Dchs1-Fat4 regulation of osteogenic differentiation in mouse

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of Yap-Runx2 and Taz-Runx2 complexes is altered in Dchs1/Fat4 mutant osteoblasts. In conclusion, these data identify Dchs1-Fat4 as a signalling pathway in osteoblast differentiation, reveal its crucial role within the early Runx2 progenitors, and identify distinct requirements for Yap and Taz during osteoblast differentiation

The Role of the Atypical Cadherin Fat4 during Early Mouse Kidney Induction and Mesenchymal to Epithelial Transition

Mammalian kidney development is a tightly regulated process that requires proper signaling among three different cellular compartments: the ureteric bud (UB; epithelial), the condensing mesenchyme (CM) and the stroma. Improper signaling between these three cell lineages can result in renal hypoplasia and/or dysplasia. Fat4 is an atypical cadherin involved in planar cell polarity (PCP), growth and cell adhesion. It has been proposed that Fat4 might work with another atypical cadherin Dachsous1 (Dchs1) and the Golgi-localized kinase Four-jointed1 (Fjx1). My thesis examined Fat4 and novel genetic interactions with Fat4 during two stages of mouse kidney development: nephron progenitor renewal and early kidney induction. I determined that Fat4-/- single mutants have an expanded renal progenitor pool (i.e. larger CM aggregates) and found that this phenotype is independent of core PCP and Hippo signaling. Loss of Dchs1 also resulted in expansion of the CM. Tissue-specific deletions show that Fat4 acts non-autonomously in the renal stroma to regulate the nephron progenitor pool. Gene expression analyses demonstrated that BMP, Notch and FGF signaling are altered in Fat4 mutants. I also found that Fat4-/-;Fjx1-/- double mutants exhibit duplex kidneys. Since increased GDNF-RET signaling also leads to duplex kidneys, I reduced GDNF signaling by examining GDNF+/-;Fat4-/-;Fjx1-/- triple mutants. Importantly, these mice did not exhibit duplex kidneys, suggesting that Fat4 and Fjx1 suppress excessive levels of GDNF-RET signaling. Overall, my thesis supports a model whereby 1) FAT4 in the stroma binds to DCHS1 in the CM to restrict renal progenitor self-renewal and, 2) Fat4 and Fjx1 restrict GDNF-RET signaling during kidney induction and subsequent branching.Ph.D

Stromal Fat4 acts non-autonomously with Dchs1/2 to restrict the nephron progenitor pool

International audienceRegulation of the balance between progenitor self-renewal and differentiation is crucial to development. In the mammalian kidney, reciprocal signalling between three lineages (stromal, mesenchymal and ureteric) ensures correct nephron progenitor self-renewal and differentiation. Loss of either the atypical cadherin FAT4 or its ligand Dachsous 1 (DCHS1) results in expansion of the mesenchymal nephron progenitor pool, called the condensing mesenchyme (CM). This has been proposed to be due to misregulation of the Hippo kinase pathway transcriptional co-activator YAP. Here, we use tissue-specific deletions to prove that FAT4 acts non-autonomously in the renal stroma to control nephron progenitors. We show that loss of Yap from the CM in Fat4-null mice does not reduce the expanded CM, indicating that FAT4 regulates the CM independently of YAP. Analysis of Six2(-/-);Fat4(-/-) double mutants demonstrates that excess progenitors in Fat4 mutants are dependent on Six2, a crucial regulator of nephron progenitor self-renewal. Electron microscopy reveals that cell organisation is disrupted in Fat4 mutants. Gene expression analysis demonstrates that the expression of Notch and FGF pathway components are altered in Fat4 mutants. Finally, we show that Dchs1, and its paralogue Dchs2, function in a partially redundant fashion to regulate the number of nephron progenitors. Our data support a model in which FAT4 in the stroma binds to DCHS1/2 in the mouse CM to restrict progenitor self-renewal

Yap- and Cdc42-dependent nephrogenesis and morphogenesis during mouse kidney development.

