Annick Turbe-Doan

THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia

Characterization of previously described intraflagellar transport (IFT) mouse mutants has led to the proposition that normal primary cilia are required for mammalian cells to respond to the sonic hedgehog (SHH) signal. Here we describe an N-ethyl-N-nitrosourea–induced mutant mouse, alien (aln), which has abnormal primary cilia and shows overactivation of the SHH pathway. The aln locus encodes a novel protein, THM1 (tetratricopeptide repeat–containing hedgehog modulator-1), which localizes to cilia. aln-mutant cilia have bulb-like structures at their tips in which IFT proteins (such as IFT88) are sequestered, characteristic of Chlamydomonas reinhardtii and Caenorhabditis elegans retrograde IFT mutants. RNA-interference knockdown of Ttc21b (which we call Thm1 and which encodes THM1) in mouse inner medullary collecting duct cells expressing an IFT88–enhanced yellow fluorescent protein fusion recapitulated the aln-mutant cilial phenotype, and live imaging of these cells revealed impaired retrograde IFT. In contrast to previously described IFT mutants, Smoothened and full-length glioblastoma (GLI) proteins localize to aln-mutant cilia. We hypothesize that the aln retrograde IFT defect causes sequestration of IFT proteins in aln-mutant cilia and leads to the overactivated SHH signaling phenotype. Specifically, the aln mutation uncouples the roles of anterograde and retrograde transport in SHH signaling, suggesting that anterograde IFT is required for GLI activation and that retrograde IFT modulates this event.

SUMO1 Haploinsufficiency Leads to Cleft Lip and Palate

The posttranslational modification sumoylation can have multiple effects on its substrate proteins. We studied a patient with isolated cleft lip and palate and a balanced chromosomal translocation that disrupts the SUMO1 (small ubiquitin-related modifier) gene, resulting in haploinsufficiency. In mouse, we found that Sumo1 is expressed in the developing lip and palate and that a Sumo1 hypomorphic allele manifests an incompletely penetrant orofacial clefting phenotype. Products of several genes implicated in clefting are sumoylated, and the Sumo1 hypomorphic allele interacts genetically with a loss-of-function allele for one of these loci. Thus, sumoylation defines a network of genes important for palatogenesis.

Apc inhibition of Wnt signaling regulates supernumerary tooth formation during embryogenesis and throughout adulthood.

The ablation of Apc function or the constitutive activation ofβ -catenin in embryonic mouse oral epithelium results in supernumerary tooth formation, but the underlying mechanisms and whether adult tissues retain this potential are unknown. Here we show that supernumerary teeth can form from multiple regions of the jaw and that they are properly mineralized, vascularized, innervated and can start to form roots. Even adult dental tissues can form new teeth in response to either epithelial Apc loss-of-function or β-catenin activation, and the effect of Apc deficiency is mediated by β-catenin. The formation of supernumerary teeth via Apc loss-of-function is non-cell-autonomous. A small number of Apc-deficient cells is sufficient to induce surrounding wild-type epithelial and mesenchymal cells to participate in the formation of new teeth. Strikingly, Msx1, which is necessary for endogenous tooth development, is dispensable for supernumerary tooth formation. In addition, we identify Fgf8, a known tooth initiation marker, as a direct target of Wnt/β-catenin signaling. These studies identify key mechanistic features responsible for supernumerary tooth formation.

PRDM16 IS REQUIRED FOR NORMAL PALATOGENESIS IN MICE

Transcriptional cofactors are essential to the regulation of transforming growth factor beta (TGFbeta) superfamily signaling and play critical and widespread roles during embryonic development, including craniofacial development. We describe the cleft secondary palate 1 (csp1) N-ethyl-N-nitrosourea-induced mouse model of non-syndromic cleft palate (NSCP) that is caused by an intronic Prdm16 splicing mutation. Prdm16 encodes a transcriptional cofactor that regulates TGFbeta signaling, and its expression pattern is consistent with a role in palate and craniofacial development. The cleft palate (CP) appears to be the result of micrognathia and failed palate shelf elevation due to physical obstruction by the tongue, resembling human Pierre Robin sequence (PRS)-like cleft secondary palate. PRDM16 should be considered a candidate for mutation in human clefting disorders, especially NSCP and PRS-like CP.

