Looking for Ideas in All the Wrong Places

Abstract

Looking for M in all the Wrong Places Iian Zipkin and Cynthia Kenyon Department of Biochemistry University of California, San Francisco, CA 94143-0554 We are primarily interested in the genetic and molecular control of directed cell migration. Towards this end, we have been looking for mutants defective in the early embryonic long-range migration of the M mesoblast, which is born in the anterior of the embryo (near the future mid-pharynx) and migrates by the comma stage to just posterior to the gonad. Only one mutation to date has been published which affects the migration of M, that is unc-39(c~73, e257, rh72)V (Manser and Wood, 1990). To facilitate screening for additional M migration-defective mutants we decided to visualize M using a GFP (Green Fluorescent Protein, thanks to Marty Chalfie) reporter construct. This construct (muIS16; courtesy Craig Hunter) is driven by the mab-5 promoter and has been shown to be expressed in M until at least mid-Ll. It is hoped that screening for a fluorescent cell in the anterior of newly- hatched larvae will be a convenient way to isolate more M migration mutants. Preliminary screening (of only 600 F1 clones) has in fact identified one new mutant, which exhibits a very low penetrance M migration defect. As a test for the premise of this screen, we examined larvae of the genotype unc 39(ct73); mulS16 to see what a fluorescent M in the anterior would look like. Manser and Wood (1990) reported that M is in an abnormal location 61% of the time in ct73. By GFP visualization, we find that number to be slightly low; in our hands M is anteriorly misplaced in approximately 80% of all Lls seen. M can end up anywhere between the posterior pharynx and V3 when misplaced. In addition, we find that M exhibits an abnormal morphology in unc-39(ct73); muIS16. In wild-type, M is a fairly large cell with a regular, rectangular shape and smooth edges. In contrast, in unc-39(ct73); muIS16 M has a variable, irregular cell shape, with multiple, filopodia-like extensions often extending quite far both anteriorly and posteriorly. These extensions look almost axonal, and in general M appears "stretched out". See Figure 1 for a typical example. Sample pictures will also appear on the WWW site "http: To further investigate this phenomenon, we looked at several mutants by GFP and scored M's position and morphology. In the wild-type muIS16 background, M had a normal position and morphology in all but one of over 200 Lls scored. In unc- 39(ct73); muIS16, M had an abnormal morphology in over 90% of animals scored, whether it was in the correct position or not. M was also seen to have an abnormal morphology in 11/40 Lls of the genotype mab-5(e2088)III; muIS16 (mab-5 being the worm HOM gene related to Antennapedia), and in two of these cases M was anteriorly displaced. It is possible that there is some linked mutation in the muIS16 background which potentiates this morphology defect, however M is significantly affected in both mab-5 and unc-39. Finally, in the new mutant, M has a normal morphology even when anteriorly displaced, further demonstrating that these characteristics are genetically separable.