Yap is a transcriptional co-activator that regulates cell proliferation and apoptosis downstream of the Hippo kinase pathway. We investigated Yap function during mouse kidney development using a conditional knockout strategy that specifically inactivated Yap within the nephrogenic lineage. We found that Yap is essential for nephron induction and morphogenesis, surprisingly, in a manner independent of regulation of cell proliferation and apoptosis. We used microarray analysis to identify a suite of novel Yap-dependent genes that function during nephron formation and have been implicated in morphogenesis. Previous in vitro studies have indicated that Yap can respond to mechanical stresses in cultured cells downstream of the small GTPases RhoA. We find that tissue-specific inactivation of the Rho GTPase Cdc42 causes a severe defect in nephrogenesis that strikingly phenocopies loss of Yap. Ablation of Cdc42 decreases nuclear localization of Yap, leading to a reduction of Yap-dependent gene expression. We propose that Yap responds to Cdc42-dependent signals in nephron progenitor cells to activate a genetic program required to shape the functioning nephron

<i>Yap</i> is required for kidney development.

<p>(A) Stages of nephrogenesis and their relationship to the UB (black) tips. Signals released from UB tips induce mesenchyme cells to condense around UB tips forming the CM (blue). Some of these CM cells aggregate forming the PA that converts into epithelial RV. The late RV fuses with UB tips and develops into comma (CSB) and S-shaped (SSB) body. (A′) Schematic diagram of the nephron components. (B) Confocal images for Yap, E-cadherin and DAPI staining in late RV at E14.5. Nuclear Yap is observed in the proximal segment of the RV (arrowheads), while Yap expression disappears in Six2:Cre expressing cells (D - arrows point to CM cells, arrowhead points to an early nephron). (C) Confocal images of p-Yap/E-cadherin/DAPI staining shows ubiquitous p-Yap expression. Individual channels images are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003380#pgen.1003380.s002" target="_blank">Figure S2</a>. (E) Immunohistochemistry using Yap/Taz antibody in RV and SSB shows a similar expression pattern observed with Yap antibody in previous panels (arrowheads). (F–F″) Confocal images for Yap/E-cadherin/DAPI staining in SSB at E14.5. Nuclear Yap is observed in proximal and distal segments of the SSB (arrowheads). (G,H) Macroscopic view of the urogenital system from wild-type and <i>Yap</i> mutant kidneys at P0. Note bilateral reduction in kidney size of mutant compared to control and empty bladder in mutant animals. (I,J) PAS staining of P0 kidneys from wild-type and <i>Yap^CM−/−</i> animals. Arrows point to the papilla. (K,L) Closer view of the cortical zone shows limited nephrogenesis in <i>Yap^CM−/−</i>. (M,N) Higher magnification shows abnormal glomeruli structure and tubules with barely discernable lumens (asterisk) in <i>Yap^CM−/−</i>. k: kidney; b: bladder; cd: collectiong duct; csb: comma-shaped body; d: distal; g: glomeruli; ic: inner cortex; ma: medulla; m: medial; nz: nephrogenic zone; p: proximal; pt: proximal tubule; ssb: S-shaped body. Scale bars represent 25 µm (B–F″; M–N), 1 mm (G–J), 200 µm (K,L).</p

Characterization of segmentation in <i>Yap</i> mutant nephrons.

<p>(A–B′) Double staining for E-cadherin and Calbindin in RV and SSB. Co-staining for Hnf1ß/WT1 (C–D′) and Sox9/WT1 (E–F′) reveals normal segmentation of the RV with both proximal and distal segments. Similarly, SSB show normal segmentation. Note the reduced size of the proximal domain in <i>Yap</i>-null SSB (compare WT1 positive segment in <i>Yap</i> mutants (D′, F′) to controls (C′, E′). This is also apparent in B′ and J′. (G–H′) Immunofluorescence for E-cadherin and Jag1 reveals no change in specification of the distal RV and the medial segment of the SSB in both genotypes. Note the aberrant morphology (asterisk) of the site where the connection occurred between the SSB and the UE (B′,D′,F′,H′ and J′). (I–J′) Immunofluorescence using antibodies to Cytokeratin (UE) and Laminin (BM) shows that fusion occurred before the comma-shaped stages. All staining performed at E15.5. CSB: comma-shaped body; RV: renal vesicle; SSB: S-shaped body. Scale bars represent 25 µm. DAPI was used to counterstain nuclei.</p