Putzke, Aaron et al. (2013) International Worm Meeting "Regulation of Post-embryonic Seam Cell Proliferation and Identity by the Non-Receptor Tyrosine Kinase, FRK-1."

Moore, Kelsey, Genzink, Katherine, Putzke, Aaron, Watson, McLane, Mila, Danielle, Askonas, Caroline

[International Worm Meeting, 2013]

A hallmark of stem cell proliferation is asymmetric division resulting in one daughter maintaining the stem cell state and the other daughter differentiating into a non-stem cell identity. These divisions are crucial for building tissues and have become an important area of study for human disease. We have characterized a non-receptor tyrosine kinase, FRK-1, and hypothesize that it is required for maintenance of the asymmetric divisions in the stem cell-like seam cells of the hypodermis. Our data show 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 the asymmetry in the stem cell-like divisions of the seam cell lineage. frk-1(ok760) mutants show that the lateral hypodermal cells are consistent in size to the anterior daughter of a normal asymmetric seam cell division. Using scm::gfp and elt-5::gfp we observe a decrease in seam cell numbers, as well as an intermittent loss of alae. Furthermore, crossing frk-1(ok760) with transgenic lines containing non-seam hypodermal GFP markers, such as elt-3, dpy-7 and later markers such as col-19, show a proportion of lateral hypodermal cells appear to precociously differentiate when compared to wildtype. Interestingly, our data also show a role for FRK-1 in seam cell proliferation, as eliminating FRK-1 during the L3-L4 transition results in supernumerary seam cells that are dependent on asymmetric Wnt signaling. Finally, we have observed a significant change in the expression of heterochronic regulators in frk-1(ok760) homozygous larvae, including up-regulation of let-7 and lin-4 and down-regulation of lin-14, lin-28 and hbl-1, supporting a requirement for FRK-1 in preventing precocious differentiation. We are currently investigating whether there is a dependence on FRK-1 kinase activity and translocation to the nucleus during the stem cell-like divisions of seam cells. In conclusion, our data suggest a requirement for FRK-1 during larval development in maintaining the asymmetric divisions of seam cells, thereby preventing precocious differentiation prior to adulthood.

Calderon, Adriana et al. (2015) International Worm Meeting "Asymmetric Wnt Pathway Regulation of Post-embryonic Seam Cell Proliferation and Identity via the Non-receptor Tyrosine Kinase FRK-1."

Watson, McLane, Putzke, Aaron, Genzink, Katherine, Phillips, Bryan, Calderon, Adriana, Mila, Danielle, Baldwin, Austin

[International Worm Meeting, 2015]

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, we have also detected a general loss of alae formation. Additionally, we have observed a significant change in the expression of key heterochronic regulators in frk-1(ok760) mutants. However, 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. 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. Specifically, we observe aberrant POP-1 and WRM-1 localization that is dependent on the presence of FRK-1 and APR-1. 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. 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 signaling mechanism.