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.

Role and regulation of Yap in KrasG12D-induced lung cancer.

The Hippo pathway and its downstream transcriptional co-activator Yap influence lung cancer, but the nature of the Yap contribution has been unclear. Using a genetically engineered mouse lung cancer model, we show that Yap deletion completely blocks KrasG12D and p53 loss-driven adenocarcinoma initiation and progression, whereas heterozygosity for Yap partially suppresses lung cancer growth and progression. We also characterize Yap expression during tumor progression and find that nuclear Yap can be detected from the earliest stages of lung carcinogenesis, but at levels comparable to that in aveolar type II cells, which are a cell of origin for lung adenocarcinoma. At later stages of tumorigenesis, variations in Yap levels are detected, which correlate with differences in cell proliferation within tumors. Our observations imply that Yap is not directly activated by oncogenic Kras during lung tumorigenesis, but is nonetheless absolutely required for this tumorigenesis, and support Yap as a therapeutic target in lung adenocarcinoma.

Fat4-Dchs1 signalling controls cell proliferation in developing vertebrae.

The protocadherins Fat4 and Dchs1 act as a receptor-ligand pair to regulate many developmental processes in mice and humans, including development of the vertebrae. Based on conservation of function between Drosophila and mammals, Fat4-Dchs1 signalling has been proposed to regulate planar cell polarity (PCP) and activity of the Hippo effectors Yap and Taz, which regulate cell proliferation, survival and differentiation. There is strong evidence for Fat regulation of PCP in mammals but the link with the Hippo pathway is unclear. In Fat4(-/-) and Dchs1(-/-) mice, many vertebrae are split along the midline and fused across the anterior-posterior axis, suggesting that these defects might arise due to altered cell polarity and/or changes in cell proliferation/differentiation. We show that the somite and sclerotome are specified appropriately, the transcriptional network that drives early chondrogenesis is intact, and that cell polarity within the sclerotome is unperturbed. We find that the key defect in Fat4 and Dchs1 mutant mice is decreased proliferation in the early sclerotome. This results in fewer chondrogenic cells within the developing vertebral body, which fail to condense appropriately along the midline. Analysis of Fat4;Yap and Fat4;Taz double mutants, and expression of their transcriptional target Ctgf, indicates that Fat4-Dchs1 regulates vertebral development independently of Yap and Taz. Thus, we have identified a new pathway crucial for the development of the vertebrae and our data indicate that novel mechanisms of Fat4-Dchs1 signalling have evolved to control cell proliferation within the developing vertebrae.

Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis.

Skeletal shape varies widely across species as adaptation to specialized modes of feeding and locomotion, but how skeletal shape is established is unknown. An example of extreme diversity in the shape of a skeletal structure can be seen in the sternum, which varies considerably across species. Here we show that the Dchs1-Fat4 planar cell polarity pathway controls cell orientation in the early skeletal condensation to define the shape and relative dimensions of the mouse sternum. These changes fit a model of cell intercalation along differential Dchs1-Fat4 activity that drives a simultaneous narrowing, thickening and elongation of the sternum. Our results identify the regulation of cellular polarity within the early pre-chondrogenic mesenchyme, when skeletal shape is established, and provide the first demonstration that Fat4 and Dchs1 establish polarized cell behaviour intrinsically within the mesenchyme. Our data also reveal the first indication that cell intercalation processes occur during ventral body wall elongation and closure.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Fat4/Dchs1 signaling between stromal and cap mesenchyme cells influences nephrogenesis and ureteric bud branching.

