Debora Bogani

Asymmetric Localization of Vangl2 and Fz3 Indicate Novel Mechanisms for Planar Cell Polarity in Mammals

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell–cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.

SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia

Loss of cellular polarity is a hallmark of epithelial cancers, raising the possibility that regulators of polarity have a role in suppressing tumorigenesis. The Scribble complex is one of at least three interacting protein complexes that have a critical role in establishing and maintaining epithelial polarity. In human colorectal, breast, and endometrial cancers, expression of the Scribble complex member SCRIB is often mislocalized and deregulated. Here, we report that Scrib is indispensable for prostate homeostasis in mice. Scrib heterozygosity initiated prostate hyperplasia, while targeted biallelic Scrib loss predisposed mice to prostate intraepithelial neoplasia. Mechanistically, Scrib was shown to negatively regulate the MAPK cascade to suppress tumorigenesis. Further analysis revealed that prostate-specific loss of Scrib in mice combined with expression of an oncogenic Kras mutation promoted the progression of prostate cancer that recapitulated the human disease. The clinical significance of the work in mice was highlighted by our observation that SCRIB deregulation strongly correlated with poor survival in human prostate cancer. These data suggest that the polarity network could provide a new avenue for therapeutic intervention.

Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) reveals a requirement for MAPK signalling in mouse sex determination.

The boygirl (byg) mouse mutant reveals that MAP3K4-mediated signaling is necessary for normal SRY expression and testis specification in the developing mouse gonad.

The PCP genes Celsr1 and Vangl2 are required for normal lung branching morphogenesis

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1Crsh and Vangl2Lp mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.

Pkd1l1 establishes left-right asymmetry and physically interacts with Pkd2

In mammals, left-right (L-R) asymmetry is established by posteriorly oriented cilia driving a leftwards laminar flow in the embryonic node, thereby activating asymmetric gene expression. The two-cilia hypothesis argues that immotile cilia detect and respond to this flow through a Pkd2-mediated mechanism; a putative sensory partner protein has, however, remained unidentified. We have identified the Pkd1-related locus Pkd1l1 as a crucial component of L-R patterning in mouse. Systematic comparison of Pkd1l1 and Pkd2 point mutants reveals strong phenocopying, evidenced by both morphological and molecular markers of sidedness; both mutants fail to activate asymmetric gene expression at the node or in the lateral plate and exhibit right isomerism of the lungs. Node and cilia morphology were normal in mutants and cilia demonstrated typical motility, consistent with Pkd1l1 and Pkd2 activity downstream of nodal flow. Cell biological analysis reveals that Pkd1l1 and Pkd2 localise to the cilium and biochemical experiments demonstrate that they can physically interact. Together with co-expression in the node, these data argue that Pkd1l1 is the elusive Pkd2 binding partner required for L-R patterning and support the two-cilia hypothesis.

Gadd45γ and Map3k4 Interactions Regulate Mouse Testis Determination via p38 MAPK-Mediated Control of Sry Expression

Summary Loss of the kinase MAP3K4 causes mouse embryonic gonadal sex reversal due to reduced expression of the testis-determining gene, Sry. However, because of widespread expression of MAP3K4, the cellular basis of this misregulation was unclear. Here, we show that mice lacking Gadd45γ also exhibit XY gonadal sex reversal caused by disruption to Sry expression. Gadd45γ is expressed in a dynamic fashion in somatic cells of the developing gonads from 10.5 days postcoitum (dpc) to 12.5 dpc. Gadd45γ and Map3k4 genetically interact during sex determination, and transgenic overexpression of Map3k4 rescues gonadal defects in Gadd45γ-deficient embryos. Sex reversal in both mutants is associated with reduced phosphorylation of p38 MAPK and GATA4. In addition, embryos lacking both p38α and p38β also exhibit XY gonadal sex reversal. Taken together, our data suggest a requirement for GADD45γ in promoting MAP3K4-mediated activation of p38 MAPK signaling in embryonic gonadal somatic cells for testis determination in the mouse.

Scribble is required for normal epithelial cell-cell contacts and lumen morphogenesis in the mammalian lung

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.

