MIG-13 Positions Migrating Cells along the Anteroposterior Body Axis of C. elegans

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1 Cell, Vol. 98, 25 36, July 9, 1999, Copyright 1999 by Cell Press MIG-13 Positions Migrating Cells along the Anteroposterior Body Axis of C. elegans Mary Sym, Naomi Robinson, and Cynthia Kenyon* Department of Biochemistry and Biophysics University of California, San Francisco San Francisco, California Summary The C. elegans Q neuroblasts and their descendants migrate along the anteroposterior (A/P) body axis to positions that are not associated with any obvious landmarks. We find that a novel protein, MIG-13, is required to position these cells correctly. MIG-13 is a transmembrane protein whose expression is restricted to the anterior and central body regions by Hox gene activity. MIG-13 functions non cell autonomously within these regions to promote migration toward the anterior: loss of mig-13 activity shifts the Q descendants toward the posterior, whereas increasing the level of MIG-13 shifts them anteriorly in a dose-dependent manner. Our findings suggest that MIG-13 is a component of a global A/P migration system, and that the level of MIG-13 determines where along the body axis these migrating cells stop. Introduction During development, migrating cells and growth cones navigate long distances along the dorsoventral (D/V) and anteroposterior (A/P) body axes to their final desti- nations (for reviews, Tessier-Lavigne and Goodman, 1996; Varela-Echavarria and Guthrie, 1997). A number of proteins that guide cells and growth cones along the D/V axis have now been identified. For example, the netrin and slit ligands can act as attractants or repellants to guide cells and growth cones to ventral or dorsal positions (Ishii et al., 1992; Serafini et al., 1994; Colama- rino and Tessier-Lavigne, 1995; Wadsworth et al., 1996; Battye et al., 1999; Brose et al., 1999; Kidd et al., 1999; Li et al., 1999). In contrast to migration along the D/V axis, little is known about the mechanisms that guide and position cells along the A/P body axis. Some cells that migrate along the A/P body axis migrate toward chemoattrac- tants secreted by their target tissue. For example, the C. elegans sex myoblasts are thought to migrate up a gradient of FGF whose production is regulated by their target tissue, the gonad (Burdine et al., 1998). However, many cells and growth cones that migrate along the A/P body axis stop reproducibly at positions that are not * To whom correspondence should be addressed ( ckenyon@ biochem.ucsf.edu). Present address: Axys Pharmaceuticals, 100 Kimball Way, South San Francisco, California Present address: Department of Biology, Texas Wesleyan University, Fort Worth, Texas associated with any obvious landmarks. The mechanisms that determine these stopping points are unknown. We have been studying the migrations of the Q neuroblasts and their descendants along the A/P axis of C. elegans. The two Q cells, QL and QR, are born on opposite sides of the animal, but they and their descendants migrate in opposite directions, stopping at positions that are not associated with any obvious structures or cell types. QL and its descendants migrate toward the pos- terior, whereas QR and its descendants migrate toward the anterior. Both cells divide several times during the course of their migrations, and their descendants each stop at specific locations that together span the entire A/P body axis. Thus, by studying these cells, one can investigate the mechanism by which different cells in a lineage can be programmed to migrate to different locations along the A/P axis. Some of the information these cells require to migrate correctly is provided by the Hox genes mab-5 and lin- 39, which function cell autonomously within the Q de- scendants to program their migratory behaviors (Kenyon, 1986; Salser and Kenyon, 1992; Clark et al., 1993; Wang et al., 1993; Harris et al., 1996). mab-5, which patterns the posterior body region of the animal, is expressed within the QL lineage and is required for the descendants of QL to migrate posteriorly (Salser and Kenyon, 1992). In mab-5( ) mutants, the descendants of QL migrate toward the anterior, and conversely, if mab-5 is expressed specifically within an anteriorly migrating Q descendant, the cell will reverse direction and migrate pos- teriorly (Harris et al., 1996). lin-39, which specifies cell fates in the central body region, promotes anterior cell migration. In the absence of lin-39, the QR descendants stop prematurely, whereas overexpression of lin-39 causes the cells to migrate too far anteriorly (Clark et al., 1993; Wang et al., 1993; Hunter and Kenyon, 1995). Since Hox genes encode transcriptional regulators, they are likely to regulate genes that encode receptors or other factors that determine how the cells respond to extracellular cues. Little is known about the extracellular guidance factors that provide the Q cell descendants with positional information, or about the underlying logic that deter- mines where the migrating cells stop. In this study, we show that a key determinant in their positioning is the novel transmembrane protein MIG-13, which functions outside migrating cells in a dose-dependent fashion to determine their final stopping points. Results mig-13 Activity Is Required for Anterior Migrations in the QR Lineage The mig-13(mu31) mutation was shown previously to shorten the anterior migrations of the QR descendants (Harris et al., 1996). We isolated two additional mig- 13 alleles, mu225 and mu294, in a screen for mutants affecting the Q lineage migrations. In both of these mutants, two of the QR descendants, QR.paa and QR.pap,

