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Resident progenitor and/or stem cell populations in the adult adrenal cortex enable cortical cells to undergo homeostatic renewal and regeneration after injury. Renewal occurs predominantly in the outer layers of the adrenal gland but newly formed cells undergo centripetal migration, differentiation and lineage conversion in the process of forming the different functional steroidogenic zones. Over the past 10 years, advances in the genetic characterization of adrenal diseases and studies of mouse models with altered adrenal phenotypes have helped to elucidate the molecular pathways that regulate adrenal tissue renewal, several of which are fine-tuned via complex paracrine and endocrine influences. Moreover, the adrenal gland is a sexually dimorphic organ, and testicular androgens have inhibitory effects on cell proliferation and progenitor cell recruitment in the adrenal cortex. This Review integrates these advances, including the emerging role of sex hormones, into existing knowledge on adrenocortical cell renewal. An in-depth understanding of these mechanisms is expected to contribute to the development of novel therapies for severe endocrine diseases, for which current treatments are unsatisfactory.

The role of WNT/β-catenin signalling in mouse neocortex development remains ambiguous. Most studies demonstrate that WNT/β-catenin regulates progenitor self-renewal but others suggest it can also promote differentiation. Here we explore the role of WNT/STOP signalling, which stabilizes proteins during G2/M by inhibiting glycogen synthase kinase (GSK3)-mediated protein degradation. We show that mice mutant for cyclin Y and cyclin Y-like 1 (Ccny/l1), key regulators of WNT/STOP signalling, display reduced neurogenesis in the developing neocortex. Specifically, basal progenitors, which exhibit delayed cell cycle progression, were drastically decreased. Ccny/l1-deficient apical progenitors show reduced asymmetric division due to an increase in apical–basal astral microtubules. We identify the neurogenic transcription factors Sox4 and Sox11 as direct GSK3 targets that are stabilized by WNT/STOP signalling in basal progenitors during mitosis and that promote neuron generation. Our work reveals that WNT/STOP signalling drives cortical neurogenesis and identifies mitosis as a critical phase for neural progenitor fate., The EMBO Journal, 40 (19), ISSN:0261-4189, ISSN:1460-2075

Significance In the mammalian ovary, primordial germ cells maintain a genomic program associated with pluripotency until they stop proliferating, move toward oogenesis, and enter meiosis. The molecular mechanisms that enable primordial germ cells to exit pluripotency and enter meiosis in a timely manner are unclear, and their identification represents a major challenge in reproductive biology because the fertility of each individual depends on this. Evidence that cessation of germ cell proliferation is a cell-autonomous event, unrelated to the number of cell divisions, led to a search for an intrinsic timing mechanism that has long remained elusive. We describe here that WNT/β-catenin signaling regulates this timing and coordinates the transition from pluripotent to gametogenesis-competent germ cells., Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell–intrinsic β-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in β-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of β-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/β-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that β-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.

Retinoic acid (RA) is an essential signaling molecule for cardiac development and plays a protective role in the heart after myocardial infarction (MI). In both cases, the effect of RA signaling on cardiomyocytes, the principle cell type of the heart, has been reported to be indirect. Here we have developed an inducible murine transgenic RA-reporter line using CreER(T2) technology that permits lineage tracing of RA-responsive cells and faithfully recapitulates endogenous RA activity in multiple organs during embryonic development. Strikingly, we have observed a direct RA response in cardiomyocytes during mid-late gestation and after MI. Ablation of RA signaling through deletion of the Aldh1a1/a2/a3 genes encoding RA-synthesizing enzymes leads to increased cardiomyocyte apoptosis in adults subjected to MI. RNA sequencing analysis reveals Tgm2 and Ace1, two genes with well-established links to cardiac repair, as potential targets of RA signaling in primary cardiomyocytes, thereby providing novel links between the RA pathway and heart disease.

During kidney development, WNT/β-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of nephron progenitor cells. Here, we show in mice that the signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/β-catenin signalling and their genetic deletion leads to a rapid decline of nephron progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and reveal Lgr-dependent and independent functions for these ligands during kidney formation., eLife digest Kidneys filter waste out of the bloodstream to produce urine. Each kidney contains many structures called nephrons which separate the waste from the blood. The number of nephrons in a kidney varies between people, and those with low numbers have a higher risk of chronic kidney disease. Nephrons are formed before birth from a specific group of so-called progenitor cells. Each of these cells can either divide to make others like itself, or it can specialize to make nephron cells. At the end of embryonic kidney development, all the progenitor cells become nephron cells. Cells that specialize to become part of a nephron first go through a change called a mesenchyme-to-epithelial transition. Epithelial cells move less than mesenchymal cells, and also develop a clear structure where the two ends of the cell adapt to different roles. Evidence suggests that a cell communication process called WNT/β-catenin signaling controls this transition. Yet the details of how this transition is controlled are not fully understood. One way to activate WNT/β-catenin signaling is with R-spondin proteins, which have been found in developing kidneys. Vidal et al. studied R-spondins during the embryonic development of kidneys in mice. Removing R-spondins stopped the progenitor cells from producing more of themselves and increased the number that died. The R-spondins were also needed for the progenitor cells to specialize as nephron cells through the mesenchyme-to-epithelial transition. Further results revealed that R-spondins activate WNT/β-catenin signaling in these cells, even though the proteins that usually act as R-spondin receptors (called LGR4/5/6) could be removed without affecting the results. This suggests that R-spondins interact with different receptor proteins during kidney development. These findings highlight the role of R-spondins and WNT/β-catenin signaling in kidney development. Future studies will seek the receptor proteins that R-spondins interact with in kidneys. They may also look to understand how R-spondins balance their different roles in progenitor cells and during cell specialization. These results in mice could also be extended to determine their relevance in human health and disease, including chronic kidney disease, which is responsible for more deaths than breast or prostate cancer.

