Co-operative roles for E-cadherin and N-cadherin during lens vesicle separation and lens epithelial cell survival

The classical cadherins are known to have both adhesive and signaling functions. It has also been proposed that localized regulation of cadherin activity may be important in cell assortment during development. In the context of eye development, it has been suggested that cadherins are important for separation of the invaginated lens vesicle from the surface ectoderm. To test this hypothesis, we conditionally deleted N-cadherin or E-cadherin from the presumptive lens ectoderm of the mouse. Conditional deletion of either cadherin alone did not produce a lens vesicle separation defect. However, these conditional mutants did exhibit common structural deficits, including microphthalmia, severe iris hyperplasia, persistent vacuolization within the fibre cell region, and eventual lens epithelial cell deterioration. To assess the co-operative roles of E-cadherin and N-cadherin within the developing lens, double conditional knockout embryos were generated. These mice displayed distinct defects in lens vesicle separation and persistent expression of another classical cadherin, P-cadherin, within the cells of the persistent lens stalk. Double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival. Finally, the severity of the lens phenotype was shown to be sensitive to the number of wild-type E- and N-cadherin alleles. These data suggest that the co-operative expression of both E- and N-cadherin during lens development is essential for normal cell sorting and subsequent lens vesicle separation.     

Cadherins in development and cancer

Proper embryonic development is guaranteed under conditions of regulated cell-cell and cell-matrix adhesion. The cells of an embryo have to be able to distinguish their neighbours as being alike or different. Cadherins, single-pass transmembrane, Ca(2+)-dependent adhesion molecules that mainly interact in a homophilic manner, are major contributors to cell-cell adhesion. Cadherins play pivotal roles in important morphogenetic and differentiation processes during development, and in maintaining tissue integrity and homeostasis. Changes in cadherin expression throughout development enable differentiation and the formation of various organs. In addition to these functions, cadherins have strong implications in tumourigenesis, since frequently tumour cells show deregulated cadherin expression and inappropriate switching among family members. In this review, I focus on E- and N-cadherin, giving an overview of their structure, cellular function, importance during development, role in cancer, and of the complexity of Ecadherin gene regulation.     

SEC14 and Spectrin Domains 1 (Sestd1) and Dapper Antagonist of Catenin 1 (Dact1) Scaffold Proteins Cooperatively Regulate the Van Gogh-like 2 (Vangl2) Four-pass Transmembrane Protein and Planar Cell Polarity (PCP) Pathway during Embryonic Development in Mice

The planar cell polarity (PCP) pathway is a conserved non-canonical (β-catenin-independent) branch of Wnt signaling crucial to embryogenesis, during which it regulates cell polarity and polarized cell movements. Disruption of PCP components in mice, including Vangl2 and Dact1, results in defective neural tube closure and other developmental defects. Here, we show that Sestd1 is a novel binding partner of Vangl2 and Dact1. The Sestd1-Dact1 interface is formed by circumscribed regions of Sestd1 (the carboxyl-terminal region) and Dact1 (the amino-terminal region). Remarkably, we show that loss of Sestd1 precisely phenocopies loss of Dact1 during embryogenesis in mice, leading to a spectrum of birth malformations, including neural tube defects, a shortened and/or curly tail, no genital tubercle, blind-ended colons, hydronephrotic kidneys, and no bladder. Moreover, as with Dact1, a knock-out mutation at the Sestd1 locus exhibits reciprocal genetic rescue interactions during development with a semidominant mutation at the Vangl2 locus. Consistent with this, examination of Wnt pathway activities in Sestd1 mutant mouse embryonic tissue reveals disrupted PCP pathway biochemistry similar to that characterized in Dact1 mutant embryos. The Sestd1 protein is a divergent member of the Trio family of GTPase regulatory proteins that lacks a guanine nucleotide exchange factor domain. Nonetheless, in cell-based assays the Sestd1-Dact1 interaction can induce Rho GTPase activation. Together, our data indicate that Sestd1 cooperates with Dact1 in Vangl2 regulation and in the PCP pathway during mammalian embryonic development.     

