Andreas Schedl

R-spondin1 is essential in sex determination, skin differentiation and malignancy

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.

Two Splice Variants of the Wilms' Tumor 1 Gene Have Distinct Functions during Sex Determination and Nephron Formation

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.

Functional analysis of Sox8 and Sox9 during sex determination in the mouse

Sex determination in mammals directs an initially bipotential gonad to differentiate into either a testis or an ovary. This decision is triggered by the expression of the sex-determining gene Sry, which leads to the activation of male-specific genes including the HMG-box containing gene Sox9. From transgenic studies in mice it is clear that Sox9 is sufficient to induce testis formation. However, there is no direct confirmation for an essential role for Sox9 in testis determination. The studies presented here are the first experimental proof for an essential role for Sox9 in mediating a switch from the ovarian pathway to the testicular pathway. Using conditional gene targeting, we show that homozygous deletion of Sox9 in XY gonads interferes with sex cord development and the activation of the male-specific markers Mis and P450scc, and leads to the expression of the female-specific markers Bmp2 and follistatin. Moreover, using a tissue specific knock-out approach, we show that Sox9 is involved in Sertoli cell differentiation, the activation of Mis and Sox8, and the inactivation of Sry. Finally, double knock-out analyses suggest that Sox8 reinforces Sox9 function in testis differentiation of mice.

Activation of β-catenin signaling by Rspo1 controls differentiation of the mammalian ovary

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 beta-catenin signaling pathway. beta-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 beta-catenin activation determines the fate of the gonad, with Rspo1 acting as a crucial regulator of canonical beta-catenin signaling required for female development.

Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment

BACKGROUND The mammalian hair represents an unparalleled model system to understand both developmental processes and stem cell biology. The hair follicle consists of several concentric epithelial sheaths with the outer root sheath (ORS) forming the outermost layer. Functionally, the ORS has been implicated in the migration of hair stem cells from the stem cell niche toward the hair bulb. However, factors required for the differentiation of this critical cell lineage remain to be identified. Here, we describe an unexpected role of the HMG-box-containing gene Sox9 in hair development. RESULTS Sox9 expression can be first detected in the epithelial component of the hair placode but then becomes restricted to the outer root sheath (ORS) and the hair stem cell compartment (bulge). Using tissue-specific inactivation of Sox9, we demonstrate that this gene serves a crucial role in hair differentiation and that skin deleted for Sox9 lacks external hair. Strikingly, the ORS acquires epidermal characteristics with ectopic expression of GATA3. Moreover, Sox9 knock hair show severe proliferative defects and the stem cell niche never forms. Finally, we show that Sox9 expression depends on sonic hedgehog (Shh) signaling and demonstrate overexpression in skin tumors in mouse and man. CONCLUSIONS We conclude that although Sox9 is dispensable for hair induction, it directs differentiation of the ORS and is required for the formation of the hair stem cell compartment. Our genetic analysis places Sox9 in a molecular cascade downstream of sonic hedgehog and suggests that this gene is involved in basal cell carcinoma.

Renal abnormalities and their developmental origin

Congenital abnormalities of the kidney and urinary tract (CAKUT) occur in 1 out of 500 newborns, and constitute approximately 20–30% of all anomalies identified in the prenatal period. CAKUT has a major role in renal failure, and there is increasing evidence that certain abnormalities predispose to the development of hypertension and cardiovascular disease in adult life. Moreover, defects in nephron formation can predispose to Wilms tumour, the most frequent solid tumour in children. To understand the basis of human renal diseases, it is essential to consider how the kidney develops.

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source

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.

Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease

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.

Cross-talk in kidney development

As in most organs, the emerging theme in kidney development is the importance of cross-talk between several tissues and cell lineages to allow morphogenesis to proceed in a complex but highly regulated way. Over the past few years, knock-out and transgenic analyses in mice and evolutionary comparison with non-mammalian species have been particularly instrumental in identifying molecules with crucial functions for tissue-tissue interactions. The transcription factors Wt1 and Eya1, the signalling molecules Gdnf and LIF and the receptors c-Ret and GdnfRalpha have been demonstrated to fulfil fundamental roles in the first step of metanephric induction, the outgrowth of the ureter. Signalling by members of the Wnt, BMP and FGF families, regulated by transcription factors such as Pax2, mediates nephrogenesis by adjusting the balance between the ureteric bud epithelium, stromal and nephrogenic tissues. The stromal tissue, neglected for many years, has been shown to serve important functions in regulating the growth of nephrons. Finally, we have also begun to gain insight into the molecular events underlying patterning of the nephron into distinct functional units including glomerulus, proximal and distal tubule.

Oncogenicity of the Developmental Transcription Factor Sox9

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.