Formation of the kidney requires reciprocal signaling among the ureteric tubules, cap mesenchyme and surrounding stromal mesenchyme to orchestrate complex morphogenetic events. The protocadherin Fat4 influences signaling from stromal to cap mesenchyme cells to regulate their differentiation into nephrons. Here, we characterize the role of a putative binding partner of Fat4, the protocadherin Dchs1. Mutation of Dchs1 in mice leads to increased numbers of cap mesenchyme cells, which are abnormally arranged around the ureteric bud tips, and impairment of nephron morphogenesis. Mutation of Dchs1 also reduces branching of the ureteric bud and impairs differentiation of ureteric bud tip cells into trunk cells. Genetically, Dchs1 is required specifically within cap mesenchyme cells. The similarity of Dchs1 phenotypes to stromal-less kidneys and to those of Fat4 mutants implicates Dchs1 in Fat4-dependent stroma-to-cap mesenchyme signaling. Antibody staining of genetic mosaics reveals that Dchs1 protein localization is polarized within cap mesenchyme cells, where it accumulates at the interface with stromal cells, implying that it interacts directly with a stromal protein. Our observations identify a role for Fat4 and Dchs1 in signaling between cell layers, implicate Dchs1 as a Fat4 receptor for stromal signaling that is essential for kidney development, and establish that vertebrate Dchs1 can be molecularly polarized in vivo.

Regulation of neuronal migration by Dchs1-Fat4 planar cell polarity.

Planar cell polarity (PCP) describes the polarization of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for postnatal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood [1]. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways [1-3]. Fz-PCP is conserved in vertebrates, but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 and DCHS1, key components of Fat-PCP signaling, cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration [4]. Here, we investigate the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor (FBM) neurons as a model. We find that Fat4 and Dchs1 are expressed in complementary gradients and are required for the collective tangential migration of FBM neurons and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM neurons and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM neuron migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM neuron polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. Our results also identify Fat-PCP as a novel neuronal guidance system and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes.

Characterization of a Dchs1 mutant mouse reveals requirements for Dchs1-Fat4 signaling during mammalian development.

The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs.

Drosophila lowfat, a novel modulator of Fat signaling.

The Fat-Hippo-Warts signaling network regulates both transcription and planar cell polarity. Despite its crucial importance to the normal control of growth and planar polarity, we have only a limited understanding of the mechanisms that regulate Fat. We report here the identification of a conserved cytoplasmic protein, Lowfat (Lft), as a modulator of Fat signaling. Drosophila Lft, and its human homologs LIX1 and LIX1-like, bind to the cytoplasmic domains of the Fat ligand Dachsous, the receptor protein Fat, and its human homolog FAT4. Lft protein can localize to the sub-apical membrane in disc cells, and this membrane localization is influenced by Fat and Dachsous. Lft expression is normally upregulated along the dorsoventral boundary of the developing wing, and is responsible for elevated levels of Fat protein there. Levels of Fat and Dachsous protein are reduced in lft mutant cells, and can be increased by overexpression of Lft. lft mutant animals exhibit a wing phenotype similar to that of animals with weak alleles of fat, and lft interacts genetically with both fat and dachsous. These studies identify Lft as a novel component of the Fat signaling pathway, and the Lft-mediated elevation of Fat levels as a mechanism for modulating Fat signaling.

Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila.

The dachs gene was first identified almost a century ago based on its requirements for appendage growth, but has been relatively little studied. Here, we describe the phenotypes of strong dachs mutations, report the cloning of the dachs gene, characterize the localization of Dachs protein, and investigate the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. Our results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development.

Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1.

The Arabidopsis GCN5, ADA2a and ADA2b proteins are homologs of components of several yeast and animal transcriptional coactivator complexes. Previous work has implicated these plant coactivator proteins in the stimulation of cold-regulated gene expression by the transcriptional activator protein CBF1. Surprisingly, protein interaction studies demonstrate that the DNA-binding domain of CBF1 (and of a related protein, TINY), rather than its transcriptional activation domain, can bind directly to the Arabidopsis ADA2 proteins. The ADA2a and ADA2b proteins can also bind directly to GCN5 through their N-terminal regions (comparable to a region previously defined in yeast Ada2) and through previously unmapped regions in the middle of the ADA2 proteins, which bind to the HAT domain of GCN5. The ADA2 proteins enhance the ability of GCN5 to acetylate histones in vitro and enable GCN5 to acetylate nucleosomal histones. Moreover, GCN5 can acetylate the ADA2 proteins at a motif unique to the plant homologs and absent from fungal and animal homologs. We speculate that this modification may represent a novel autoregulatory mechanism for the plant SAGA-like transcriptional coactivator complexes.