Genes Necessary for C. Elegans Cell and Growth Cone Migrations

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Genes Necessary for C. Elegans Cell and Growth Cone Migrations Development 124, 1831-1843 (1997) 1831 Printed in Great Britain © The Company of Biologists Limited 1997 DEV9531 Genes necessary for C. elegans cell and growth cone migrations Wayne C. Forrester* and Gian Garriga Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA *Author for correspondence (e-mail: [email protected]) SUMMARY The migrations of cells and growth cones contribute to cell interactions between the CAN and HSN, a neuron that form and pattern during metazoan development. To study migrates anteriorly to a position adjacent to the CAN, the mechanisms that regulate cell motility, we have control the final destination of the HSN cell body. Third, C. screened for C. elegans mutants defective in the posteriorly elegans larval development requires the CANs. In the directed migrations of the canal-associated neurons absence of CAN function, larvae arrest development, with (CANs). Here we describe 14 genes necessary for CAN cell excess fluid accumulating in their pseudocoeloms. This migration. Our characterization of the mutants has led to phenotype may reflect a role of the CANs in osmoregula- three conclusions. First, the mutations define three gene tion. classes: genes necessary for cell fate specification, genes necessary for multiple cell migrations and a single gene Key words: cell migration, axonal outgrowth, C. elegans, canal necessary for final positioning of migrating cells. Second, associated neurons INTRODUCTION by regulating actin dynamics, perhaps by regulating the pro- trusion of actin-rich structures at the leading edge of the cell Cell migration and axonal outgrowth play important roles in or growth cone to drive forward movement (reviewed by Hall, shaping form and pattern during development. The migrations 1994; Mitchison and Cramer, 1996; Lauffenburger and of growth cones, for example, contribute to the final pattern of Horwitz, 1996). connectivity that is essential for nervous system function. To identify genes necessary for cell and growth cone migra- Although much is known about the patterns of cell and growth tions in C. elegans, we isolated mutants defective in the migra- cone migrations, both genetic and molecular approaches are tions of the canal-associated neurons (CANs). The CANs are only beginning to define the molecules that regulate these cell a pair of bilaterally symmetric neurons that are born in the head movements. and migrate posteriorly to the middle of the embryo (Fig. 1A, Extracellular matrix molecules and cell-surface ligands can Sulston et al., 1983). After migrating, each CAN extends two function to guide cell and growth cone migrations (reviewed axons along the lateral body wall, one anteriorly to the head by Hynes, 1992; Goodman and Shatz, 1993; Luo and Raper, and one posteriorly to the tail (Fig. 1B). During their develop- 1994; Tessier-Lavigne, 1994). The product of the C. elegans ment, the CANs express two differentiation markers, the gene unc-6, for example, encodes a secreted protein that guides homeodomain proteins CEH-10 and CEH-23 (Svendsen and both cell and growth cone migrations along the dorsal-ventral McGhee, 1995; Wang et al., 1993). axis, acting through the UNC-5 and UNC-40 cell surface In this paper, we present a phenotypic characterization of the receptors (Hedgecock et al., 1990; Leung-Hagesteijn et al., CAN abnormal migration (Cam) mutants. This analysis has led 1992; McIntire et al., 1992; Chan et al., 1996). Vertebrate to three conclusions. First, the mutations define three gene homologs of UNC-6, the netrins, function similarly to guide classes. In the first class, ceh-10 and possibly cam-2 regulate growth cone migrations along the dorsal-ventral axis of the ver- CAN cell differentiation. For example, ceh-10 mutations tebrate spinal cord (Serafini et al., 1994; Kennedy et al., 1994). disrupt CAN migration, axonal outgrowth and expression of Furthermore, a netrin receptor, DCC, is an UNC-40 homolog CEH-23. In the second class, eight genes may function directly (Keino-Masu et al., 1996). in cell migration and axonal outgrowth. Mutations in these Signals from extracellular guidance cues are integrated by genes disrupt multiple cell and growth cone migrations. In the intracellular signaling molecules. Members of the Rho family third class, cam-1 may function to define the precise destina- of Ras-like small GTPases, Cdc42, Rac and Rho, are intracel- tions of certain migrating cells. Mutations in cam-1 disrupt lular signaling molecules that regulate cell motility and attach- final positioning of certain migrating cells, resulting in both ment (Ridley and Hall, 1992; Ridley et al., 1992; Nobes and incomplete and excessive migrations. In addition to these Hall, 1995), and recent genetic analysis of the unc-73 gene has genes, mutations in three syc (synthetic Cam) genes result in suggested