Deletion of Gremlin1 increases cell proliferation and migration responses in mouse embryonic fibroblasts

Gremlin1 (Grem1) is an antagonist of bone morphogenetic proteins (BMPs) that plays a critical role in embryonic and postnatal development. Grem1 has been implicated as both a promoter and an inhibitor of cell proliferation driven by BMP-4 and other mitogens in a diverse range of cell types. Recent data showed that Grem1 can trigger angiogenesis via vascular endothelial growth factor receptor (VEGFR2) binding, highlighting that the precise modalities of Grem1 signalling require further elucidation.In an attempt to enhance our understanding of the role of Grem1 in cell proliferation, mouse embryonic fibroblasts lacking grem1 (grem1−/−) were generated. Grem1−/− cells showed elevated levels of proliferation in vitro compared to wild-type and grem1+/−, with accelerated scratch wound repair but no obvious changes in cell cycle profile. Modest increases in BMP-4-stimulated Smad1/5/8 phosphorylation were detected in grem1−/− cells, with concomitant modest changes in Smad-dependent gene expression. Surprisingly, levels of ERK phosphorylation were reduced in grem1−/− cells compared to wild-type.These data suggest Grem1 is an inhibitor of embryonic fibroblast proliferation in vitro. Furthermore, the signalling pathways causing increased cell proliferation in the absence of Grem1 may involve other pathways distinct from canonical Smad and non-canonical ERK signalling.     

Integrating cellular dimensions with cell differentiation during early development

Early embryo development is characterized by alteration of cellular dimensions and fating of blastomeres. An emerging concept is that cell size and shape drive cellular differentiation during early embryogenesis in a variety of model organisms. In this review, we summarize recent advances that elucidate the contribution of the physical dimensions of a cell to major embryonic transitions and cell fate specification in vivo. We also highlight techniques and newly evolving methods for manipulating the sizes and shapes of cells and whole embryos in situ and ex vivo. Finally, we provide an outlook for addressing fundamental questions in the field and more broadly uncovering how changes to cell size control decision making in a variety of biological contexts.     

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.     

Characterization of the Skeletal Fusion with Sterility (sks) Mouse Showing Axial Skeleton Abnormalities Caused by Defects of Embryonic Skeletal Development

The development of the axial skeleton is a complex process, consisting of segmentation and differentiation of somites and ossification of the vertebrae. The autosomal recessive skeletal fusion with sterility (sks) mutation of the mouse causes skeletal malformations due to fusion of the vertebrae and ribs, but the underlying defects of vertebral formation during embryonic development have not yet been elucidated. For the present study, we examined the skeletal phenotypes of sks/sks mice during embryonic development and the chromosomal localization of the sks locus. Multiple defects of the axial skeleton, including fusion of vertebrae and fusion and bifurcation of ribs, were observed in adult and neonatal sks/sks mice. In addition, we also found polydactyly and delayed skull ossification in the sks/sks mice. Morphological defects, including disorganized vertebral arches and fusions and bifurcations of the axial skeletal elements, were observed during embryonic development at embryonic day 12.5 (E12.5) and E14.5. However, no morphological abnormality was observed at E11.5, indicating that defects of the axial skeleton are caused by malformation of the cartilaginous vertebra and ribs at an early developmental stage after formation and segmentation of the somites. By linkage analysis, the sks locus was mapped to an 8-Mb region of chromosome 4 between D4Mit331 and D4Mit199. Since no gene has already been identified as a cause of malformation of the vertebra and ribs in this region, the gene responsible for sks is suggested to be a novel gene essential for the cartilaginous vertebra and ribs.     

Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity

Signalling mediated by Notch receptors is known to have multiple functions during vertebrate neural development, regulating processes like progenitor differentiation and cell type diversification. Various Notch ligands are expressed in the developing nervous system and their activities might contribute to this multiplicity of functions. Here, we show that two Delta-like genes, Dll1 and Dll4, are sequentially expressed in differentiating neurons of the embryonic mouse retina and spinal cord's pV2 domain, with Dll1 starting to be expressed before Dll4. Analysis of Dll1 mutants reveals this gene is necessary and sufficient to maintain a pool of progenitors in the embryonic neuroepithelium. Accordingly, in the spinal cord domains where Dll1 is the only expressed Notch ligand, its inactivation leads to an increased rate of neurogenesis and premature differentiation of neural progenitors. In contrast, in the pV2 domain and retina where Dll1 is co-expressed with Dll4, progenitors are not exhausted and cell diversity is maintained. Together, our results support a model where Dll1 and Dll4 are part of a unique genetic circuitry that regulates subsequent steps of neurogenesis in the retina and pV2 domain: while Dll1 serves to prevent the untimely differentiation of neural progenitors, Dll4 might function to generate diversity within the population of differentiating neurons.     

p600 Plays Essential Roles in Fetal Development

p600 is a multifunctional protein implicated in cytoskeletal organization, integrin-mediated survival signaling, calcium-calmodulin signaling and the N-end rule pathway of ubiquitin-proteasome-mediated proteolysis. While push, the Drosophila counterpart of p600, is dispensable for development up to adult stage, the role of p600 has not been studied during mouse development. Here we generated p600 knockout mice to investigate the in vivo functions of p600. Interestingly, we found that homozygous deletion of p600 results in lethality between embryonic days 11.5 and 13.5 with severe defects in both embryo and placenta. Since p600 is required for placental development, we performed conditional disruption of p600, which deletes selectively p600 in the embryo but not in the placenta. The conditional mutant embryos survive longer than knockout embryos but ultimately die before embryonic day 14.5. The mutant embryos display severe cardiac problems characterized by ventricular septal defects and thin ventricular walls. These anomalies are associated with reduced activation of FAK and decreased expression of MEF2, which is regulated by FAK and plays a crucial role in cardiac development. Moreover, we observed pleiotropic defects in the liver and brain. In sum, our study sheds light on the essential roles of p600 in fetal development.     

NgBR is essential for endothelial cell glycosylation and vascular development

NgBR is a transmembrane protein identified as a Nogo‐B‐interacting protein and recently has been shown to be a subunit required for cis‐prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial‐specific NgBR knockout embryos. Here, we show that endothelial‐specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE‐cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo‐B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.     

Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s,Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis

scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.     

Hedgehog and Dpp signaling induce cadherin Cad86C expression in the morphogenetic furrow during Drosophila eye development

During Drosophila eye development, cell differentiation is preceded by the formation of a morphogenetic furrow, which progresses across the epithelium from posterior to anterior. Cells within the morphogenetic furrow are apically constricted and shortened along their apical-basal axis. However, how these cell shape changes and, thus, the progression of the morphogenetic furrow are controlled is not well understood. Here we show that cells simultaneously lacking Hedgehog and Dpp signal transduction fail to shorten and do not enter the morphogenetic furrow. Moreover, we have identified a gene, cadherin Cad86C, which is highly expressed in cells of the leading flank of the morphogenetic furrow. Ectopic activation of either the Hedgehog or Dpp signal transduction pathway results in elevated Cad86C expression. Conversely, simultaneous loss of both Hedgehog and Dpp signal transduction leads to decreased Cad86C expression. Finally, ectopic expression of Cad86C in either eye-antennal imaginal discs or wing imaginal discs results in apical constriction and shortening of cells. We conclude that Hedgehog and Dpp signaling promote the shortening of cells within the morphogenetic furrow. Induction of Cad86C expression might be one mechanism through which Hedgehog and Dpp promote these cell shape changes.     