2 Cell 26 Figure 1. Cell Migration Defects in mig-13( ) Mutants (A) QL and QR each generate three neurons (colored circles) and two cells that die (X s). Arrows indicate migratory paths, and shaded symbols represent cell divisions. (B) Migrations of cells in the QR lineage in the wild type and in three representative mig-13(mu31) mutant animals. In mig-13 mutants, QR and its descendants stop migrating prematurely. The QR.ap cell (green circle) can migrate to positions located anywhere along the A-P axis, including posterior to the birthplace of QR. (C) The final positions of the QR.pax cells are graphed using the V cell daughters as reference points along the x axis. The mig-13 positions are significantly posterior to those found in wild type (n 100). (D) Final positions of QR.ap in wild-type and mig-13(mu31) mutant animals (n 200). (E) The embryonic migration of BDU starts from the region between V1 and V2 and ends just anterior to V1. In about 60% 70% of mig- 13(mu31) animals, this migration is blocked (n 100). (Figure 1) were located significantly posterior to their normal stopping points, as in the original mig-13(mu31) mutant. By following these migrations in live animals, we found that QR itself migrated a short distance anteriorly, as it does in wild type. However, the subsequent migrations of the QR.p descendants were severely shortened (Figures 1B and 1C). In addition, the QR.ap cell, which normally migrates into the head region, often failed to complete its migration and remained in the central body region (Figures 1B and 1D). In some animals, this cell reversed direction and migrated into the posterior body region or the tail (Figures 1B and 1D). Mutations in mig-13 also affected the anterior migration of BDU, a neuron whose cell body migrates a short distance anteriorly during embryogenesis. In 60% 70% of the mig-13 mutants, this migration was blocked (Figure 1E). Many other cell migrations were examined, including those of the QL descendants, HSN, ALM, CAN, juvenile coelomocytes (ccla/p and ccra/p), M, distal tip cells, and sex myoblasts; however, none of these cells were affected (data not shown). In addition, no other obvious phenotypes were detected in these mutants; body morphology, egg laying, movement, and mating behaviors all appeared to be normal. All three mig-13 alleles were recessive and showed similar degrees of penetrance. At least one allele, mu225, was predicted to be a null mutation based on molecular criteria. In addition, when placed in trans to the deficiency sydf1, mu31 did not have a stronger phenotype than mu31 homozygotes (data not shown). Thus, the migration defects seen in these mutants are most likely due to a loss of mig-13 activity. mig-13 Influences the Positioning of Cells along the A/P Axis The finding that the QR.ap cell often migrated toward the posterior rather than the anterior in mig-13 mutants

3 MIG-13 Positions Migrating Cells along the A/P Axis 27 Figure 2. mig-13 Affects Cell Positioning along the A/P Axis The histograms shown in the left column show the distributions of cells in egl-20(n585), mig- 13(mu31), and mab-5(e1751) single mutants. In the double mutants mig-13(mu31); mab- 5(e1751gf) and mig-13(mu31); egl-20(n585), the cells are displaced further posteriorly. In many of these double mutant animals, the cells actively migrated toward the posterior. The right column shows a schematic representation of the direction and extent of migration in each of these mutant backgrounds. The vertical dashed line is used as a common reference point. n 100 for each strain. suggested that MIG-13 is required for cell guidance tations also shortened the anterior migrations of the QL rather than for the act of migration itself. In addition, we descendants in a mab-5( ) background (Figure 3A). It found that mig-13 mutations could reverse their direc- was also possible that mig-13 mutations shortened the tion of migration when combined with other mutations anterior migrations of the QR descendants by interfering that affect anterior migration. Mutations in the Wnt ho- with the expression of lin-39, the Hox gene that promotes molog egl-20 (Maloof et al., 1999) cause the QR descendants anterior cell migration. However, we found that to stop somewhat prematurely (Harris et al., 1996 a lin-39::lacz fusion (Wang et al., 1993) was expressed and Figure 2). In egl-20(n585); mig-13(mu31) double mutants, normally in a mig-13(mu31) background (data not shown), the QR.p descendants often reversed direction suggesting that this was not the case. In lin-39 mutants, and migrated toward the posterior (Harris et al., 1996; the anterior migration of the QR descendants is short- Figure 2). Likewise, in animals carrying a gain-of-func- ened, although their final positions are more variable tion (gf) mutation in the Hox gene mab-5, cells in the than in mig-13 mutants (Figure 3B). In mig-13; lin-39 QR lineage remain stationary rather than migrating ante- double mutants, the migration defect was more severe riorly (Salser and Kenyon, 1992). In mab-5(gf); mig- than in