Retinoic acid synthesis is not essential for meiosis in fetal ovaries, challenging the dogma about the meiosis-inducing factors., In mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells.

Coronary arteries are essential to support the heart with oxygen, and coronary heart disease is one of the leading causes of death worldwide. The coronary arteries form at highly stereotyped locations and are derived from the primitive vascular plexus of the heart. How coronary arteries are remodeled and the signaling molecules that govern this process are poorly understood. Here, we have identified the Wnt-signaling modulator Rspo3 as a crucial regulator of coronary artery formation in the developing heart. Rspo3 is specifically expressed around the coronary stems at critical time points in their development. Temporal ablation of Rspo3 at E11.5 leads to decreased β-catenin signaling and a reduction in arterial-specific proliferation. As a result, the coronary stems are defective and the arterial tree does not form properly. These results identify a mechanism through which localized expression of RSPO3 induces proliferation of the coronary arteries at their stems and permits their formation.

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions –290/–274 and –57/–41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position –217/–206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1 , including Denys–Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.

International audience; Liver zonation, the spatial separation of different metabolic pathways along the liver sinusoids, is fundamental for proper functioning of this organ, and its disruption can lead to the development of metabolic disorders such as hyperammonemia. Metabolic zonation involves the induction of β-catenin signaling around the central veins, but how this patterned activity is established and maintained is unclear. Here, we show that the signaling molecule Rspondin3 is specifically expressed within the endothelial compartment of the central vein. Conditional deletion of Rspo3 in mice disrupts activation of central fate, demonstrating its crucial role in determining and maintaining β-catenin-dependent zonation. Moreover, ectopic expression of Rspo1, a close family member of Rspo3, induces the expression of pericentral markers, demonstrating Rspondins to be sufficient to imprint a more central fate. Thus, Rspo3 is a key angiocrine factor that controls metabolic zonation of liver hepatocytes.

The X-linked WTX/AMER1 protein constitutes an important component of the β-catenin destruction complex that can both enhance and suppress canonical β-catenin signaling. Somatic mutations in WTX/AMER1 have been found in a proportion of the pediatric kidney cancer Wilms' tumor. By contrast, germline mutations cause the severe sclerosing bone dysplasia osteopathia striata congenita with cranial sclerosis (OSCS), a condition usually associated with fetal or perinatal lethality in male patients. Here we address the developmental and molecular function of WTX by generating two novel mouse alleles. We show that in addition to the previously reported skeletal abnormalities, loss of Wtx causes severe midline fusion defects including cleft palate and ectopic synostosis at the base of the skull. By contrast, deletion of the C-terminal part of the protein results in only mild developmental abnormalities permitting survival beyond birth. Adult analysis, however, revealed skeletal defects including changed skull morphology and an increased whole-body bone density, resembling a subgroup of male patients carrying a milder, survivable phenotype. Molecular analysis in vitro showed that while β-catenin fails to co-immunoprecipitate with the truncated protein, partial recruitment appears to be achieved in an indirect manner using AXIN/AXIN2 as a molecular bridge. Taken together our analysis provides a novel model for WTX-caused bone diseases and explains on the molecular level how truncation mutations in this gene may retain some of WTX-protein functions. © 2018 American Society for Bone and Mineral Research.

Coronary artery anomalies are common congenital disorders with serious consequences in adult life. Coronary circulation begins when the coronary stems form connections between the aorta and the developing vascular plexus. We recently identified the WNT signaling modulator R-spondin 3 (Rspo3), as a crucial regulator of coronary stem proliferation. Using expression analysis and tissue-specific deletion we now demonstrate that Rspo3 is primarily produced by cardiomyocytes. Moreover, we have employed CRISPR/Cas9 technology to generate novel Lgr4-null alleles that showed a significant decrease in coronary stem proliferation and thus phenocopied the coronary artery defects seen in Rspo3 mutants. Interestingly, Lgr4 mutants displayed slightly hypomorphic right ventricles, an observation also made after myocardial specific deletion of Rspo3. These results shed new light on the role of Rspo3 in heart development and demonstrate that LGR4 is the principal R-spondin 3 receptor in the heart.

International audience; The Wilms' tumor suppressor WT1 is a key regulator of podocyte function that is mutated in Denys-Drash and Frasier syndromes. Here we have used an integrative approach employing ChIP, exon array, and genetic analyses in mice to address general and isoform-specific functions of WT1 in podocyte differentiation. Analysis of ChIP-Seq data showed that almost half of the podocyte-specific genes are direct targets of WT1. Bioinformatic analysis further identified coactivator FOXC1-binding sites in proximity to WT1-bound regions, thus supporting coordinated action of these transcription factors in regulating podocyte-specific genes. Transcriptional profiling of mice lacking the WT1 alternative splice isoform (+KTS) had a more restrictive set of genes whose expression depends on these alternatively spliced isoforms. One of these genes encodes the membrane-associated guanylate kinase MAGI2, a protein that localizes to the base of the slit diaphragm. Using functional analysis in mice, we further show that MAGI2α is essential for proper localization of nephrin and the assembly of the slit diaphragm complex. Finally, a dramatic reduction of MAGI2 was found in an LPS mouse model of glomerular injury and in genetic cases of human disease. Thus, our study highlights the central role of WT1 in podocyte differentiation, identifies that WT1 has a central role in podocyte differentiation, and identifies MAGI2α as the crucial isoform in slit diaphragm assembly, suggesting a causative role of this gene in the etiology of glomerular disorders.

