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First published online 15 June 2005
doi: 10.1242/dev.01900

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

Neuroscience Research Institute and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA

View larger version (101K): [in a new window]   Fig. 1. Zygotic loss of FRK-1 blocks epidermal enclosure and elongation during embryogenesis. Differential interference contrast images of frk-1(RNAi) (A), mDf7 homozygous (D), and wild-type (G) embryos are shown at approximately 8.5 hours post-fertilization. frk-1(RNAi) and mDf7 embryos arrest without enclosing. The epidermal cells on the surface of the embryo round up and appear to be defective in cell adhesion. Epidermal cells do not migrate properly and become clustered together on the dorsal side of the embryo, as revealed with the nuclear marker LIN-26 (B,E,H). Apical junctions in the epidermis, revealed with antibody MH27, constrict and cluster dorsally (C,F,I). Embryos, as shown in this and subsequent figures, are 50 µm along the anteroposterior axis.

View larger version (92K): [in a new window]   Fig. 2. frk-1 rescues the morphogenetic and differentiation defects of mDf7 homozygous embryos. Embryos homozygous for mDf7 (A) do not express the seam-cell-specific marker recognized by antibody NE2-1B4 (B). Expression of FRK-1 from an extrachromosomal array restores enclosure and elongation (C) and expression of the seam-specific NE2-1B4 antigen (D) to mDf7 embryos. Mouse FerT similarly rescues mDf7 embryos (E,F). A wild-type embryo is shown at the three-fold stage (G) with the NE2-1B4 marker (H).

View larger version (44K): [in a new window]   Fig. 3. FRK-1 is required for late epidermal differentiation. (A) Percent of embryos with a wild-type (empty bars) and frk-1(RNAi) (filled bars) genotype expressing the indicated marker. Numbers of frk-1(RNAi) embryos scored are shown above each bar. More than 100 embryos were scored for all markers in wild-type embryos. Seam cell marker (SCM) is a transgenic reporter that expresses a GFP-ß-galactosidase fusion protein specifically in seam cells driven by an uncharacterized promoter (Terns et al., 1997). (B) Nomarski images of mutant and rescued embryos. Expression of the frk-1 coding region from a pan-epidermal promoter, elt-1p, restores enclosure and elongation to homozygous mDf7 embryos. Expression in non-seam epidermis only, using the elt-3 promoter, is not sufficient to allow enclosure or elongation. (C) Genetic model for the pathway of epidermal differentiation, showing position of FRK-1 action based on gene expression data.

View larger version (14K): [in a new window]   Fig. 4. Quantification of rescue experiments with homozygous mDf7 embryos. Percent embryonic lethality (empty bars) and enclosure defective (filled bars) in progeny from mDf7 heterozygotes is shown. All mDf7 homozygous embryos (25% of the progeny for this fully recessive deficiency) fail to enclose and die. Both phenotypes are efficiently rescued with cosmid T04B2 or a fragment containing frk-1. The kinase-dead FRK-1(D308R) mutant, Mouse FerT, and elt-1p-driven frk-1 all rescue enclosure effectively and embryonic lethality partially. Rescue of both phenotypes apparently requires expression in the lateral seam cells, as expression of frk-1 from the elt-3 promoter does not rescue mDf7. More than 300 embryos were scored for each independent array assayed in the rescue experiments.

View larger version (62K): [in a new window]   Fig. 5. FRK-1 kinase activity is not required for enclosure and elongation. (A) In vitro assays demonstrate the autophosphorylation activity of FRK-1, which is eliminated in the FRK-1(D308R) mutant. The enclosure and elongation defects of mDf7 homozygous embryos (B) are rescued by expression of the kinase-dead form of FRK-1 (C). The rescued embryos express the seam-cell-specific marker, NE2-1B4 (C), as is seen with rescue by wild-type FRK-1.

View larger version (111K): [in a new window]   Fig. 6. FRK-1 is expressed ubiquitously in all cells during embryogenesis, and becomes nuclear excluded just before enclosure. FRK-1 is localized to the nucleus and at regions of cell-cell contacts in early embryos, as evident in 4-cell (A) and 40-cell (B) embryos. Immediately before enclosure, FRK-1 becomes nuclear-excluded, as seen in an embryo with approximately 550 cells (C). The protein remains nuclear-excluded throughout elongation (e.g. 1.25-fold stage; D), and cell surface localization is especially pronounced in the seam cells of elongated embryos (e.g. 3-fold embryo, E; overlay with MH27 apical junction antigen in F). Arrows point to apical junctions surrounding seam cells. Although both antibodies result in the same staining pattern, the images in this figure show localization using the antibody elicited to the FRK-1b peptide (see Materials and methods).

View larger version (68K): [in a new window]   Fig. 7. FRK-1 mislocalizes to the nucleus in mutants defective in cell adhesion components. In wild-type embryos, FRK-1 localizes to the plasma membrane of all cells immediately before enclosure (A), and remains nuclear-excluded throughout elongation (e.g. 1.5-fold embryo; B). In a mutant lacking the HMP-2 ß-catenin, FRK-1 becomes mislocalized to the nucleus, as is particularly evident in the seam cells (D). FRK-1 is similarly mislocalized to nuclei in a mutant lacking the PAT-3 ß-integrin (F), albeit to a less pronounced extent. In both cases, FRK-1 is localized to the plasma membrane before enclosure (C,E), demonstrating that the shift in localization takes place after enclosure is completed. Arrowheads indicate seam cells (B) or seam cell nuclei (D,F). Co-immunoprecipitation demonstrates that HMP-2 and FRK-1 physically interact in vitro (G). The Western analysis shows a single band at 45 kD when probed with the anti-FRK-1 antibody and a band at approximately 75 kD (HMP-2) when probed with anti-FLAG.

View larger version (83K): [in a new window]   Fig. 8. Expression of FRK-1 causes loss of adhesion in cultured human cells. FRK-1 was transiently expressed from an ecdysone-inducible promoter in HEK293 cells. Cells expressing FRK-1 rounded up and detached from the laminin substrate (A), whereas expression of ß-galactosidase from the same promoter had virtually no effect on adhesion of the cells to the substrate (B). Insets show higher magnification phase contrast micrographs. (C) Quantification of cell adhesion defects resulting from FRK-1 expression. The number of cells remaining attached to substrate was quantified as described in Materials and methods and the values normalized for transformation frequencies (50%) and to the values determined from cells transformed with the empty pIND vector (defined as 100% attached, or 0% floating). Experiments were performed with either fibronectin (Fn) or poly-D-lysine as a substrate. Scale bars: 50 µm.

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