either single mutant. Since both mutations are 13(mu31) double mutants, these cells often migrated thought to be null alleles (Wang et al., 1993; Figure 4), a short distance posteriorly (Figure 2). Together these this finding suggested that lin-39 and mig-13 act in inde- findings indicate that mig-13 activity is not required for pendent or partially overlapping pathways. migration per se, but rather for the guidance or positioning of cells along the A/P axis. mig-13 Encodes a Novel Transmembrane Protein We cloned mig-13 by positional mapping followed by The mig-13 Phenotype Does Not Result transformation rescue. mig-13 encodes a predicted from Inappropriate Hox Gene Activity single-pass transmembrane protein consisting of 362 One possible explanation for why the QR descendants amino acids (Figure 4B). The N terminus of MIG-13 conwere shifted posteriorly in mig-13 mutants was that the tains a signal sequence, implying that it resides outside cells inappropriately expressed the Hox gene mab-5.to of the cell. There does not appear to be a true mig-13 test this, we first asked whether the mig-13 phenotype ortholog in the current databases; however, the N-terminal would be suppressed by a mab-5 loss-of-function mutation. domain contains two conserved motifs implicated We found that the positions of the QR.p descen- in protein protein interactions: a CUB domain and an dants in mab-5(e2088); mig-13(mu31) double mutants LDL receptor repeat. Each of these motifs has a distinct were indistinguishable from those of mig-13(mu31) sin- pattern of cysteines (Yamamoto et al., 1984; Bork and gle mutants (data not shown). Furthermore, mig-13 mu- Beckmann, 1993). The predicted cytoplasmic domain

4 Cell 28 Figure 3. The mig-13 Phenotype Is Not Caused by Misexpression of the Hox Genes mab-5 or lin-39 (A) The final positions of the QL.pax cells are shown in wild type, mig-13(mu31), mab- 5(e2088), and mig-13(mu31); mab-5(e2088) double mutants. (B) The final positions of the QR.pax cells are shown in wild type, mig-13(mu31), lin-39(mu26), and mig-13(mu31); lin-39(mu26) double mutants. The vertical dashed line is used as a common reference point. n 100 for each strain. does not have any obvious motifs, but it appears to be the cell-autonomous marker ncl-1 (Hedgecock and Herrich in proline residues. man, 1995; Miller et al., 1996). This array was carried in We found that all three mig-13 mutants contained a strain bearing homozygous mutations in both of these base changes within the mig-13 coding region (Figure genes. Spontaneous mitotic loss of the array generated 4A). All these mutations are predicted to reduce or elimi- mosaic animals that could be identified and classified nate gene function, consistent with the genetic analysis according to their Ncl phenotype. These animals were described above. then scored for their Mig phenotypes to determine which cells required mig-13 activity. Expression of mig-13::gfp The first embryonic cell division gives rise to the AB To learn where mig-13 was expressed, we constructed and P 1 cells. QR is derived from ABp, the posterior a mig-13::gfp translational fusion (Chalfie et al., 1994) daughter of AB. Among the cells expressing mig- containing all mig-13 upstream and downstream regulaand 13::GFP were the ventral cord neurons (ABp-derived) tory sequences in addition to the entire coding region. the pharyngeal-intestinal valve cells (P 1 -derived). This construct completely rescued the Mig-13 mutant Each of five animals in which the array was lost in the phenotype, suggesting that the fusion protein was exas P 1 lineage had a wild-type QR migration phenotype, pressed in all the cells that require mig-13 activity. As did mosaics containing smaller losses within the P 1 predicted from the sequence, MIG-13 appeared to be lineage (MS, C, and D losses), indicating that mig-13 is localized to the plasma membrane of all mig-13- not required in the P 1 lineage (Figure 6). This eliminates expressing cells. mig-13::gfp was first seen during emof a significant number of cell types as possible sources bryogenesis in pharyngeal, hypodermal, and neuronal mig-13 activity, including muscle, intestine, and the precursors located in the anterior body region (Figure pharyngeal-intestinal valve cells. In contrast, seven ani- 5A). At hatching, mig-13::gfp was expressed in the pha- mals in which the array was lost in the AB lineage were ryngeal-intestinal valve cells (Figure 5B), as well as in all phenotypically mutant, indicating that mig-13 was the cell bodies and axons of a number of neurons in the required in this branch of the lineage. Further analysis retrovesicular ganglion and the ventral cord (Figures showed that losses in ABp resulted in a mutant pheno- 5C 5G). Expression appeared to be restricted to cells type, whereas losses in ABa did not perturb cell migrain the anterior half of the body with the exception of tion (Figure 6). DA9, which is located in the preanal ganglion. In later Since both the QR descendants and the ventral cord larval and adult stages, the same cells continued to neurons are generated by ABp, smaller losses were reexpress mig-13::gfp, although additional staining was quired to separate these cell types. QR is derived from seen in the postembryonically derived ventral cord neu- ABpra, whereas the ventral cord neurons are generated rons located in the anterior half of the animal. Signifi- by two branches, ABplp and ABprp. Losses in ABpra cantly, no expression was seen in the migrating QR or ABprap both produced phenotypically wild-type anidescendants. mals, indicating that mig-13 is not required in QR or its descendants (Figure 6). In a direct screen for specific mig-13 Acts Non Cell Autonomously mosaic classes, 31 animals were found in which the QR To determine which cells require mig-13 activity, we descendants had either lost the array but were phenocarried out genetic mosaic analysis using an extrachromosomal typically wild type, or carried the array but were pheno- array bearing wild-type copies of mig-13 and typically mutant. These results indicated that mig-13