International audience; WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain-of-function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele.

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce β-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life.

Nephrin (NPHS1) has been described as an important structural protein of kidney podocytes. Mutations in this gene lead to the Finnish-type congenital nephrotic syndrome. More recently, a role of nephrin as a signalling molecule in kidney podocytes has been identified. Here, we show that nephrin not only has a function in kidney podocytes, but is also required for cardiovascular development. Nephrin is expressed in the epicardium and coronary vessels during human and mouse embryonic development. Nephrin knockout embryos showed abnormal epicardial cell morphology and, at later stages of development, a reduced number of coronary vessels due to increased apoptosis, and in addition, cardiac fibrosis. Connexin 43, which is required for coronary vessel formation, was downregulated in nephrin knockout embryos. Expression of the p75NTR neurotrophin receptor, a known mediator of apoptosis, was increased in mutants. Furthermore, co-immunoprecipitation studies demonstrated a direct interaction of nephrin with p75NTR. Primary nephrin-deficient cardiac cells showed a 5-fold higher rate of apoptosis in response to progenitor of nerve growth factor compared with wild-type cells, which could be rescued by RNAi against p75NTR. Taken together, our data demonstrate that nephrin directly interacts with p75NTR and reveal an important role for nephrin in murine cardiac development by permitting survival of cardiovascular progenitor cells.

Congenital abnormalities of the kidney and urinary tract are some of the most common defects detected in the unborn child. Kidney growth is controlled by the GDNF/RET signalling pathway, but the molecular events required for the activation of RET downstream targets are still poorly understood. Here we show that SOX9, a gene involved in campomelic dysplasia (CD) in humans, together with its close homologue SOX8, plays an essential role in RET signalling. Expression of SOX9 can be found from the earliest stages of renal development within the ureteric tip, the ureter mesenchyme and in a segment-specific manner during nephrogenesis. Using a tissue-specific knockout approach, we show that, in the ureteric tip, SOX8 and SOX9 are required for ureter branching, and double-knockout mutants exhibit severe kidney defects ranging from hypoplastic kidneys to renal agenesis. Further genetic analysis shows that SOX8/9 are required downstream of GDNF signalling for the activation of RET effector genes such as Sprouty1 and Etv5. At later stages of development, SOX9 is required to maintain ureteric tip identity and SOX9 ablation induces ectopic nephron formation. Taken together, our study shows that SOX9 acts at multiple steps during kidney organogenesis and identifies SOX8 and SOX9 as key factors within the RET signalling pathway. Our results also explain the aetiology of kidney hypoplasia found in a proportion of CD patients.

WTX/AMER1 is a novel negative regulator of the WNT/β-catenin pathway with mutations detected in Wilms' tumors and an X-linked sclerosing bone dysplasia. WTX/AMER1 (Fam123b) shares several domains of homology with two other recently identified proteins: AMER2 (Fam123a) and AMER3 (Fam123c). Here, we describe an in-depth expression analysis of all three members of this gene family during mouse embryonic development. All genes were strongly expressed in the central as well as the peripheral nervous system, thus suggesting important roles of this gene family during neurogenesis. Specific expression was found in the retina, inner ear, and nasal epithelium. Outside of the nervous system Wtx/Amer1 showed the broadest expression domains including cephalic and limb mesenchyme, skeletal muscle, bladder, gonads, lung bud, salivary glands, and the kidneys. The widespread expression pattern of Wtx/Amer1, together with its role as a modulator of the Wnt signaling pathway, suggest that Wtx/Amer1 serves pleiotropic roles during mammalian organogenesis. Developmental Dynamics 239:1867–1878, 2010. © 2010 Wiley-Liss, Inc.

Human patients with Frasier syndrome express reduced levels of the +KTS isoforms of the developmental regulator WT1 and exhibit complete XY gonadal dysgenesis and male-to-female sex reversal. Mice with a targeted mutation that blocks production of these isoforms show a reduction in Sry mRNA in the gonad, but the molecular and cellular basis of this reduction has not been established. Using immunofluorescence analysis, we found a significantly lower level of SRY protein per cell in XY Wt1(+KTS)-null mouse gonads. We also found a reduced number of SRY-expressing cells, correlating with a decrease in cell proliferation at and near the coelomic epithelium at 11.5 dpc. No reduction in somatic cell numbers was seen in XX Wt1(+KTS)-null gonads, indicating that the effect of WT1 on cell proliferation is mediated by Sry. Sertoli cell differentiation was blocked in XY Wt1(+KTS)-null mouse gonads, as indicated by the loss of SOX9 and Fgf9 expression, but the addition of recombinant FGF9 to ex vivo gonad cultures rescued the mutant phenotype, as indicated by the induction of the Sertoli-cell specific marker anti-Müllerian hormone. Our data suggest that WT1(+KTS) is involved in the cell-autonomous regulation of Sry expression, which in turn influences cell proliferation and Sertoli cell differentiation via FGF9. Thus, sex reversal in Wt1(+KTS)-null mice and Frasier syndrome patients results from a failure of Sertoli cells both to fully differentiate and to reach sufficient numbers to direct testis development.

International audience; The sex of an individual is determined by the fate of the gonad. While the expression of Sry and Sox9 is sufficient to induce male development, we here show that female differentiation requires activation of the canonical b-catenin signaling pathway. b-catenin activation is controlled by Rspo1 in XX gonads and Rspo1 knockout mice show masculinized gonads. Molecular analyses demonstrate an absence of female-specific activation of Wnt4 and as a consequence XY-like vascularization and steroidogenesis. Moreover, germ cells of XX knockout embryos show changes in cellular adhesions and a failure to enter XX specific meiosis. Sex cords develop around birth, when Sox9 becomes strongly activated. Thus, a balance between Sox9 and b-catenin activation determines the fate of the gonad, with Rspo1 acting as a crucial regulator of canonical b-catenin signaling required for female development.

