb-null versus wild-type post-implantation ExE, and identified 30 genes down-regulated in Esrrb-mutants. Among them is Bmp4, which is produced by the ExE and known to be critical for primordial germ cell (PGC) specification in vivo. We further identified an enhancer region bound by Esrrb at the Bmp4 locus by performing Esrrb ChIP-seq and luciferase reporter assay using TS cells. Finally, we established a knockout mouse line in which the enhancer region was deleted using CRISPR/Cas9 technology. Both Esrrb-null embryos and enhancer knockout embryos expressed lower levels of Bmp4 in the ExE, and had reduced numbers of PGCs. These results suggested that Esrrb functions as an upstream factor of Bmp4 in the ExE, regulating proper PGC development in mice.

Radial glia of the mouse cerebral cortex emerge from neuroepithelial stem cells around embryonic day 11 and produce excitatory cortical neurons until a few days before birth. The molecular mechanisms that regulate the end of cortical neurogenesis remain largely unknown. Here we investigated if the Dicer-dependent microRNA (miRNA) pathway is involved. By electroporating a cre-recombinase expression vector into the cortex of E13.5 embryos carrying a conditional allele of Dicer1, we induced mosaic recombination causing Dicer1 deletion and reporter activation in a subset of radial glia. We analysed the long-term fates of their progeny. We found that mutant radial glia produced abnormally large numbers of Cux1-positive neurons, many of which populated the superficial cortical layers. Injections of the S-phase marker bromodeoxyuridine between postnatal days 3 and 14 showed that much of this population was generated postnatally. Our findings suggest a role for Dicer-dependent processes in limiting the timespan of cortical neurogenesis.

During vesicular transport between the endoplasmic reticulum and the Golgi, members of the TMED/p24 protein family form hetero-oligomeric complexes that facilitate protein-cargo recognition as well as vesicle budding. In addition, they regulate each other's level of expression. Despite analyses of TMED/p24 protein distribution in mammalian cells, yeast, and C. elegans, little is known about the role of this family in vertebrate embryogenesis. We report the presence of a single point mutation in Tmed2/p24beta(1) in a mutant mouse line, 99J, identified in an ENU mutagenesis screen for recessive developmental abnormalities. This mutation does not affect Tmed2/p24beta(1) mRNA levels but results in loss of TMED2/p24beta(1) protein. Prior to death at mid-gestation, 99J homozygous mutant embryos exhibit developmental delay, abnormal rostral-caudal elongation, randomized heart looping, and absence of the labyrinth layer of the placenta. We find that Tmed2/p24beta(1) is normally expressed in tissues showing morphological defects in 99J mutant embryos and that these affected tissues lack the TMED2/p24beta(1) oligomerization partners, TMED7/p24gamma(3) and TMED10/p24delta(1). Our data reveal a requirement for TMED2/p24beta(1) protein in the morphogenesis of the mouse embryo and placenta.

During embryogenesis, lymphatic endothelial progenitor cells first arise from a subset of blood vascular endothelial cells in the dorsolateral aspects of the cardinal veins. The molecular cues responsible for defining the regionalisation of such a discrete pool of progenitors remain uncharacterised. Here we identify a novel function for CYP26B1, an enzyme known to play a role in tissue morphogenesis by fine-tuning retinoic acid (RA) concentration, in regulating lymphangiogenesis. Cyp26b1-null mice, in which RA levels are elevated, exhibited an increased number of lymphatic endothelial progenitor cells in the cardinal veins, together with hyperplastic, blood filled lymph sacs and hyperplastic dermal lymphatic vessels. Conversely, mice over-expressing Cyp26b1 had hypoplastic lymph sacs and lymphatic vessels. Our data suggest that RA clearance by CYP26B1 in the vicinity of lymphatic endothelial progenitor cells is important for determining the position and size of the progenitor pool specified. Our studies identify a genetic pathway that underpins the architecture of the developing lymphatics and define CYP26B1 as a novel modulator of lymphatic vascular patterning.