5 MIG-13 Positions Migrating Cells along the A/P Axis 29 we examined lin-12(n137) mutants, in which this cell is transformed to another cell type (Greenwald et al., 1983). Migration of the QR descendants in these mutants was normal; thus, DA9 is not required for mig-13-mediated anterior guidance (data not shown). Figure 4. mig-13 Is Predicted to Encode a Novel Transmembrane Protein (A) The eight exons of mig-13 are numbered and spaced relative to the intervening introns. The three mig-13 mutations are indicated by arrows. mu225 is a single G base pair deletion in exon 2 that is predicted to cause a premature STOP codon soon after the deletion. The position of this deletion is denoted by an arrow in the amino acid sequence shown in (B). mu31 is a G-to-A transition in a splice- acceptor site. mu294 is a G-to-A transition in exon 5 that changes a conserved cysteine to a tyrosine (asterisk in [B]). (B) mig-13 is predicted to encode a protein of 362 amino acids. The putative signal sequence is indicated by the boxed region. The four conserved cysteines in the CUB domain are indicated by the shaded circles. The six conserved cysteines in the LDL receptor repeat are indicated by the open circles. In addition, the invariant isoleucine residue in this repeat is boxed. The putative transmembrane domain (shaded box) is followed by a proline-rich cytoplasmic tail. The pro- line residues in this region are underlined. (C) A schematic diagram of MIG-13 illustrates several features of the predicted protein structure, including the CUB domain, the LDL receptor repeat, followed by a transmembrane domain and a proline-rich cytoplasmic tail (not to scale). expression within the QR lineage was neither necessary nor sufficient for wild-type cell migration. Unfortunately, the types of double losses required to produce animals specifically lacking mig-13( ) activity in the ventral cord neurons (ABprp and ABplp) were not found. However, since these cells were the only ABp-derived cells that expressed the rescuing mig-13::gfp fusion, they were the most likely candidates for the source of mig-13 activity. To test the possibility that expression in the posteriorly located DA9 cell was important for mig-13 function, MIG-13 Promotes Anterior Migration in a Dose-Dependent Fashion In wild-type animals, mig-13::gfp is expressed by cells located in the anterior and central body regions (Figure 5). To ask whether the correct location and/or levels of MIG-13 protein were important, the mig-13 cdna was expressed under the control of a heat shock promoter (hsp16-1), which induces high levels of expression in nearly all cells (Stringham et al., 1992). We confirmed that this treatment resulted in widespread overexpression of MIG-13 using a polyclonal antiserum raised against recombinant MIG-13 protein (see Experimental Procedures). Staged populations of L1 larvae were heat shocked and examined at the end of L1, after the migrations of the Q descendants had been completed. We found that the hs-mig-13 construct was able to rescue the mig- 13( ) migration defect (Figure 7A). The same strain without heat shock treatment did not show any rescue. In addition, the mig-13(mu31) strain without the hs-mig- 13 construct did not show any rescue following heat shock. Thus, spatially restricted expression of mig-13 is not necessary for the QR descendants to migrate normally. Although some rescue was observed with hsmig-13 when heat shock occurred at hatching, the highest degree of rescue was found in animals that were heat shocked at 2 3 hr after hatching. After this point, the degree of rescue started to decline and essentially no rescue was observed after 7 hr, when the migrations within the Q lineage are nearly completed (data not shown). This suggests that mig-13 expression is required at the time of migration (approximately when Q divides). All the data described below and in Figure 7 are from animals that were heat shocked 2 3 hr after hatching. In mig-13 mutant strains carrying hs-mig-13, most of the animals were either partially or fully rescued after a 30 min heat shock treatment. In some animals, however, the QR descendants were located significantly anterior to their wild-type positions (Figure 7A). In a wild-type background, hs-mig-13 caused an even greater fraction of the cells to migrate too far anteriorly (Figure 7B). This surprising observation suggested that the level of MIG- 13 protein can influence how far anteriorly the QR de- scendants migrate. To test this hypothesis directly, we carried out a MIG-13 dose response analysis using the length of heat shock to vary the level of MIG-13. We found that progressive increases in the dose of MIG-13 caused progressive increases in the extent of anterior migration (Figure 7E), with the longest pulses of heat shock causing the cells to overshoot their normal stop- ping points. Expression of hs-mig-13 could also cause the QL de- scendants, which normally remain in the posterior, to migrate anteriorly a short distance (Figure 7D). Thus, it appeared that hs-mig-13 could partially counteract the effect of mab-5, which acts within the QL descendants