International audience; After acute kidney injury (AKI), surviving cells within the nephron proliferate and repair. We identify Sox9 as an acute epithelial stress response in renal regeneration. Translational profiling after AKI revealed a rapid upregulation of Sox9 within proximal tubule (PT) cells, the nephron cell type most vulnerable to AKI. Descendants of Sox9(+) cells generate the bulk of the nephron during development and regenerate functional PT epithelium after AKI-induced reactivation of Sox9 after renal injury. After restoration of renal function post-AKI, persistent Sox9 expression highlights regions of unresolved damage within injured nephrons. Inactivation of Sox9 in PT cells pre-injury indicates that Sox9 is required for the normal course of post-AKI recovery. These findings link Sox9 to cell intrinsic mechanisms regulating development and repair of the mammalian nephron.

R-spondins are a recently characterized small family of growth factors. Here we show that human R-spondin1 (RSPO1) is the gene disrupted in a recessive syndrome characterized by XX sex reversal, palmoplantar hyperkeratosis and predisposition to squamous cell carcinoma of the skin. Our data show, for the first time, that disruption of a single gene can lead to complete female-to-male sex reversal in the absence of the testis-determining gene, SRY.

Nestin is an intermediate filament protein originally described in neural stem cells and a variety of progenitor cells. More recently, nestin was detected in rat kidney podocytes. We show here that nestin is expressed in a developmentally regulated pattern in the kidney. Nestin was detected by immunohistochemistry in the condensing mesenchyme surrounding the ureter, in developing glomeruli, in podocytes of the adult kidney, and in a podocyte cell line. Nestin shared a striking overlap in expression with the Wilms' tumor suppressor Wt1. Nestin was significantly upregulated in a cell line with inducible Wt1 expression upon induction of Wt1. Cotransfection experiments in human embryonic kidney cells (HEK293) revealed stimulation of a nestin intron 2 enhancer element up to six-fold by the Wt1(-KTS) splice variant. Nestin expression was significantly reduced in an inducible mouse model of glomerular disease. This model is based on podocyte-specific overexpression of Pax2 and associated with a loss of Wt1 expression ( 33 ). Furthermore, also in the developing heart, nestin was found in an overlapping pattern with Wt1 in the epicardium and the forming coronary vessels. Strikingly, in the hearts of Wt1 knockout mice, nestin was barely detectable compared with the hearts of wild-type embryos. Our results show that nestin is expressed at different stages of kidney and cardiac development and suggest that its expression in these organs might be regulated by the Wilms' tumor suppressor Wt1.

Bone marrow (BM) transplantation has been shown to provide beneficial effects in injured organs, including heart, liver, and kidney. We explored the therapeutic potential of BM transplantation (BMT) in Wilms' tumor suppressor 1 (Wt1) heterozygous mice, which represent a model of mesangial sclerosis. After transplantation of wild-type BM, there is statistically significantly lower urinary albumin and increased survival in Wt1+/− recipients. Control BMT using Wt1+/− donors showed no significant beneficial effects. The long-term beneficial effect of BMT was dependent on the dose of irradiation applied to the recipients before BMT. At a lethal dose of 1,000 cGy, the decrease in albuminuria and prolongation of lifespan in Wt1+/− mice were transient, with maximal amelioration at 12 weeks and resumption of albuminuria by 24 weeks after BMT. This was, at least in part, due to irradiation and not Wt1 heterozygosity because wild-type recipients also developed albuminuria within 24 weeks of BMT with 1,000 cGy. In contrast, Wt1+/− mice transplanted after 400 cGy showed long-term improvement in albuminuria and lifespan. Approximately 0.4% of podocytes were marrow derived, a level that is unlikely to be responsible for the therapeutic effects. In addition, donor BM cells formed rings surrounding the glomeruli, and approximately one third of the cells in these rings were macrophages. In conclusion, transplantation of wild-type BM attenuates progression of mesangial sclerosis in the Wt1+/− model of renal disease, and the mechanism by which this occurs may involve engraftment of BM-derived cells in the renal parenchyma.

WT1 has been implicated in human leukemia and hematopoiesis, but its role in stem cell differentiation is not yet fully defined. We show that Wt1-null murine fetal liver cells are capable of reconstituting functional hematopoiesis following transplantation into irradiated recipients. There was also no significant difference between the in vitro colony-forming ability of wild-type and Wt1-null cells. Using a reporter gene assay in a transgenic mouse system, expression from the WT1 promoter was detectable in adult bone marrow, but undetectable in subsets of different hematopoietic cells. We conclude that Wt1 is not essential for murine hematopoiesis and that there may be significant differences in its role between mouse and man.

SummaryTrunk neural crest cells are generated at the border between the neural plate and nonneural ectoderm, where they initiate a distinct program of gene expression, undergo an epithelial-mesenchymal transition (EMT), and delaminate from the neuroepithelium. Here, we provide evidence that members of three families of transcription induce these properties in premigratory neural crest cells. Sox9 acts to provide the competence for neural crest cells to undergo an EMT and is required for trunk neural crest survival. In the absence of Sox9, cells apoptose prior to or shortly after delamination. Slug/Snail, in the presence of Sox9, is sufficient to induce an EMT in neural epithelial cells, while FoxD3 regulates the expression of cell-cell adhesion molecules required for neural crest migration. Together, the data suggest a model in which a combination of transcription factors regulates the acquisition of the diverse properties of neural crest cells.

