Claude Van Campenhout

Phenotypic annotation of the mouse X chromosome

Mutational screens are an effective means used in the functional annotation of a genome. We present a method for a mutational screen of the mouse X chromosome using gene trap technologies. This method has the potential to screen all of the genes on the X chromosome without establishing mutant animals, as all gene-trapped embryonic stem (ES) cell lines are hemizygous null for mutations on the X chromosome. Based on this method, embryonic morphological phenotypes and expression patterns for 58 genes were assessed, approximately 10% of all human and mouse syntenic genes on the X chromosome. Of these, 17 are novel embryonic lethal mutations and nine are mutant mouse models of genes associated with genetic disease in humans, including BCOR and PORCN. The rate of lethal mutations is similar to previous mutagenic screens of the autosomes. Interestingly, some genes associated with X-linked mental retardation (XLMR) in humans show lethal phenotypes in mice, suggesting that null mutations cannot be responsible for all cases of XLMR. The entire data set is available via the publicly accessible website (http://xlinkedgenes.ibme.utoronto.ca/).

The Postsynaptic Density 95/Disc-Large/Zona Occludens Protein Syntenin Directly Interacts with Frizzled 7 and Supports Noncanonical Wnt Signaling

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.

Dlg3 Trafficking and Apical Tight Junction Formation Is Regulated by Nedd4 and Nedd4-2 E3 Ubiquitin Ligases

Summary The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation.

The Notch-effector HRT1 gene plays a role in glomerular development and patterning of the Xenopus pronephros anlagen.

Notch signaling has been shown to play a role in cell fate decisions in the Xenopus pronephros anlagen. Here, we show that the Xenopus Hairy-related transcription factor (HRT) gene XHRT1, and the Hairy/Enhancer of split (HES) genes Xhairy1, Xhairy2b, esr9 and esr10, have distinct restricted dynamic expression patterns during pronephros development, and that their expression is regulated by Notch. XHRT1, which is the earliest and strongest gene expressed in the pronephric region, is initially transcribed predominantly in the forming glomus, where it is downregulated by antisense morpholino oligonucleotide inhibition of xWT1. Later, it is activated in the most dorsoanterior part of the pronephros anlagen that gives rise to the proximal tubules. In agreement with this dynamic expression profile, we found that early activation of Notch favors glomus, whereas only later activation promotes proximal tubule formation. We show that, among the bHLH-O factors tested, only XHRT1 efficiently inhibits distal tubule and duct formation, and that only its translational inhibition causes a reduction of the expression of proximal tubule and glomus markers. Using domain swap experiments, we found that the XHRT1 C-terminal region is crucial for its activity. Together, our results provide evidence that XHRT1 plays an important role in glomerular development and early proximodistal patterning that is distinct from those of the other pronephric bHLH repressors.

The Xenopus doublesex-related gene Dmrt5 is required for olfactory placode neurogenesis.

The Dmrt (doublesex and mab-3 related transcription factor) genes encode a large family of evolutionarily conserved transcription factors whose function in sex specific differentiation has been well studied in all animal lineages. In vertebrates, their function is not restricted to the developing gonads. For example, Xenopus Dmrt4 is essential for neurogenesis in the olfactory system. Here we have isolated and characterized Xenopus Dmrt5 and found that it is coexpressed with Dmrt4 in the developing olfactory placodes. As Dmrt4, Dmrt5 is positively regulated in the ectoderm by neural inducers and negatively by proneural factors. Both Dmrt5 and Dmrt4 genes are also activated by the combined action of the transcription factor Otx2, broadly transcribed in the head ectoderm and of Notch signaling, activated in the anterior neural ridge. As for Dmrt4, knockdown of Dmrt5 impairs neurogenesis in the embryonic olfactory system and in neuralized animal caps. Conversely, its overexpression promotes neuronal differentiation in animal caps, a property that requires the conserved C-terminal DMA and DMB domains. We also found that the sea anenome Dmrt4/5 related gene NvDmrtb also induces neurogenesis in Xenopus animal caps and that conversely, its knockdown in Nematostella reduces elav-1 positive neurons. Together, our data identify Dmrt5 as a novel important regulator of neurogenesis whose function overlaps with that of Dmrt4 during Xenopus olfactory system development. They also suggest that Dmrt may have had a role in neurogenesis in the last common ancestor of cnidarians and bilaterians.

Flattop regulates basal body docking and positioning in mono- and multiciliated cells

Planar cell polarity (PCP) regulates basal body (BB) docking and positioning during cilia formation, but the underlying mechanisms remain elusive. In this study, we investigate the uncharacterized gene Flattop (Fltp) that is transcriptionally activated during PCP acquisition in ciliated tissues. Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells. Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells. In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton. Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear. Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.03842.001

The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception

PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus

V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome-wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages. Summary: In vertebrates, V1 interneuron specification requires Prdm12, whose expression depends on Pax6 and retinoic acid and is restricted to the p1 domain by Dbx1 and Nkx6.1/2, themselves repressed by Prdm12.

Evolution of the Discs large gene family provides new insights into the establishment of apical epithelial polarity and the etiology of mental retardation.

Cell polarity is essential to the function of many cell types, such as epithelial cells and neurons. The Discs large (Dlg) scaffolding protein was identified in Drosophila as a major regulator of basolateral epithelial identity. Four Dlg orthologs (Dlg1 through 4) are found in vertebrates, and mutations in the human Dlg3 gene are associated with X-linked mental retardation. We recently found that Dlg3 controls apical epithelial polarity and tight junction formation and contributes to neural induction in mouse development.1 During evolution, Dlg3 acquired specific PPxY motifs, which bind to the WW domains of the E3 ubiquitin ligases, Nedd4 and Nedd4-2. This interaction results in monoubiquitination of Dlg3, leading to directed microtubule-dependent protein trafficking, via the exocyst complex, in different polarized cell types. Directed trafficking of Dlg3 plays an important role, during both mammalian development and in adulthood, in the establishment and maintenance of specialized apical cell junctions, such as tight junctions in epithelial cells and synapses in neurons.