Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.     

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.     

A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis

Smith-Lemli-Opitz syndrome (SLOS), desmosterolosis and lathosterolosis are human syndromes caused by defects in the final stages of cholesterol biosynthesis. Many of the developmental malformations in these syndromes occur in tissues and structures whose embryonic patterning depends on signaling by the Hedgehog (Hh) family of secreted proteins. Here we report that response to the Hh signal is compromised in mutant cells from mouse models of SLOS and lathosterolosis and in normal cells pharmacologically depleted of sterols. We show that decreasing levels of cellular sterols correlate with diminishing responsiveness to the Hh signal. This diminished response occurs at sterol levels sufficient for normal autoprocessing of Hh protein, which requires cholesterol as cofactor and covalent adduct. We further find that sterol depletion affects the activity of Smoothened (Smo), an essential component of the Hh signal transduction apparatus.     

Deficiency of PORCN, a regulator of Wnt signaling, is associated with focal dermal hypoplasia

Focal dermal hypoplasia (FDH) is an X-linked dominant multisystem birth defect affecting tissues of ectodermal and mesodermal origin. Using a stepwise approach of (i) genetic mapping of FDH, (ii) high-resolution comparative genome hybridization to seek deletions in candidate chromosome areas and (iii) point mutation analysis in candidate genes, we identified PORCN, encoding a putative O-acyltransferase and potentially crucial for cellular export of Wnt signaling proteins, as the gene mutated in FDH. The findings implicate FDH as a developmental disorder caused by a deficiency in PORCN.     

Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling

The vertebrate planar cell polarity (PCP) pathway has previously been found to control polarized cell behaviors rather than cell fate. We report here that disruption of Xenopus laevis orthologs of the Drosophila melanogaster PCP effectors inturned (in) or fuzzy (fy) affected not only PCP-dependent convergent extension but also elicited embryonic phenotypes consistent with defective Hedgehog signaling. These defects in Hedgehog signaling resulted from a broad requirement for Inturned and Fuzzy in ciliogenesis. We show that these proteins govern apical actin assembly and thus control the orientation, but not assembly, of ciliary microtubules. Finally, accumulation of Dishevelled and Inturned near the basal apparatus of cilia suggests that these proteins function in a common pathway with core PCP components to regulate ciliogenesis. Together, these data highlight the interrelationships between cell polarity, cellular morphogenesis, signal transduction and cell fate specification.     

Dnmt3a is essential for hematopoietic stem cell differentiation

Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.     

The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia

Anonychia and hyponychia congenita (OMIM 206800) are rare autosomal recessive conditions in which the only presenting phenotype is the absence or severe hypoplasia of all fingernails and toenails. After determining linkage to chromosome 20p13, we identified homozygous or compound heterozygous mutations in the gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, in eight affected families. Rspo4 expression was specifically localized to developing mouse nail mesenchyme at embryonic day 15.5, suggesting a crucial role in nail morphogenesis.     

Conservation and diversification of Wnt signaling function during the evolution of nematode vulva development

Cell-fate specification and cell-cell signaling have been well studied during vulva development in Caenorhabditis elegans and provide a paradigm in evolutionary developmental biology. Pristionchus pacificus has been developed as a 'satellite' organism with an integrated physical and genetic map that allows detailed comparisons to C. elegans. A common aspect of vulva formation in both species is the polarization of the P7.p lineage, which is responsible for vulval symmetry. In C. elegans, Wnt signaling is crucial for P7.p cell-fate patterning; nothing is known about vulval symmetry in P. pacificus. We isolated mutations that disrupt polarization of the P7.p lineage in P. pacificus and found that the corresponding gene encodes a Frizzled-like molecule. In addition, mutations in Ppa-lin-17 (encoding Frizzled) and morpholino knock-down of Ppa-lin-44 (encoding Wnt), Ppa-egl-20 (encoding Wnt), Ppa-mig-5 (encoding Dsh), Ppa-apr-1 (encoding APC) and Ppa-bar-1 (encoding beta-catenin) results in gonad-independent vulva differentiation, indicating that these genes have a role in a negative signaling process. In contrast, in C. elegans, Wnt signaling has a positive role in vulva induction, and mutations in bar-1 result in a hypoinduced phenotype. Therefore, whereas the molecular mechanisms that generate vulval symmetry are conserved, the genetic control of vulva induction diversified during evolution.     