Early mammalian embryogenesis is controlled by mechanisms governing the balance between pluripotency and differentiation. The expression of early lineage-specific genes can vary significantly between species, with implications for developmental control and stem cell derivation. However, the mechanisms involved in patterning the human embryo are still unclear. We analyzed the appearance and localization of lineage-specific transcription factors in staged preimplantation human embryos from the zygote until the blastocyst. We observed that the pluripotency-associated transcription factor OCT4 was initially expressed in 8-cell embryos at 3 days post-fertilization (dpf), and restricted to the inner cell mass (ICM) in 128-256 cell blastocysts (6dpf), approximately 2 days later than the mouse. The trophectoderm (TE)-associated transcription factor CDX2 was upregulated in 5dpf blastocysts and initially coincident with OCT4, indicating a lag in CDX2 initiation in the TE lineage, relative to the mouse. Once established, the TE expressed intracellular and cell-surface proteins cytokeratin-7 (CK7) and fibroblast growth factor receptor-1 (FGFR1), which are thought to be specific to post-implantation human trophoblast progenitor cells. The primitive endoderm (PE)-associated transcription factor SOX17 was initially heterogeneously expressed in the ICM where it co-localized with a sub-set of OCT4 expressing cells at 4-5dpf. SOX17 was progressively restricted to the PE adjacent to the blastocoel cavity together with the transcription factor GATA6 by 6dpf. We observed low levels of Laminin expression in the human PE, though this basement membrane component is thought to play an important role in mouse PE cell sorting, suggesting divergence in differentiation mechanisms between species. Additionally, while stem cell lines representing the three distinct cell types that comprise a mouse blastocyst have been established, the identity of cell types that emerge during early human embryonic stem cell derivation is unclear. We observed that derivation from plating intact human blastocysts resulted predominantly in the outgrowth of TE-like cells, which impairs human embryonic stem cell derivation. Altogether, our findings provide important insight into developmental patterning of preimplantation human embryos with potential consequences for stem cell derivation.

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.

MicroRNAs are small non-coding RNAs that regulate protein expression by binding 3'UTRs of target mRNAs, thereby inhibiting translation. Similar to siRNAs, miRNAs are cleaved by Dicer. Mouse and ES cell Dicer mutants demonstrate that microRNAs are necessary for embryonic development and cellular differentiation. However, technical obstacles and the relative infancy of this field have resulted in few data on the functional significance of individual microRNAs. We present evidence that miR-17 family members, miR-17-5p, miR-20a, miR-93, and miR-106a, are differentially expressed in developing mouse embryos and function to control differentiation of stem cells. Specifically, miR-93 localizes to differentiating primitive endoderm and trophectoderm of the blastocyst. We also observe high miR-93 and miR-17-5p expression within the mesoderm of gastrulating embryos. Using an ES cell model system, we demonstrate that modulation of these miRNAs delays or enhances differentiation into the germ layers. Additionally, we demonstrate that these miRNAs regulate STAT3 mRNA in vitro. We suggest that STAT3, a known ES cell regulator, is one target mRNA responsible for the effects of these miRNAs on cellular differentiation.

Mesenchyme to epithelium transitions are crucial to embryonic development. The early mouse embryo offers an excellent model to study epithelium formation as during the first three days of development two epithelia are formed, the trophectoderm (TE) and the primitive endoderm (PrE). We have previously shown that PrE cells are determined within the blastocyst ICM long before epithelium formation. In this work, we isolated Lrp2 as a novel PrE precursor (pre-PrE) marker by using a microarray strategy that combines a transcriptome analysis of three stem cell lines and early embryos. A detailed expression analysis shows that Lrp2 expression is induced in late E3.5 embryos indicating that pre-PrE cells are progressively maturing prior to polarization into an epithelium. Furthermore, the subcellular location of Lrp2, Disabled-2 (Dab2) and Collagen-IV shows that the epithelial structure is acquired in individual cells through successive steps.

During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 h period consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 h, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3a(tm1Amc) mutants but not in Dll3(pu) mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro.