Fat-Dachsous Signaling Coordinates Cartilage Differentiation and Polarity during Craniofacial Development

Organogenesis requires coordinated regulation of cellular differentiation and morphogenesis. Cartilage cells in the vertebrate skeleton form polarized stacks, which drive the elongation and shaping of skeletal primordia. Here we show that an atypical cadherin, Fat3, and its partner Dachsous-2 (Dchs2), control polarized cell-cell intercalation of cartilage precursors during craniofacial development. In zebrafish embryos deficient in Fat3 or Dchs2, chondrocytes fail to stack and misregulate expression of sox9a. Similar morphogenetic defects occur in rerea/atr2a ^−/− mutants, and Fat3 binds REREa, consistent with a model in which Fat3, Dchs2 and REREa interact to control polarized cell-cell intercalation and simultaneously control differentiation through Sox9. Chimaeric analyses support such a model, and reveal long-range influences of all three factors, consistent with the activation of a secondary signal that regulates polarized cell-cell intercalation. This coordinates the spatial and temporal morphogenesis of chondrocytes to shape skeletal primordia and defects in these processes underlie human skeletal malformations. Similar links between cell polarity and differentiation mechanisms are also likely to control organ formation in other contexts.     

The Zebrafish Homologue of the Human DYT1 Dystonia Gene Is Widely Expressed in CNS Neurons but Non-Essential for Early Motor System Development

DYT1 dystonia is caused by mutation of the TOR1A gene, resulting in the loss of a single glutamic acid residue near the carboxyl terminal of TorsinA. The neuronal functions perturbed by TorsinA[ΔE] are a major unresolved issue in understanding the pathophysiology of dystonia, presenting a critical roadblock to developing effective treatments. We identified and characterized the zebrafish homologue of TOR1A, as a first step towards elucidating the functions of TorsinA in neurons, in vivo, using the genetically-manipulable zebrafish model. The zebrafish genome was found to contain a single alternatively-spliced tor1 gene, derived from a common ancestral locus shared with the dual TOR1A and TOR1B paralogues found in tertrapods. tor1 was expressed ubiquitously during early embryonic development and in multiple adult tissues, including the CNS. The 2.1 kb tor1 mRNA encodes Torsin1, which is 59% identical and 78% homologous to human TorsinA. Torsin1 was expressed as major 45 kDa and minor 47 kDa glycoproteins, within the cytoplasm of neurons and neuropil throughout the CNS. Similar to previous findings relating to human TorsinA, mutations of the ATP hydrolysis domain of Torsin1 resulted in relocalization of the protein in cultured cells from the endoplasmic reticulum to the nuclear envelope. Zebrafish embryos lacking tor1 during early development did not show impaired viability, overt morphological abnormalities, alterations in motor behavior, or developmental defects in the dopaminergic system. Torsin1 is thus non-essential for early development of the motor system, suggesting that important CNS functions may occur later in development, consistent with the critical time window in late childhood when dystonia symptoms usually emerge in DYT1 patients. The similarities between Torsin1 and human TorsinA in domain organization, expression pattern, and cellular localization suggest that the zebrafish will provide a useful model to understand the neuronal functions of Torsins in vivo.     

Maintenance of mating cell integrity requires the adhesin Fig2p

Fungal adhesins represent a large family of serine/threonine-rich secreted glycoproteins. Adhesins have been shown to play roles in heterotypic and homotypic cell-cell adhesion processes, morphogenetic pathways and invasive/pseudohyphal growth, frequently in response to differentiation cues. Here we address the role of the Saccharomyces cerevisiae mating-specific adhesin Fig2p. Cells lacking FIG2 possess a variety of mating defects that relate to processes involving the cell wall, including morphogenetic defects, cell fusion defects, and alterations in agglutination activities. We found that mating-specific morphogenetic defects caused by the absence of FIG2 are suppressible by increased external osmolarity and that, during mating, fig2Δ cells display reduced viability relative to wild-type cells. These defects result from alterations in signaling activated by the mating and cell integrity pathways. Finally, we show that fig2Δ zygotes also have defects in zygotic spindle positioning that are osmoremedial, whereas the requirements for FIG2 in normal cell-cell agglutination and cell fusion during mating are insensitive to changes in the extracellular osmotic environment. We conclude that FIG2 performs distinct functions in the mating cell wall that are separable with respect to their ability to be suppressed by changes in external osmolarity and that a fundamental role of FIG2 in mating cells is the maintenance of cell integrity.