Brain 4 (Brn4/Pou3f4) and Pax6 are POU-homeodomain and paired-homeodomain transcription factors, respectively, that are expressed in the brain and the glucagon-expressing cells in the pancreas. Brn4 expression begins at embryonic day 10 in the pancreas, just before pax6 and both appear in the glucagon immunoreactive cells. At a later time point, E19, no Brn4 co-localization is observed with insulin or somatostatin but a rare pancreatic polypeptide (PP)-producing cell can be found, while Pax6 is found in all endocrine cells. These data suggest that brn4 is the only alpha-cell specific transcription factor yet identified; therefore, we sought to analyze alpha-cell development and function in mice with a targeted disruption of the brn4 gene. In homozygous brn4(-/-) mice, pancreatic bud formation, glucagon cell numbers and physiological measurements all appear normal. Examination of other transcription factors found in the glucagon cells showed normal Pax6 and Nkx2.2 immunoreactivity, suggesting that Brn4 does not regulate these transcription factors. Pax6 mutant mice (pax6(Sey/Sey)), with a natural inactivating mutation in pax6, have few endocrine cells but normal numbers of Brn4 and Nkx2.2 cells. The pancreatic phenotype of the pax6 mutants can be rescued with a YAC clone containing the human Pax6 gene.

The Wilms' Tumour 1 (WT1) gene plays an important role at three different stages of kidney development. The onset of kidney formation, the progression of kidney formation and the maintenance of normal kidney function. Disruption of WT1 may lead to a whole spectrum of kidney diseases ranging from tumour development to mild forms of renal failure. However, the underlying mechanisms are largely unknown. The WT1 proteins have been implicated in various cellular processes like proliferation, differentiation and apoptosis and in agreement with these diverse functions, the number of target genes is still mounting. The development of mouse models in recent years has contributed considerably to a better understanding of the biological activities of WT1, and in this article we will discuss the role of WT1 during kidney formation and kidney function.

The mechanism that causes neural stem cells in the central nervous system to switch from neurogenesis to gliogenesis is poorly understood. Here we analyzed spinal cord development of mice in which the transcription factor Sox9 was specifically ablated from neural stem cells by the CRE/loxPrecombination system. These mice exhibit defects in the specification of oligodendrocytes and astrocytes, the two main types of glial cells in the central nervous system. Accompanying an early dramatic reduction in progenitors of the myelin-forming oligodendrocytes, there was a transient increase in motoneurons. Oligodendrocyte progenitor numbers recovered at later stages of development, probably owing to compensatory actions of the related Sox10 and Sox8, both of which overlap with Sox9 in the oligodendrocyte lineage. In agreement, compound loss of Sox9 and Sox10 led to a further decrease in oligodendrocyte progenitors. Astrocyte numbers were also severely reduced in the absence of Sox9 and did not recover at later stages of spinal cord development. Taking the common origin of motoneurons and oligodendrocytes as well as V2 interneurons and some astrocytes into account, stem cells apparently fail to switch from neurogenesis to gliogenesis in at least two domains of the ventricular zone, indicating that Sox9 is a major molecular component of the neuron–glia switch in the developing spinal cord.

International audience; : Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes.

Alternative splicing of Wt1 results in the insertion or omission of the three amino acids KTS between zinc fingers 3 and 4. In vitro experiments suggest distinct molecular functions for + and −KTS isoforms. We have generated mouse strains in which specific isoforms have been removed. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. Homozygous mutants of both strains die after birth due to kidney defects. Strikingly, mice lacking +KTS isoforms show a complete XY sex reversal due to a dramatic reduction of Sry expression levels. Our data demonstrate distinct functions for the two splice variants and place the +KTS variants as important regulators for Sry in the sex determination pathway.

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9. Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5′ to SOX9 are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9 , we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9 . We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5′ to SOX9 .

We describe the localization of the gene encoding reticulocalbin, a Ca 2+ -binding protein of the endoplasmic reticulum, on human chromosome 11p13 midway between the WT1 and the PAX6 genes and show that it is hemizygously deleted in WAGR individuals. The mouse reticulocalbin gene is also shown to map to the region of conserved synteny on mouse chromosome 2 and to be deleted in the Small eye Harwell ( Sey H ) mutation. Loss of the reticulocalbin gene could contribute to the early lethality of Sey H and Sey Dey homozygotes.

International audience; Kidney organogenesis requires the tight control of proliferation, differentiation and apoptosis of renal progenitor cells. How the balance between these cellular decisions is achieved remains elusive. The Wilms' tumour suppressor Wt1 is required for progenitor survival, but the molecular cause for renal agenesis in mutants is poorly understood. Here we demonstrate that lack of Wt1 abolishes fibroblast growth factor (FGF) and induces BMP/pSMAD signalling within the metanephric mesenchyme. Addition of recombinant FGFs or inhibition of pSMAD signalling rescues progenitor cell apoptosis induced by the loss of Wt1. We further show that recombinant BMP4, but not BMP7, induces an apoptotic response within the early kidney that can be suppressed by simultaneous addition of FGFs. These data reveal a hitherto unknown sensitivity of early renal progenitors to pSMAD signalling, establishes FGF and pSMAD signalling as antagonistic forces in early kidney development and places WT1 as a key regulator of pro-survival FGF signalling pathway genes.