Symplastic spermatids (sys) male mice are sterile due to a recessive mutation that causes defective adhesion between spermatids and Sertoli cells within the seminiferous epithelium. We show that the mutation in sys mice involves a deletion of 1.24 Mb of chromosome 14. Comparative genomic analysis suggests that this region contains only one gene, Fndc3a. A genetic complementation analysis using mice with a specific mutation within Fndc3a verifies that mutation of Fndc3a is the cause of male sterility in sys mice. Fndc3a is a member of a three-gene family in mice. Fndc3a, which is expressed in several tissues including testis, encodes a novel protein composed of a proline-rich amino-terminus, nine fibronectin type-III domains, and a hydrophobic carboxy-terminus. The proline-rich region of each family member contains conserved amino acids that include a PPGY consensus binding site for type I WW domain containing proteins. The hydrophobic carboxy-terminus is similar to that found in 'tail-anchored' proteins, integral membrane proteins that are localized to the cytosolic face of the endoplasmic reticulum. Immunohistochemical staining indicated that FNDC3A localizes to the acrosome of spermatids, as well as to Leydig cells in the mouse testis. Acrosomal localization of FNDC3A is observed in spermatids between step 2 and step 10 inclusive. In step 12 spermatids, FNDC3A is largely absent from the acrosomal region with immunostaining being localized to vesicular structures located within the cytoplasm of elongate spermatids. Models are presented for the function of FNDC3A in mediating spermatid-Sertoli adhesion during mouse spermatogenesis.

The Sin3a/HDAC co-repressor complex is a critical regulator of transcription networks that govern cell cycle control and apoptosis throughout development. Previous studies have identified Sin3a as essential for embryonic development around the time of implantation, during which the epiblast cell cycle is uniquely structured to achieve very rapid divisions with little tolerance of DNA damage. This study investigates the specific requirement for Sin3a in the early mouse embryo and shows that embryos lacking Sin3a suffer unresolved DNA damage and acute p53-independent apoptosis specifically in the E3.5-4.5 epiblast. Surprisingly, Myc and E2F targets in Sin3a-null ICMs are downregulated, suggesting a central but non-canonical role for Sin3a in regulating the pluripotent embryonic cell cycle. ES cells deleted for Sin3a mount a DNA damage response indicative of unresolved double-strand breaks, profoundly arrest at G2, and undergo apoptosis. These results indicate that Sin3a protects the genomic integrity of pluripotent embryonic cells and governs their unusual cell cycle.

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele.

Myf5 is a member of the muscle-specific determination genes and plays a critical role in skeletal muscle development. Whereas the expression of Myf5 during embryonic and fetal myogenesis has been extensively studied, its expression in progenitors that will ultimately give rise to adult satellite cells, the stem cells responsible for muscle repair, is still largely unexplored. To investigate this aspect, we have generated a mouse strain carrying a CreER coding sequence in the Myf5 locus. In this strain, Tamoxifen-inducible Cre activity parallels endogenous Myf5 expression. Combining Myf5(CreER) and Cre reporter alleles, we were able to evaluate the contribution of cells expressing Myf5 at distinct developmental stages to the pool of satellite cells in adult hindlimb muscles. Although it was possible to trace back the origin of some rare satellite cells to a subpopulation of Myf5(+ve) progenitors in the limb buds at the late embryonic stage (∼E12), a significant number of satellite cells arise from cells which expressed Myf5 for the first time at the fetal stage (∼E15). These studies provide direct evidence that adult satellite cells derive from progenitors that first express the myogenic determination gene Myf5 during fetal stages of myogenesis.

Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4-5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite-like cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse.

Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal-spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.

During pancreatic development insulin(+) cells co-express the transcription factors MafB and Pax6, and transition from a MafA(-) to MafA(+) state. To examine the role of Pax6 and MafB in the development of beta-cells, we analyzed embryonic pancreata from Pax6- and MafB-deficient mice. Pax6 deficiency, as manifest in the Pax6(Sey-Neu) allele, reduced not only the number of cells expressing insulin or glucagon, but also the number of MafB, PDX-1 and MafA expressing cells. We show that MafB can directly activate expression of insulin and glucagon, and a MafB protein engineered to contain N248S mutation in the MafB (kr(ENU)) results in significantly reduced activation. Furthermore, pancreata from MafB deficient (kr(ENU)/kr(ENU)) mice exhibited reduced number of cells expressing insulin, glucagon, PDX-1 and MafA, with only a minor reduction in MafB expressing cells. MafB deficiency does not affect endocrine specification but does affect the lineage commitment of the endocrine cells and their maturation. Similar to Pax6 deficient mice, MafB deficient mice showed reductions both in insulin and glucagon expressing cells and in the ability of MafB and PDX-1 expressing cells to activate expression of these hormones. However, MafB deficient mice exhibited no effect on Pax6 expression. These results suggest that MafB may function as a downstream mediator of Pax6 in regulating the specification of insulin and glucagon expressing cells. Interestingly, the remaining insulin(+) cells in these knockouts preferentially express Hb9, suggesting the existence of an alternate pathway for the generation of insulin expressing cells, even in the absence of Pax6 and MafB function. Thus, Pax6 acts upstream of MafB, which in turn may trigger the expression of insulin and regulate the PDX-1 and MafA expression required for beta-cell maturation.