Mutations in X-linked PORCN, a putative regulator of Wnt signaling, cause focal dermal hypoplasia

Focal dermal hypoplasia is an X-linked dominant disorder characterized by patchy hypoplastic skin and digital, ocular and dental malformations. We used array comparative genomic hybridization to identify a 219-kb deletion in Xp11.23 in two affected females. We sequenced genes in this region and found heterozygous and mosaic mutations in PORCN in other affected females and males, respectively. PORCN encodes the human homolog of Drosophila melanogaster porcupine, an endoplasmic reticulum protein involved in secretion of Wnt proteins.     

p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation

Stem cell function is central for the maintenance of normal tissue homeostasis. Here we show that deletion of p38alpha mitogen-activated protein (MAP) kinase in adult mice results in increased proliferation and defective differentiation of lung stem and progenitor cells both in vivo and in vitro. We found that p38alpha positively regulates factors such as CCAAT/enhancer-binding protein that are required for lung cell differentiation. In addition, p38alpha controls self-renewal of the lung stem and progenitor cell population by inhibiting proliferation-inducing signals, most notably epidermal growth factor receptor. As a consequence, the inactivation of p38alpha leads to an immature and hyperproliferative lung epithelium that is highly sensitized to K-Ras(G12V)-induced tumorigenesis. Our results indicate that by coordinating proliferation and differentiation signals in lung stem and progenitor cells, p38alpha has a key role in the regulation of lung cell renewal and tumorigenesis.     

Regulation of primary cilia formation and left-right patterning in zebrafish by a noncanonical Wnt signaling mediator, duboraya

Primary cilia are microtubule-based organelles that project from the surface of nearly every animal cell. Although important functions of primary cilia in morphogenesis and tissue homeostasis have been identified, the mechanisms that control the formation of primary cilia are not understood. Here we characterize a zebrafish gene, termed duboraya (dub), that is essential for ciliogenesis. Knockdown of dub in zebrafish embryos results in both defects in primary cilia formation in Kupffer's vesicle and randomization of left-right organ asymmetries. We show that, at the molecular level, the function of dub in ciliogenesis is regulated by phosphorylation, which in turn depends on Frizzled-2-mediated noncanonical Wnt signaling. We also provide evidence that, at the cellular level, dub function is essential for actin organization in the cells lining Kupffer's vesicle. Taken together, our findings identify a molecular factor that links noncanonical Wnt signaling with the control of left-right axis specification, and provide an entry point for analyzing the mechanisms that regulate primary cilia formation.     

BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling

In humans, mutations in BMPR1A, SMAD4 and PTEN are responsible for juvenile polyposis syndrome, juvenile intestinal polyposis and Cowden disease, respectively. The development of polyposis is a common feature of these diseases, suggesting that there is an association between BMP and PTEN pathways. The mechanistic link between BMP and PTEN pathways and the related etiology of juvenile polyposis is unresolved. Here we show that conditional inactivation of Bmpr1a in mice disturbs homeostasis of intestinal epithelial regeneration with an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling human juvenile polyposis syndrome. We show that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell self-renewal. Mechanistically, PTEN, through phosphatidylinosital-3 kinase-Akt, mediates the convergence of the BMP and Wnt pathways on control of beta-catenin. Thus, BMP signaling may control the duplication of intestinal stem cells, thereby preventing crypt fission and the subsequent increase in crypt number.     

Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.     

Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways

Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.     

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.