International audience; Adrenal glands and gonads share a common primordium (AGP), but the molecular events driving differentiation are poorly understood. Here we demonstrate that the Wilms tumor suppressor WT1 is a key factor defining AGP identity by inhibiting the steroidogenic differentiation process. Indeed, ectopic expression of WT1 precludes differentiation into adrenocortical steroidogenic cells by locking them into a progenitor state. Chromatin immunoprecipitation experiments identify Tcf21 and Gli1 as direct targets of WT1. Moreover, cell lineage tracing analyses identify a long-living progenitor population within the adrenal gland, characterized by the expression of WT1, GATA4, GLI1, and TCF21, that can generate steroidogenic cells in vivo. Strikingly, gonadectomy dramatically activates these WT1(+) cells and leads to their differentiation into gonadal steroidogenic tissue. Thus, our data describe a mechanism of response to organ loss by recreating hormone-producing cells at a heterotopic site.

International audience; Chondroitin sulfate proteoglycans (CSPGs) found in perineuronal nets and in the glial scar after spinal cord injury have been shown to inhibit axonal growth and plasticity. Since we have previously identified SOX9 as a transcription factor that upregulates the expression of a battery of genes associated with glial scar formation in primary astrocyte cultures, we predicted that conditional Sox9 ablation would result in reduced CSPG expression after spinal cord injury and that this would lead to increased neuroplasticity and improved locomotor recovery. Control and Sox9 conditional knock-out mice were subject to a 70 kdyne contusion spinal cord injury at thoracic level 9. One week after injury, Sox9 conditional knock-out mice expressed reduced levels of CSPG biosynthetic enzymes (Xt-1 and C4st), CSPG core proteins (brevican, neurocan, and aggrecan), collagens 2a1 and 4a1, and Gfap, a marker of astrocyte activation, in the injured spinal cord compared with controls. These changes in gene expression were accompanied by improved hind limb function and locomotor recovery as evaluated by the Basso Mouse Scale (BMS) and rodent activity boxes. Histological assessments confirmed reduced CSPG deposition and collagenous scarring at the lesion of Sox9 conditional knock-out mice, and demonstrated increased neurofilament-positive fibers in the lesion penumbra and increased serotonin immunoreactivity caudal to the site of injury. These results suggest that SOX9 inhibition is a potential strategy for the treatment of SCI. © 2012 Wiley Periodicals, Inc.

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. To investigate the role of gene dosage in more detail, we have generated yeast artificial chromosome transgenic mice carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. We provide evidence that not only reduced, but also increased levels of transcriptional regulators can cause developmental defects.

International audience; The gonad arises from the thickening of the coelomic epithelium and then commits into the sex determination process. Testis differentiation is activated by the expression of the Y-linked gene Sry, which promotes cell proliferation and differentiation of Sertoli cells, the supporting cells of the testis. In absence of Sry (XX individuals), activation of WNT/CTNNB1 signalling, via the upregulation of Rspo1 and Wnt4, promotes ovarian differentiation. However, Rspo1 and Wnt4 are expressed in the early undifferentiated gonad of both sexes, and Axin2-lacZ, a reporter of canonical WNT/CTNNB1 signalling, is expressed in the coelomic region of the E11.5 gonadal primordium, suggesting a role of these factors in early gonadal development. Here, we show that simultaneous ablation of Rspo1 and Wnt4 impairs proliferation of the cells of the coelomic epithelium, reducing the number of progenitors of Sertoli cells in XY mutant gonads. As a consequence, in XY Wnt4(-/-); Rspo1(-/-) foetuses, this leads to the differentiation of a reduced number of Sertoli cells and the formation of a hypoplastic testis exhibiting few seminiferous tubules. Hence, this study identifies Rspo1 and Wnt4 as two new regulators of cell proliferation in the early gonad regardless of its sex, in addition to the specific role of these genes in ovarian differentiation.

SOX9 [sex-determining region Y (SRY)-box 9 protein], a high mobility group box transcription factor, plays critical roles during embryogenesis and its activity is required for development, differentiation, and lineage commitment in various tissues including the intestinal epithelium. Here, we present functional and clinical data of a broadly important role for SOX9 in tumorigenesis. SOX9 was overexpressed in a wide range of human cancers, where its expression correlated with malignant character and progression. Gain of SOX9 copy number is detected in some primary colorectal cancers. SOX9 exhibited several pro-oncogenic properties, including the ability to promote proliferation, inhibit senescence, and collaborate with other oncogenes in neoplastic transformation. In primary mouse embryo fibroblasts and colorectal cancer cells, SOX9 expression facilitated tumor growth and progression whereas its inactivation reduced tumorigenicity. Mechanistically, we have found that Sox9 directly binds and activates the promoter of the polycomb Bmi1, whose upregulation represses the tumor suppressor Ink4a/Arf locus. In agreement with this, human colorectal cancers showed a positive correlation between expression levels of SOX9 and BMI1 and a negative correlation between SOX9 and ARF in clinical samples. Taken together, our findings provide direct mechanistic evidence of the involvement of SOX9 in neoplastic pathobiology, particularly, in colorectal cancer. ©2012 AACR., link_to_OA_fulltext

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1KOSox9cKO ), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1KOSox9cKO mice. Moreover, since testis development occurs in XY Rspo1KOSox9cKO mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9cKO mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised., Author Summary Mammalian sex determination is controlled by the paternal transmission of the Y-linked gene, SRY. Using mouse models, it has been shown that the main, if not the only, role of Sry is to activate the transcription factor Sox9, and these two genes are necessary and sufficient to allow male development. Indeed, defects in Sry and/or Sox9 expression result in male-to-female sex reversal of XY individuals. In XX individuals, Rspo1 is important for ovarian development as evidenced by female-to-male sex reversal of XX Rspo1 mutants. Since testicular differentiation appears concomitantly with Sox9 expression, it was assumed that Sox9 is the inducer of testicular differentiation in XX Rspo1 mutants. Our genetic study shows that i) neither Sry nor Sox9 are required for female-to-male sex reversals; ii) other masculinizing factors like Sox8 and Sox10 are activated in sex reversal conditions; iii) Rspo1 is the main effector of male-to-female sex reversal in the XY Sox9 mutants. Together these data suggest that male and female genetic pathways are both main effectors involved in sex determination and that the long-standing dogma of a default female pathway should definitively be revised.

