Regulation of Seam Cell Function by the Non-Receptor Tyrosine Kinase, FRK-1, in C. elegans
Dr Aaron Putzke
We have characterized FRK-1, a homologue of the mammalian Fer non-receptor 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. Homozygous frk-1(ok760) larvae have an excess number of lateral hypodermal cells which appear to have lost asymmetry in the stem cell-like divisions of the seam cell lineage. frk-1(ok760) mutants immunostained with the epithelial adherens junction marker, MH27, show that the lateral hypodermal cells are abnormally shaped and smaller in size (during division), similar to the anterior daughter of a normal asymmetric seam cell division. Although we have observed an increase in seam cell nuclei using scm::GFP and elt-5::GFP (egl-18), we have also detected a general loss of alae formation. Crossing frk-1(ok760) with transgenic reporter lines containing non-seam hypodermal GFP markers, such as elt-3, and later markers such as col-19, show the lateral hypodermal cells do not precociously differentiate as adult-hyp7 cells. Interestingly, we have observed a significant change in the expression of key heterochronic regulators in frk-1(ok760) mutants, including up-regulation of let-7 and lin-4 and down-regulation of lin-14, lin-28, lin-41 and hbl-1. Finally, our data also show a clear role for FRK-1 in seam cell proliferation, as eliminating FRK-1 via RNAi during the L3-L4 transition results in supernumerary seam cell nuclei, that is dependent on asymmetric Wnt signaling. We are currently investigating the dependence on FRK-1 kinase activity and FRK-1 interactions in the nucleus during mitosis for the stem cell-like self-renewal exhibited by seam cells during post-embryonic development. Our data suggest a requirement for FRK-1 in maintaining the identity and proliferation of the asymmetric, stem-cell like state of the seam cells, thereby preventing precocious differentiation prior to adulthood.
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