6 Cell 30 Figure 5. Expression Pattern of mig-13::gfp A mig-13::gfp translational fusion was integrated into the genome and examined in wildtype and mig-13(mu31) backgrounds. This fusion fully rescues the BDU and QR-descendant migration defects. All expressing cells show localization to the plasma membrane. The punctate fluorescence seen in (B) (E) is due to autofluorescent gut granules and not to mig-13::gfp. Anterior is to the left and ventral is down in all panels except for (E), which shows a ventral view. Bars, 10 m. (A) The earliest expression of mig-13::gfp is seen in the anterior region of the commastage embryo. The perimeter of the embryo is outlined in white. (B) The pharyngeal-intestinal valve cells (arrow) show expression throughout all larval and adult stages. (C) Expression in the anterior ventral cord cells (arrows) in an L1 larva. This staining persists throughout all larval and adult stages. Both the cell bodies and their axons display fluorescence, but only the focal plane of the cell bodies is shown. (D) In L1 larvae, DA9 (arrow) is the only cell in the posterior that expresses mig-13::gfp. The circumferential axon that emanates from DA9 is also visible (arrowhead). (E) A ventral view of the cord neurons allows better visualization of their circumferential axons (arrowheads). (F) A lateral view of a circumferential axon in the head of an L1 is indicated by an arrowhead. (G) A schematic summary of the mig-13::gfp expression pattern in an L1 larva is shown in green. The ventral cord neurons project circumferential axons that extend dorsally across the sides of the body. Each axon grows out on either the right (dotted lines) or left (solid lines) side of the animal in a stereotypic manner. Some axons extend directly from their cell bodies, while others emanate from longitudinal extensions along the ventral surface. to promote posterior migration. An alternative explana- 430 min) could rescue the mig-13( ) BDU migration tion might have been that hs-mig-13 promotes anterior defect (Figure 8A); however, unlike the QR descendants, migration by interfering with the Wnt signaling pathway BDU did not appear to overshoot its normal anterior which normally activates mab-5 expression within QL, stopping point. Surprisingly, we also found that several rather than promoting anterior migration directly. How- migratory cells that were not affected in mig-13( ) mutants ever, this did not appear to be the case, since hs-mig- responded to hs-mig-13. ALM and CAN are neuever, 13 could also cause the QR descendants to migrate rons that normally migrate posteriorly; however, pulses anteriorly in a mab-5 gain-of-function (e1751) back- of hs-mig-13 could either shorten their posterior migraground (Figure 7C). This mab-5(e1751) mutation renders tions or cause them to reverse direction and migrate mab-5 expression independent of Wnt signaling (Harris anteriorly (Figures 8B and 8C). HSN is a neuron that et al., 1996; Maloof et al., 1999). migrates anteriorly from the tail to the midbody. We found that expression of hs-mig-13 could cause HSN Heat-Shock-mig-13 Causes Other Migratory to migrate too far anteriorly (Figure 8D). These migratory Cells to Move Anteriorly behaviors were not observed in the same strains without We also examined the response to hs-mig-13 of several heat shock treatment, nor were they seen in wild-type cells that migrate during embryogenesis. We found that strains with heat shock treatment. Thus, hs-mig-13 had administering hs-mig-13 during embryogenesis (400 a widespread effect on cell migration, causing many

7 MIG-13 Positions Migrating Cells along the A/P Axis 31 Discussion Figure 6. mig-13 Activity Is Non Cell Autonomous Mosaic animals were found for each point in the lineage as indicated. Squares represent single losses. Circles represent losses at the point indicated from animals with additional losses elsewhere. Filled symbols represent mosaic animals with a mig-13 mutant phenotype, and open symbols represent animals that are phenotypically wild type. cells to migrate to positions located anterior to their normal stopping points. Hox Gene Activity Restricts mig-13 Expression along the A/P Axis Since mig-13 expression was primarily restricted to the anterior and central body regions (Figure 5), we hypothe- sized that mig-13 might be regulated by Hox gene activ- ity. One possibility was that lin-39, the Hox gene specific for the central body region, would be required to activate mig-13 expression. However, this was not the case since the mig-13::gfp expression pattern in a lin-39(mu26) mutant background was identical to that seen in wild type (data not shown). Alternatively, it was possible that mab-5, the Hox gene specific for the posterior body region, repressed mig- 13 expression in the posterior. To test this, we examined mig-13::gfp expression in a mab-5(e2088) loss-of- function background and found that mig-13::gfp was expressed