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields.

The embryonic brain and spinal cord initially form through the process of neural tube closure (NTC). NTC is thought to be highly similar between rodents and humans, and studies of mouse genetic mutants have greatly increased our understanding of the molecular basis of NTC with relevance for human neural tube defects. In addition, studies using amphibian and chick embryos have shed light into the cellular and tissue dynamics underlying NTC. However, the dynamics of mammalian NTC has been difficult to study due to in utero development until recently when advances in mouse embryo ex vivo culture techniques along with confocal microscopy have allowed for imaging of mouse NTC in real time. Here, we have performed live imaging of mouse embryos with a particular focus on the non-neural ectoderm (NNE). Previous studies in multiple model systems have found that the NNE is important for proper NTC, but little is known about the behavior of these cells during mammalian NTC. Here we utilized a NNE-specific genetic labeling system to assess NNE dynamics during murine NTC and identified different NNE cell behaviors as the cranial region undergoes NTC. These results bring valuable new insight into regional differences in cellular behavior during NTC that may be driven by different molecular regulators and which may underlie the various positional disruptions of NTC observed in humans with neural tube defects.

The mammalian pancreas is known to show a remarkable degree of regenerative ability. Several studies until now have demonstrated that the mammalian pancreas can regenerate in normal as well as diabetic conditions. These studies illustrate that pancreatic transcription factors that are seen to be expressed in a temporal fashion during development are re-expressed during regeneration. The only known exception to this is Neurogenin3 (NGN3). Though NGN3 protein, which marks all the pro-endocrine cells during development, is not seen during mouse pancreas regeneration, functional neo-islets are generated by 4 weeks after 70% pancreatectomy. We observed that pancreatic transcription factors upstream of ngn3 showed similar gene expression patterns during development and regeneration. However, gene transcripts of transcription factors immediately downstream of ngn3 (neuroD and nkx2.2) did not show such similarities in expression. Since NGN3 protein was not detected at any time point during regeneration, we reasoned that post-transcriptional silencing of ngn3 by microRNAs may be a possible mechanism. We carried out microRNA analysis of 283 known and validated mouse microRNAs during different stages of pancreatic development and regeneration and identified that 4 microRNAs; miR-15a, miR-15b, miR-16 and miR-195, which can potentially bind to ngn3 transcript, are expressed at least 200-fold higher in the regenerating mouse pancreas as compared to embryonic day (e) 10.5 or e 16.5 developing mouse pancreas. Inhibition of these miRNAs in regenerating pancreatic cells using anti-sense miRNA-specific inhibitors, induces expression of NGN3 and its downstream players: neuroD and nkx2.2. Similarly, overexpression of miRNAs targeting ngn3 during pancreas development shows reduction in the number of hormone-producing cells. It appears that during pancreatic regeneration in mice, increased expression of these microRNAs allows endocrine regeneration via an alternate pathway that does not involve NGN3 protein. Our studies on microRNA profiling of developing and regenerating pancreas provide us with better understanding of mechanisms that regulate post-natal islet neogenesis.

Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA treatment expanded the etv5a domain within the renal progenitor field and RA inhibition blocked etv5a in this populace, indicating that etv5a acts downstream of RA. Additionally, treatment with exogenous RA partially rescued the reduced MCC phenotype after loss of etv5a. Further, abrogation of Notch with the small molecule inhibitor DAPT increased the renal progenitor etv5a expression domain as well as MCC density in etv5a deficient embryos, suggesting Notch acts upstream to inhibit etv5a. In contrast, etv4 levels in renal progenitors were unaffected by changes in RA or Notch signaling levels, suggesting a possible non-cell autonomous role during pronephros formation. Taken together, these findings have revealed new insights about the genetic mechanisms of epithelial cell development during nephrogenesis.