A Wnt-Bmp Feedback Circuit Controls Intertissue Signaling Dynamics in Tooth Organogenesis

Computational and genetic analyses reveal a key circuit involving Wnt and Bmp in developing teeth in mice. A Toothsome Circuit Interactions between two different types of tissue, epithelium and mesenchyme, play important roles in development and cancer. Tooth development is a system for studying epithelial-mesenchymal interactions that is amenable to experimental manipulation. O’Connell et al. performed gene expression profiling of developing molar dental tissues in mice and computational analyses to construct a gene regulatory network that identified a key feedback circuit mediated by diffusible signaling molecules of the Wnt and Bmp families. This circuit controls the production of signaling molecules in other pathways and is self-sustaining. The authors validated their circuit in mice with mutations expected to disrupt the Wnt-Bmp signaling pathways. Similar feedback circuits may also operate in epithelial-mesenchymal interactions in other developing organs or in tumors. Many vertebrate organs form through the sequential and reciprocal exchange of signaling molecules between juxtaposed epithelial and mesenchymal tissues. We undertook a systems biology approach that combined the generation and analysis of large-scale spatiotemporal gene expression data with mouse genetic experiments to gain insight into the mechanisms that control epithelial-mesenchymal signaling interactions in the developing mouse molar tooth. We showed that the shift in instructive signaling potential from dental epithelium to dental mesenchyme was accompanied by temporally coordinated genome-wide changes in gene expression in both compartments. To identify the mechanism responsible, we developed a probabilistic technique that integrates regulatory evidence from gene expression data and from the literature to reconstruct a gene regulatory network for the epithelial and mesenchymal compartments in early tooth development. By integrating these epithelial and mesenchymal gene regulatory networks through the action of diffusible extracellular signaling molecules, we identified a key epithelial-mesenchymal intertissue Wnt-Bmp (bone morphogenetic protein) feedback circuit. We then validated this circuit in vivo with compound genetic mutations in mice that disrupted this circuit. Moreover, mathematical modeling demonstrated that the structure of the circuit accounted for the observed reciprocal signaling dynamics. Thus, we have identified a critical signaling circuit that controls the coordinated genome-wide expression changes and reciprocal signaling molecule dynamics that occur in interacting epithelial and mesenchymal compartments during organogenesis.

Ttc21b is required to restrict sonic hedgehog activity in the developing mouse forebrain.

Organizing centers in the developing brain provide an assortment of instructive patterning cues, including Sonic hedgehog (Shh). Here we characterize the forebrain phenotype caused by loss of Ttc21b, a gene we identified in an ENU mutagenesis screen as a novel ciliary gene required for retrograde intraflagellar transport. The Ttc21b mutant has defects in limb, eye and, most dramatically, brain development. We show that Shh signaling is elevated in the rostral portion of the mutant embryo, including in a domain in or near the zona limitans intrathalamica. We demonstrate here that ciliary defects seen in the Ttc21b mutant extend to the embryonic brain, adding forebrain development to the spectrum of tissues affected by defects in ciliary physiology. We show that development of the Ttc21b brain phenotype is modified by lowering levels of the Shh ligand, supporting our hypothesis that the abnormal patterning is a consequence of elevated Shh signaling. Finally, we evaluate Wnt signaling but do not find evidence that this plays a role in causing the perturbed neurodevelopmental phenotype we describe.

Amniocytes can serve a dual function as a source of iPS cells and feeder layers

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5–7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4+ or c-Kit+ amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

Downregulating Hedgehog Signaling Reduces Renal Cystogenic Potential of Mouse Models

Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 attenuated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target.

Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

We describe the rudolph mouse, a mutant with striking defects in both central nervous system and skeletal development. Rudolph is an allele of the cholesterol biosynthetic enzyme, hydroxysteroid (17-beta) dehydrogenase 7, which is an intriguing finding given the recent implication of oxysterols in mediating intracellular Hedgehog (Hh) signaling. We see an abnormal sterol profile and decreased Hh target gene induction in the rudolph mutant, both in vivo and in vitro. Reduced Hh signaling has been proposed to contribute to the phenotypes of congenital diseases of cholesterol metabolism. Recent in vitro and pharmacological data also indicate a requirement for intracellular cholesterol synthesis for proper regulation of Hh activity via Smoothened. The data presented here are the first in vivo genetic evidence supporting both of these hypotheses, revealing a role for embryonic cholesterol metabolism in both CNS development and normal Hh signaling.

Focusing forward genetics: a tripartite ENU screen for neurodevelopmental mutations in the mouse.

The control of growth, patterning, and differentiation of the mammalian forebrain has a large genetic component, and many human disease loci associated with cortical malformations have been identified. To further understand the genes involved in controlling neural development, we have performed a forward genetic screen in the mouse (Mus musculus) using ENU mutagenesis. We report the results from our ENU screen in which we biased our ascertainment toward mutations affecting neurodevelopment. Our screen had three components: a careful morphological and histological examination of forebrain structure, the inclusion of a retinoic acid response element-lacZ reporter transgene to highlight patterning of the brain, and the use of a genetically sensitizing locus, Lis1/Pafah1b1, to predispose animals to neurodevelopmental defects. We recovered and mapped eight monogenic mutations, seven of which affect neurodevelopment. We have evidence for a causal gene in four of the eight mutations. We describe in detail two of these: a mutation in the planar cell polarity gene scribbled homolog (Drosophila) (Scrib) and a mutation in caspase-3 (Casp3). We find that refining ENU mutagenesis in these ways is an efficient experimental approach and that investigation of the developing mammalian nervous system using forward genetic experiments is highly productive.