ectopically in three posterior ventral cord neurons, DA6, DA7, and DA8 (Figures 9E and 9F). Conversely, mig-13::gfp expression in the ventral cord neurons was greatly reduced or eliminated in a mab- 5(e1751) gain-of-function background, in which mab-5 is expressed in anterior as well as posterior cells (Figure 9C and 9D). In the wild type, MAB-5 is known to be present within the ventral cord motor neurons that express mig-13 (Salser et al., 1993; Salser, 1995), so it is conceivable that it acts directly on the mig-13 gene to repress its expression. We also examined mig-13::gfp expression in the absence of egl-5, the Hox gene that specifies cell fates in the tail region, to ask whether it might regulate expression of mig-13 in the DA9 cell. This did not appear to be the case, since no changes in mig-13::gfp expression were observed in an egl-5(u202) background (data not shown). The Role of mig-13 in Anteroposterior Cell Migration The C. elegans Q neuroblasts and their descendants migrate along the A/P body axis to specific locations that are not associated with any obvious morphological landmarks. Thus, the question of how these cells are guided to specific positions along the A/P axis is an intriguing one. Our findings suggest that these stopping points are specified, at least in part, by the level of MIG- 13 protein. MIG-13 is predicted to be a transmembrane protein containing two extracellular domains implicated in protein protein interactions, a CUB domain and an LDL receptor repeat. The CUB domain, which spans approximately 110 amino acids, is found in a wide variety of secreted and membrane-bound proteins (Bork and Beckmann, 1993). A number of proteins that influence cell migration or pattern formation contain CUB domains. For example, neuropilin, which is the receptor for the guidance molecule semaphorin, contains two CUB domains (He and Tessier-Lavigne, 1997). The Dro- sophila Tolloid protein, which is involved in establishing the Dpp gradient and is important for dorsoventral pat- terning, contains five CUB domains (Shimell et al., 1991). The LDL receptor repeat spans approximately amino acids. In the LDL receptor, seven tandem copies of this repeat mediate binding to a single LDL particle (Russell et al., 1989). Since MIG-13 has only one such repeat, it is unlikely that an LDL-like particle would be a ligand for MIG-13. This repeat is also found in a number of other proteins, such as the vitellogenin receptor and megalin (Saito et al., 1994; Schonbaum et al., 1995). The function of the LDL receptor repeat in all of these cases is to mediate binding to various ligands, although the parameters that determine ligand specificity are not well understood (Brown et al., 1997). Thus, it seems likely that MIG-13 exerts its effect on migrating cells by binding directly to other proteins in the A/P guidance system via its CUB and/or LDL receptor domains. Our findings indicate that mig-13 acts non cell autonomously, and that it is likely to be required within ventral cord neurons located in the anterior and central body regions. These neurons extend circumferential processes across the body wall into the dorsal nerve cord. Their processes lie between the hypodermal epithelium and the underlying basal lamina, where the Q descen- dants migrate (White et al., 1986). Since our temporal analysis indicates that MIG-13 acts at or near the time of cell migration, the Q cell descendants may detect MIG-13 (directly or indirectly) as they cross these pro- cesses during their migrations. Since the predicted CUB domain and LDL receptor repeat probably mediate pro- tein protein interactions, we propose that MIG-13 pro- motes anterior cell migration either by binding to a guid- ance factor present in the migratory substrate or by binding to a receptor on the migrating cells themselves. In the absence of mig-13, several cells migrate to positions that are shifted toward the posterior, sug- gesting that the wild-type function of the gene is to promote anterior migration. Surprisingly, we found that global overexpression of mig-13 causes a wide variety of cells to migrate anterior to their normal stopping points, including cells that are not affected by loss of mig-13

8 Cell 32 Figure 7. Heat-Shock-mig-13 Promotes Anterior Migration in the QR and QL Lineages (A) A hs-mig-13 construct can rescue the anterior migration defect of the QR descendants in mig-13(mu31) mutants. In addition, hs-mig-13 can cause the cells to migrate too far anteriorly (compare to [B]). (B) In an otherwise wild-type background, hs-mig-13 causes an even larger fraction of cells to migrate too far anteriorly. (C) hs-mig-13 can override the posterior program imposed by mab-5(e1751gf) on the right, as well as the normal mab-5 activity on the left (D). In both cases (C and D), cells that normally stay in the posterior are forced to migrate anteriorly in response to hs-mig-13 activity. The length of heat shock for the results shown in (A) (D) was 30 min. (E) The dose of hs-mig-13 was varied by changing the length of heat shock in a mig-13( ) background. Incremental increases in MIG-13 protein caused the QR descendants to migrate to progressively anterior stopping points. The vertical dashed line is used as a common reference point. The asterisk at the bottom indicates the wild-type position of the QR descendants (also see [B]). n 100 for each strain.

