Dual Roles of Fer Kinase Are Required for Proper Hematopoiesis and Vascular Endothelium Organization during Zebrafish Development.

Fer kinase, a protein involved in the regulation of cell-cell adhesion and proliferation, has been shown to be required during invertebrate development and has been implicated in leukemia, gastric cancer, and liver cancer. However, in vivo roles for Fer during vertebrate development have remained elusive. In this study, we bridge the gap between the invertebrate and vertebrate realms by showing that Fer kinase is required during zebrafish embryogenesis for normal hematopoiesis and vascular organization with distinct kinase dependent and independent functions. In situ hybridization, quantitative PCR and fluorescence activated cell sorting (FACS) analyses revealed an increase in both erythrocyte numbers and gene expression patterns as well as a decrease in the organization of vasculature endothelial cells. Furthermore, rescue experiments have shown that the regulation of hematopoietic proliferation is dependent on Fer kinase activity, while vascular organizing events only require Fer in a kinase-independent manner. Our data suggest a model in which separate kinase dependent and independent functions of Fer act in conjunction with Notch activity in a divergent manner for hematopoietic determination and vascular tissue organization.

Asymmetric Wnt Pathway Signaling Facilitates Stem Cell-Like Divisions via the Nonreceptor Tyrosine Kinase FRK-1 in Caenorhabditis elegans.

Asymmetric cell division is critical during development, as it influences processes such as cell fate specification and cell migration. We have characterized FRK-1, a homolog of the mammalian Fer nonreceptor tyrosine kinase, and found it to be required for differentiation and maintenance of epithelial cell types, including the stem cell-like seam cells of the hypodermis. A genomic knockout of frk-1, allele ok760, results in severely uncoordinated larvae that arrest at the L1 stage and have an excess number of lateral hypodermal cells that appear to have lost asymmetry in the stem cell-like divisions of the seam cell lineage. frk-1(ok760) mutants show that there are excess lateral hypodermal cells that are abnormally shaped and smaller in size compared to wild type, a defect that could be rescued only in a manner dependent on the kinase activity of FRK-1. Additionally, we observed a significant change in the expression of heterochronic regulators in frk-1(ok760) mutants. However, frk-1(ok760) mutants do not express late, nonseam hypodermal GFP markers, suggesting the seam cells do not precociously differentiate as adult-hyp7 cells. Finally, our data also demonstrate a clear role for FRK-1 in seam cell proliferation, as eliminating FRK-1 during the L3-L4 transition results in supernumerary seam cell nuclei that are dependent on asymmetric Wnt signaling. Specifically, we observe aberrant POP-1 and WRM-1 localization that is dependent on the presence of FRK-1 and APR-1. Overall, our data suggest a requirement for FRK-1 in maintaining the identity and proliferation of seam cells primarily through an interaction with the asymmetric Wnt pathway.

Metastatic progression of prostate cancer and e-cadherin regulation by zeb1 and SRC family kinases.

Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.

Repression of Wnt signaling by a Fer-type nonreceptor tyrosine kinase.

The Wnt signaling pathway must be properly modulated to ensure an appropriate output: pathological conditions result from either insufficient or excessive levels of Wnt signal. For example, hyperactivation of the Wnt pathway is associated with various cancers and subnormal Wnt signaling can lead to increased invasiveness of tumor cells. We found that the Caenorhabditis elegans ortholog of the Fer nonreceptor tyrosine kinase, FRK-1, limits Wnt signaling by preventing the adhesion complex-associated ß-catenin, HMP-2, from participating in Wnt-dependent specification of the endoderm during embryogenesis. Removal of FRK-1 function results in relocalization of HMP-2 to the nucleus of epidermal cells, and allows it to substitute for WRM-1, the nuclear ß-catenin that normally transduces the Wnt signal during endoderm development. APR-1, the C. elegans APC ortholog, is similarly required to prevent HMP-2 relocalization and keeps it from participating in Wnt signal transduction; this finding partially explains the paradoxical observation that APR-1 acts either negatively or positively in Wnt signaling, depending on context. The apparent hyperactivation of the Wnt response in the absence of FRK-1 leads to hyperproliferation in the endoderm, as is also seen when WRM-1 is overexpressed in wild-type embryos. The specification and proliferation activities of Wnt signaling are separable: although the Tcf/Lef factor POP-1 acts in Wnt-dependent endoderm specification, it is not apparently required for hyperproliferation resulting from excessive Wnt signaling. These findings highlight a role for a Fer-type kinase in setting the proper levels of Wnt signaling and demonstrate the importance of this modulation in ensuring appropriate cell division.

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development.

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.

Essential kinase-independent role of a Fer-like non-receptor tyrosine kinase in Caenorhabditis elegans morphogenesis.

Morphogenesis requires coordination of cell surface activity and cytoskeletal architecture. During the initial stage of morphogenesis in Caenorhabditis elegans, the concerted movement of surface epithelial cells results in enclosure of the embryo by the epidermis. We report that Fer-related kinase-1 (FRK-1), an ortholog of the mammalian non-receptor tyrosine kinase Fer, is necessary for embryonic enclosure and morphogenesis in C. elegans. Expression of FRK-1 in epidermal cells is sufficient to rescue a chromosomal deficiency that removes the frk-1 locus, demonstrating its autonomous requirement in the epidermis. The essential function of FRK-1 is independent of its kinase domain, suggesting a non-enzymatic role in morphogenesis. Localization of FRK-1 to the plasma membrane requires beta-catenin, but not cadherin or alpha-catenin, and muscle-expressed beta-integrin is non-autonomously required for this localization; in the absence of these components FRK-1 becomes nuclear. Mouse FerT rescues the morphogenetic defects of frk-1 mutants and expression of FRK-1 in mammalian cells results in loss of adhesion, implying a conserved function for FRK-1/FerT in cell adhesion and morphogenesis. Thus, FRK-1 performs a kinase-independent function in differentiation and morphogenesis of the C. elegans epidermis during embryogenesis.

Gastrulation: PARtaking of the bottle.

Gastrulation in C. elegans embryos involves ingression of individual cells, but is driven by apical constriction of the kind that promotes migration of epithelial cell sheets. Recent work shows that PAR proteins, known for their role in polarization and unequal cell division, are also associated with the polarization that establishes this apical constriction.