that these molecules may function in C. elegans cell Cam defects only in the presence of a ceh-23-gfp reporter that and growth cone migrations (Run et al., 1996). Signaling expresses GFP in the CANs. molecules ultimately influence cell and growth cone migrations Second, cell interactions between the CANs and HSNs 1832 W. C. Forrester and G. Garriga A. epi-1(gm139), epi-1(gm146), fam-2(gm94), kyIs5 [ceh-23-unc-76- gfp::lin-15] CAN LGV: unc-34(gm104), unc-34(gm114), unc-34(gm115), unc- Head 34(gm134), vab-8(gm99), vab-8(gm138) LGX: mig-2(gm38), mig-2(gm103sd), kyIs4 [ceh-23-unc-76- Tail gfp::lin-15] The isolation and genetic characterization of Cam mutants epi-1 HSN (epithelialization defective), ina-1 (integrin, α-subunit), unc-34 (uncoordinated), mig-2 (cell migration defective), unc-73, vab-8 Gonad (variable abnormal), ceh-10 (C. elegans homeobox), cam-1, cam-2 embryo (CAN abnormal migration), syc-1, syc-2, syc-3, (synthetic Cam), fam- 1 and fam-2 (fasciculation and cell migration defective) will be described in a subsequent paper by Forrester et al. (unpublished data). The ceh-10 alleles gm71, gm100, gm131, and gm133 all appear B. identical to gm58 for all phenotypes examined, and therefore only data for gm58 are presented in this report. dpy-5(e61) and unc-30(e191) CAN were described by Brenner (1974) and dpy-20(e1282ts) was described by Hosono et al. (1982). The chromosome III balancer qC1 was described by Austin and Kimble (1989). kyIs5 is a ceh-23-gfp reporter larva transgene that is integrated on LGIV (J. Zallen and C. Bargmann, personal communication) and kyIs4 is the same reporter integrated on Fig. 1. CANs and HSNs. (A) CAN and HSN migrations. Schematic LGX (J. Zallen and C. Bargmann, personal communication). lateral view of an embryo 400 minutes after first cleavage, the Because most of the mutants were isolated in a strain containing approximate time of the CAN and HSN migrations. Only one of each the kyIs5 reporter, we removed this transgene from the mutants by of the two bilaterally symmetric CANs and HSNs is shown. Arrows crossing to wild type. The epi-1 alleles gm121, gm139 and gm146, indicate routes of CAN and HSN migration. By the time the CANs and the fam-2(gm94) allele are tightly linked to kyIs5 and have not and HSNs reach their final destinations flanking the gonad been separated from the reporter. For quantitative determinations of primordium, however, the embryo is much longer than shown here. CAN axonal morphologies in mutants using green fluorescent protein After Sulston et al. (1983). (B) CAN morphology. Schematic lateral (GFP) (Chalfie et al., 1994), the kyIs5 reporter was crossed into the view of a newly hatched first larval stage animal. Anterior is to the mutant backgrounds. For epi-1(gm57), which was isolated in a back- left. The CAN cell body is located near the middle of the animal ground lacking kyIs5, the gm57 mutation was crossed into a kyIs4 along the anterior-posterior axis. Only one of each of the two CANs background. is shown. Each CAN extends one axon anteriorly to the head and one Other than in ceh-10(gm58) mutants, all cell migrations were axon posteriorly to the tail. scored in homozygous mutant animals derived from homozygous parents. Because ceh-10(gm58) is lethal, we examined cell positions appear to control the extent of the anteriorly directed HSN cell in homozygous animals derived from ceh-10(gm58)/qC1 parents. body migrations. The HSNs are born in the tail and migrate Because unc-73(gm67) and unc-73(gm123) progeny of homozygous anteriorly to positions adjacent to the CANs (Fig. 1A). In parents rarely survive to adulthood, we examined axonal morpholo- animals with missing or anteriorly displaced CANs, the HSNs gies of homozygous mutant animals derived from unc-73/dpy-5(e61) often migrate beyond their normal destinations, suggesting that hermaphrodites. Similarly, because epi-1(gm139) animals are sterile, the CANs signal the HSNs to stop migrating. we examined axonal morphologies in mutant animals derived from epi-1(gm139)/dpy-20(e1282ts) unc-30(e191) hermaphrodites. Third, C. elegans postembryonic development requires CAN function. In mutants that lack functional CANs, embryogen- Scoring of axons and cells esis proceeds at a normal rate, but larval development arrests. Axons were scored as defective in Table 2 if they deviated from wild- Furthermore, withering of the posterior body region displayed type morphology. This morphology was defined by electron micro- by many Cam mutants can be explained by a defect in scopic reconstructions of the C. elegans nervous system (White et al., outgrowth of posteriorly directed CAN axons that results in a 1986), immunocytochemical staining of wild-type animals (Desai et lack of CAN function in the tail. We speculate that the larval al., 1988; McIntire et al., 1992) and analysis of GFP expression in arrest and tail withering phenotypes of the mutants reflect a transgenic animals (J.
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