9 MIG-13 Positions Migrating Cells along the A/P Axis 33 activity. Some of these cells even reverse direction and migrate anteriorly rather than posteriorly. The fact that many cells can respond to MIG-13 suggests that it may be a component of a global A/P guidance system. Another component of this system is likely to be the VAB-8 protein, which acts cell autonomously within migrating cells (Wolf et al., 1998). In vab-8 loss-of-function mutants, many posteriorly directed cell and axon migrations along the A/P axis are shortened (Wightman et al., 1996). If MIG-13 promotes anterior migration as part of a global A/P guidance system, it is puzzling that most of these cells are not affected by mig-13 loss-of-function mutations. Some cells, such as HSN, probably do not encounter MIG-13 protein during their migrations, since they migrate within the posterior body region. However, others, such as CAN and ALM, do migrate in the vicinity of cells that express mig-13. It is possible that the wildtype level or precise positioning of MIG-13 is not adequate to influence their migrations. Alternatively, mig- 13 may function redundantly with other factors. The fact that mig-13 and egl-20 have partially overlapping functions is consistent with this possibility (discussed below). One of the most intriguing aspects of A/P cell migration is that many cells stop at precise positions that are not associated with any obvious morphological targets. Our dose response analysis of MIG-13 showed that the extent of migration toward the anterior was correlated with the level of MIG-13 protein. As the MIG-13 dose was increased, the QR descendants migrated progressively further into the anterior, with many cells migrating past their normal stopping points at the highest doses. Conversely, removing mig-13 activity shifted the stopping points of migrating cells toward the posterior, in some cases causing cells to migrate posteriorly rather than anteriorly. For example, in both egl-20/wnt and mab- 5(e1751gf) mutants alone, the QR descendants migrate anteriorly but stop prematurely. When mig-13( ) mutations were introduced into either strain, the QR descendants often reversed direction and migrated posteriorly. Taken together, these results indicate that lowering the level of MIG-13 shifts cells posteriorly, and raising it shifts cells anteriorly. Moreover, the precise level of MIG- 13 can determine the extent of anterior migration. Thus, we conclude that this protein plays a key role in defining the stopping points of migrating cells during wild-type development. How might the quantity of MIG-13 change the positions of the QR descendants? MIG-13 does not provide cells with the information they need to distinguish anterior from posterior since they migrate in a directionspecific fashion even when MIG-13 is expressed along the entire A/P axis. Thus, there must be a source of guidance information that operates independently of mig-13 to orient cell migrations along the A/P body axis. The positional shifts of migrating cells caused by changes in the level of MIG-13 are reminiscent of the positional shifts in pattern elements caused by changes in the levels of graded morphogens such as the Dro- sophila Bicoid, Dorsal, or Dpp proteins (Driever and Nüsslein-Volhard, 1988; Roth et al., 1989; Struhl et al., 1989; Wharton et al., 1993; Nellen et al., 1996; Marqués et al., 1997). Similarly, MIG-13 may be part of a gradient Figure 8. Heat-Shock-mig-13 Shifts the Stopping Points of Many Migrating Cells toward the Anterior The diagram at the top depicts the wild-type embryonic migrations of BDU, ALM, CAN, and HSN relative to the V cells. (A) hs-mig-13 can rescue the BDU migration defect in mig-13(mu31) animals. (B) In an otherwise wild-type background, hs-mig-13 can promote the anterior migration of ALM, a cell that normally migrates posteriorly. (C) hs-mig-13 can also shorten the posterior migration of CAN and cause HSN to migrate too far anteriorly (D). n 100 for each strain.

10 Cell 34 Figure 9. mig-13::gfp Expression Is Inhibited by mab-5 Activity (A) In wild-type L1 animals, mig-13::gfp is expressed in the ventral cord neurons located in the anterior half of the body (arrows). (C) In a mab-5 gain-of-function mutant (e1751), these cells no longer express mig-13::gfp. Pharyngeal-intestinal valve staining was still present in this background. (E) In a mab-5 loss-of-function mutant (e2088), ventral cord neurons located in the posterior (arrows) show ectopic expression of mig-13::gfp. The arrowhead points to DA9, which normally expresses mig-13::gfp. DA8 is the most posterior cell that ectopically expresses mig-13::gfp, and it lies near DA9. DA8 is indicated by an asterisk to distinguish it from DA9. Schematic diagrams of each expression pattern are also depicted (B, D, and F). Brackets indicate approximate body regions that are shown in the corresponding fluorescent images. The punctate fluorescence seen in (A), (C), and (E) is due to autofluorescent gut granules and not to mig-13::gfp. Bar, 10 m. system that orients and positions migrating cells along the A/P body axis. In this model, the Q cell descendants seek out specific positions defined by the level of a factor present in a concentration gradient along the A/P axis. Changes in the level of MIG-13 could shift the positions of the migrating cells along the A/P axis by changing the concentration of the graded factor itself or by changing the cell s perception of the graded factor. Gradient models are attractive because they can explain how both the direction and extent of cell migration can be altered by changing the level of MIG-13. However, models that do not involve gradients are also plausible. For example, one or more components of the guidance system could have a defined A/P polarity that orients the cells along the body axis. In this model, MIG- 13 could position cells by enhancing or antagonizing an intrinsic migratory response that the cells have to the polarized signal. Hox Gene Control of mig-13 Expression In the wild type, the Hox gene mab-5 restricts mig-13 expression to the anterior half of