Sfrp1 and Sfrp2 are required for normal male sexual development in mice

Secreted frizzled-related proteins (Sfrps) are antagonists of WNT signalling implicated in a variety of biological processes. However, there are no reports of a direct role for Sfrps in embryonic organogenesis in mammals. Using in vivo loss-of-function studies we report here for the first time a redundant role for Sfrp1 and Sfrp2 in embryonic sexual development of the mouse. At 16.5 dpc, male embryos lacking both genes exhibit multiple defects in gonad morphology, reproductive tract maturation and gonad positioning. Abnormal positioning of the testis appears to be due to failed gubernaculum development and an unusually close association between the cranial end of the reproductive tract and the kidney. The testes of double homozygotes are smaller than controls, contain fewer cords from the earliest stages, but still express Insl3, which encodes the hormone required for gubernacular masculinisation. Lgr8, which encodes the Insl3 receptor, is also expressed in the mutant gubernaculum, suggesting that Sfrp1/Sfrp2 signalling is not required for expression of the ligand or receptor that controls transabdominal testicular descent. Similarities between the abnormalities of embryonic sexual development in Sfrp1(-/-)Sfrp2(-/-) embryos with those exhibited by the Looptail and Wnt5a mutants suggest that disrupted non-canonical Wnt signalling may cause these defects.

Role of the Transcription Factor Sox4 in Insulin Secretion and Impaired Glucose Tolerance

OBJECTIVES— To identify, map, clone, and functionally validate a novel mouse model for impaired glucose tolerance and insulin secretion. RESEARCH DESIGN AND METHODS— Haploinsufficiency of the insulin receptor and associated mild insulin resistance has been used to sensitize an N-ethyl-N-nitrosourea (ENU) screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. The new impaired glucose tolerance 4 (IGT4) model was selected using an intraperitoneal glucose tolerance test and inheritance of the phenotype confirmed by generation of backcross progeny. Segregation of the phenotype was correlated with genotype information to map the location of the gene and candidates sequenced for mutations. The function of the SRY-related high mobility group (HMG)-box 4 (Sox4) gene in insulin secretion was tested using another ENU allele and by small interfering RNA silencing in insulinoma cells. RESULTS— We describe two allelic autosomal dominant mutations in the highly conserved HMG box of the transcription factor Sox4. Previously associated with pancreas development, Sox4 mutations in the adult mouse result in an insulin secretory defect, which exhibits impaired glucose tolerance in association with insulin receptor+/−–induced insulin resistance. Elimination of the Sox4 transcript in INS1 and Min6 cells resulted in the abolition of glucose-stimulated insulin release similar to that observed for silencing of the key metabolic enzyme glucokinase. Intracellular calcium measurements in treated cells indicate that this defect lies downstream of the ATP-sensitive K+ channel (KATP channel) and calcium influx. CONCLUSIONS— IGT4 represents a novel digenic model of insulin resistance coupled with an insulin secretory defect. The Sox4 gene has a role in insulin secretion in the adult β-cell downstream of the KATP channel.

Stringent requirement of a proper level of canonical WNT signalling activity for head formation in mouse embryo

In mouse embryos, loss of Dickkopf-1 (DKK1) activity is associated with an ectopic activation of WNT signalling responses in the precursors of the craniofacial structures and leads to a complete truncation of the head at early organogenesis. Here, we show that ENU-induced mutations of genes coding for two WNT canonical pathway factors, the co-receptor LRP6 and the transcriptional co-activator β-catenin, also elicit an ectopic signalling response and result in loss of the rostral tissues of the forebrain. Compound mutant embryos harbouring combinations of mutant alleles of Lrp6, Ctnnb1 and Dkk1 recapitulate the partial to complete head truncation phenotype of individual homozygous mutants. The demonstration of a synergistic interaction of Dkk1, Lrp6 and Ctnnb1 provides compelling evidence supporting the concepts that (1) stringent regulation of the level of canonical WNT signalling is necessary for head formation, (2) activity of the canonical pathway is sufficient to account for the phenotypic effects of mutations in three different components of the signal cascade and (3) rostral parts of the brain and the head are differentially more sensitive to canonical WNT signalling and their development is contingent on negative modulation of WNT signalling activity.