SUMMARY Wilms’ tumour is a paediatric kidney malignancy that arises through aberrant differentiation of nephric stem cells. We are studying the role of one Wilms’ tumour pre-disposition gene, WT1. This is a tumour suppressor gene whose function is required for normal development of the genitourinary system. WT1 encodes a putative transcriptional repressor of the zinc finger family. Here we discuss how one of the normal functions of WT1 may be to suppress myogenesis during kidney development. Furthermore, we describe how we are proposing to use YAC (yeast artificial chromosome) transgenesis to analyse WT1 regulation and function in mice. We also discuss the evolution of the WT1 gene amongst different vertebrate classes and how this may provide insights into genitourinary evolution.

Mice lacking the function of the polycomb group protein CBX2 (chromobox homolog 2; also known as M33) show defects in gonadal, adrenal, and splenic development. In particular, XY knockout (KO) mice develop ovaries but not testes, and the gonads are hypoplastic in both sexes. However, how CBX2 regulates development of these tissues remains largely unknown. In the present study, we used microarray, RT-PCR, and immunohistochemical analyses to show that the expression of Sry, Sox9, Lhx9, Ad4BP/SF-1, Dax-1, Gata4, Arx, and Dmrt1, genes encoding transcription factors essential for gonadal development, is affected in Cbx2 KO gonads. Male-to-female sex reversal in Cbx2 KO mice was rescued by crossing them with transgenic mice displaying forced expression of Sry or Sox9. However, testes remained hypoplastic in these mice, indicating that the size and the sex of the gonad are determined by different sets of genes. Our study implicates Cbx2 in testis differentiation through regulating Sry gene expression.

During corticogenesis, late-born callosal projection neurons (CPNs) acquire their laminar position through glia-guided radial migration and then undergo final differentiation. However, the mechanisms controlling radial migration and final morphology of CPNs are poorly defined. Here, we show that in COUP-TFI mutant mice CPNs are correctly specified, but are delayed in reaching the cortical plate and have morphological defects during migration. Interestingly, we observed that the rate of neuronal migration to the cortical plate normally follows a low-rostral to high-caudal gradient, similar to that described for COUP-TFI. This gradient is strongly impaired in COUP-TFI–/– brains. Moreover, the expression of the Rho-GTPase Rnd2, a modulator of radial migration, is complementary to both these gradients and strongly increases in the absence of COUP-TFI function. We show that COUP-TFI directly represses Rnd2 expression at the post-mitotic level along the rostrocaudal axis of the neocortex. Restoring correct Rnd2 levels in COUP-TFI–/– brains cell-autonomously rescues neuron radial migration and morphological transitions. We also observed impairments in axonal elongation and dendritic arborization of COUP-TFI-deficient CPNs, which were rescued by lowering Rnd2 expression levels. Thus, our data demonstrate that COUP-TFI modulates late-born neuron migration and favours proper differentiation of CPNs by finely regulating Rnd2 expression levels.

Expression of transgenes in mice often fails to follow the normal temporal and spatial pattern and to reach the same level as the endogenous copies. Only in exceptional cases has position-independent and copy number-dependent expression been reproduced. The size constraint of standard constructs may prevent the inclusion of important remote regulatory elements. Yeast artificial chromosomes (YACs) provide a means of cloning large DNA fragments and the transfer of YAC DNA into somatic cells has been reported. We have previously produced transgenic mice carrying a 35 kilobase YAC construct. Here we report the transfer of a 250 kilobase YAC covering the mouse tyrosinase gene into mice by pronuclear injection of gel-purified YAC DNA. The YAC was inserted into the mouse genome without major rearrangements and expression of the YAC-borne tyrosinase gene resulted in complete rescue of the albino phenotype of the recipient mice. Expression from the transgene reached levels comparable to that of the endogenous gene and showed copy number dependence and position independence.

Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications., Author Summary In mammals, many genes execute a unique set of distinctive and common functions in different cell types. Strategies to address these individual roles often involve the generation of series of transgenic animals. Here, we present a novel approach that combines a single transgenic mouse line with tissue-specific transfection protocols and organotypic cultures to enable the quick analysis of numerous genes in a cell-specific manner. As a proof of principle, we analyzed the function of transcription factors in tangentially migrating neurons of the developing vertebrate hindbrain. We identified a temporary halt in migration as a novel mechanism for neurons to decide whether to cross or not cross the midline. Our model may serve as a general tool to quickly study axonal pathfinding, neuronal cell migration, or patterning processes in a well-defined population of neurons.

Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke in humans. Dominant mutations in NOTCH3 cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a genetic archetype of cerebral ischemic SVD. Progress toward understanding the pathogenesis of this disease and developing effective therapies has been hampered by the lack of a good animal model. Here, we report the development of a mouse model for CADASIL via the introduction of a CADASIL-causing Notch3 point mutation into a large P1-derived artificial chromosome (PAC). In vivo expression of the mutated PAC transgene in the mouse reproduced the endogenous Notch3 expression pattern and main pathological features of CADASIL, including Notch3 extracellular domain aggregates and granular osmiophilic material (GOM) deposits in brain vessels, progressive white matter damage, and reduced cerebral blood flow. Mutant mice displayed attenuated myogenic responses and reduced caliber of brain arteries as well as impaired cerebrovascular autoregulation and functional hyperemia. Further, we identified a substantial reduction of white matter capillary density. These neuropathological changes occurred in the absence of either histologically detectable alterations in cerebral artery structure or blood-brain barrier breakdown. These studies provide in vivo evidence for cerebrovascular dysfunction and microcirculatory failure as key contributors to hypoperfusion and white matter damage in this genetic model of ischemic SVD.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.

Previous complementation analyses with five (c11DSD, c5FR60Hg, c2YPSj, c4FR60Hd, c6H) of the mouse albino deletions defined at least two genes on chromosome 7, known as eed and exed, which are necessary for development of the embryonic and extraembryonic ectoderm, respectively, of early postimplantation embryos. The region of chromosome 7 containing these two genes has now been accessed at the molecular level by cloning two of the deletion breakpoint-fusion fragments. The c2YPSj breakpoints were isolated by cloning an EcoRI fragment containing a copy of an albino region-specific repeat unique to c2YPSj DNA. Similarly, the c11DSD breakpoints were isolated by cloning a c11DSD EcoRI fragment detected by a unique-sequence probe mapping proximal to the albino-coat-color locus. By mapping the cloned breakpoints relative to the remaining three deletions, the c11DSD distal breakpoint was found to define the distal limit of the region containing eed, whereas the c2YPSj and c6H distal breakpoints were found to define the proximal and distal limits, respectively, of the region containing exed.

Sox9 is an Sry-box-containing gene that encodes a transcriptional activator. During mouse gonadogenesis, Sox9 is detected in the male gonad at 11.5 days postcoitus (dpc). At 12.5 dpc, testicular cords form, morphologically distinguishing the male gonad from the ovary. From this stage onwards, Sox9 expression is restricted to the Sertoli cell lineage and persists in the adult. Humans with heterozygous mutations in SOX9 develop a skeletal syndrome known as campomelic dysplasia. Furthermore, most XY SOX9 heterozygotes show variable male-to-female sex reversal, implicating SOX9 in testis development. Sox9 heterozygous knockout mice die at birth with a syndrome similar to that of human campomelic dysplasia. In contrast to humans, XY Sox9+/- mice form normal appearing testes. Germ-line knockout of Sox9 using a conditional null allele provides a tool for generating Sox9-/- mice by simple genetic crosses. However, Sox9-/- mice die soon after 11.5 dpc because of cardiovascular defects. In vitro culture of the urogenital ridges of XY Sox9-/- results in gonads lacking testicular cords and Sertoli cell marker expression, but with the expression of ovarian-specific markers. Therefore, Sox9 is essential for diverting an intrinsically ovarian program of organogenesis toward testis formation.

The formation of intramyocardial blood vessels is critical for normal heart development and tissue repair after infarction. We report here expression of the Wilms' tumor gene-1, Wt1, in coronary vessels, which could contribute to the defective cardiac vascularization in Wt1-/- mice. Furthermore, the high-affinity neurotrophin receptor TrkB, which is expressed in the epicardium and subepicardial blood vessels, was nearly absent from Wt1-deficient hearts. Activation of Wt1 in an inducible cell line significantly enhanced TrkB expression. The promoter of NTRK2, the gene encoding TrkB, was stimulated ∼10-fold by transient cotransfection of a Wt1 expression construct. The critical DNA-binding site for activation of the NTRK2 promoter by Wt1 was delineated by DNase I footprint analysis and electrophoretic mobility shift assay. Transgenic experiments revealed that the identified Wt1 consensus motif in the NTRK2 promoter was necessary to direct expression of a reporter gene to the epicardium and the developing vasculature of embryonic mouse hearts. Finally, mice with a disrupted Ntrk2 gene lacked a significant proportion of their intramyocardial blood vessels. These findings demonstrate that transcriptional activation of the TrkB neurotrophin receptor gene by the Wilms' tumor suppressor Wt1 is a crucial mechanism for normal vascularization of the developing heart.

Neuronal lineage formation in the developing olfactory epithelium has been extensively studied at the cellular level, but little is known about the genes that control proliferation and differentiation of neuronal progenitor cells. Here, we report that the Wilms9 tumour zinc-finger protein, Wt1, is required for normal formation of the olfactory epithelium. Wt1 was detected by immunohistochemistry in the developing olfactory epithelium of wild-type embryos between gestational days E9.5 and E18.5. Embryos with complete lack of Wt1 and embryos with selective ablation of the alternatively spliced Wt1( + KTS) isoform both had thinner olfactory epithelia and fewer neuronal progenitor cells than do normal animals. Mash1 and neurogenin 1, two basic helix-loop-helix transcription factors with critical functions during olfactory neuron development, were reduced in the Wt1( + KTS) -/- mutants compared with the wild-type mice. Stable expression of the Wt1( + KTS) isoform, but not of the Wt1( - KTS) variant, upregulated Mash1 mRNA and protein in vitro. The olfactory epithelia of mouse embryos, which lacked the Wt1(-KTS) protein, appeared normal. However, formation of the neural retina was severely impaired in the Wt1( - KTS) -/- mutants. These findings demonstrate that the Wt1(+KTS) protein, which has been proposed to play a role in mRNA processing, acts upstream of Mash1 to promote the development of the olfactory epithelium. Furthermore, neuron formation depends on distinct functions of alternatively spliced Wt1 products in the embryonic retina and the olfactory epithelium.