the animal by repressing its expression in the posterior. It is likely that this spatial restriction of mig-13 expression is functionally significant, since misexpression of mig-13 within the posterior body region causes the QL descendants to migrate anteriorly. Hox gene regulation of mig-13 expression appears to define a local environment in which anterior cell migration is facilitated. This, in turn, allows the QR descendants, which migrate within this region, to move further anteriorly than they would otherwise. The finding that mab-5 positions an extracellular guidance factor is particularly intriguing because mab-5 has already been shown to act cell autonomously within the migrating QL descendants to program their migratory behaviors (Kenyon, 1986; Harris et al., 1996). Thus, control of Q cell migration along the A/P axis is affected by Hox gene function in two very different but mutually reinforcing ways: mab-5 acts within the QL descendants to promote their migration toward the posterior, and in parallel, it acts outside the migrating cells to prevent the expression of a protein that promotes anterior migration. mab-5 and mig-13 appear to represent two antagonistic forces that can move cells in one direction or the other depending upon the relative strengths of their activities. Segregating these two forces through region-specific regulation is likely to be important in generating the asymmetry of the Q cell migrations. Experimental Procedures Genetics and Phenotypic Analysis Standard methods for culturing and genetic analysis were used (Brenner, 1974; Sulston and Hodgkin, 1988). Strains were maintained at 20 C unless otherwise indicated. Alleles used are listed by linkage group as follows: LGIII: lin-39(mu26), lin-39(n1760), mab-5(e2088), mab-5(e1751), egl-5(u202), ncl-1(e1865); LGIV: egl-20(n585), dpy- 20(e1282); LGV: him-5(e1490); LGX: mig-13(mu31), mig-13(mu225), mig-13(mu294). Transgenic arrays were generated using standard techniques (Mello et al., 1991). Genomic integrants and extrachromosomal arrays are designated as follows: muis42 [pms77 (mig-13::gfp) pmh86 (dpy-20)], muis37 [pms114 (hs-mig-13) pmh86 (dpy-20)], muex42 [pmh86 (dpy-20) C33C3 (ncl-1) pms37 (mig-13)]. The

11 MIG-13 Positions Migrating Cells along the A/P Axis 35 resulting plasmid allowed expression and purification of amino acids of the MIG-13 protein in E. coli using a 6-His tag system (Qiagen Qiaexpressionist). The recombinant protein was then injected into rabbits for antibody production (Caltag Laboratories). Affinity-purified antibodies were used to stain worms carrying the hs-mig-13 construct. In the absence of heat shock, membrane stain- ing was seen on a variety of cells, including those observed in the rescuing mig-13::gfp strain. The antibodies were of limited analytical value, since they appeared to cross-react with other proteins. Therefore, we did not use these antibodies to assess the wild-type mig-13 expression pattern. However, they allowed us to confirm that pulses of hs-mig-13 caused widespread expression of MIG-13 protein, since heat-shocked animals carrying a hs-mig-13 fusion showed a dramatic increase in staining in nearly all cells. This high level of staining was not observed in either the hs-mig- 13 strain without heat shock or in wild-type animals with heat shock, or in heat-shocked animals carrying unrelated heat shock con- structs. positions of the Q.pa daughters were determined directly using Nomarski optics at the end of L1. At this stage, the hypodermal V cells have divided once and the P nuclei have all descended into the cord. The positions of the Q.pa daughters were scored relative to the V cell daughters since these are stationary landmarks. Since the Q.ap cell can migrate into the head or tail ganglia, it is difficult to identify directly under Nomarski optics. Therefore, its position was determined using a tax-4::gfp fusion, which labels only a small number of neurons including QL.ap and QR.ap. This fusion was made by inserting 12 kb of the tax-4 promoter (Komatsu et al., 1996) just upstream of GFP in the vector ppd95.75 (A. Fire et al., personal communication) and integrated into the genome using irradiation. mig-13 Mutant Isolation and Cloning The mig-13(mu31) mutation was identified in a Nomarski screen for mutants with misplaced Q cell descendants. The mig-13 (mu225 and mu294) mutations were isolated in a screen for misplaced Q cell descendants using a mec-7::gfp fusion, which labels a number of neurons including the two Q cell descendants AVM (QR.paa) and PVM (QL.paa) (M. S. et al., unpublished data; Hamelin et al., 1992; Mitani et al., 1993). All three alleles were generated with ethyl methanesulfonate (EMS). Genetic mapping placed mig-13 in a small inter- val (0.6 map units) between lon-2 and dpy-8 on the X chromosome. Further mapping using physical markers (RFLPs) and subsequent transformation rescue narrowed the region to a single cosmid, F15F4. Subcloning of this cosmid followed by transformation rescue experiments localized the mig-13 gene to a9kbnrui-kpni fragment. Sequence information from the C. elegans genome project predicted a single full-length gene in this region (based on the GeneFinder algorithm). A 1.5 kb deletion in the 5 end of this gene was made in the 9 kb rescuing fragment. This construct failed to rescue the mig-13( ) phenotype. Multiple cdnas were isolated from a mixedstage cdna library (Barstead and Waterston, 1989) and from RT PCR experiments using SL1 (Krause and Hirsh, 1987) and internal primers. Sequencing of these cdnas indicated that mig-13 contains eight exons. Acknowledgments We thank members of the Kenyon lab for technical advice, lively discussions, and critical comments on this manuscript. We also thank Rebecca Smith for construction of the lin-39; mig-13 double mutant, Robert Barstead for his C. elegans cdna library, Lindsay Hinck for mec-7::gfp plasmid construction, and Queelim Ch ng for his invaluable help in screening. This work was supported by a grant to C. K. from the National Institutes of Health. M. 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