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First published online March 4, 2005
doi: 10.1242/10.1242/dev.01569

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The roles of two C. elegans HOX co-factor orthologs in cell migration and vulva development

Lucie Yang^*, Mary Sym and Cynthia Kenyon

Department of Biochemistry and Biophysics, University of California, San Francisco, Mission Bay Genentech Hall, 600 16th Street, Room S312A, San Francisco, CA 94143-2200, USA

View larger version (18K): [in a new window]   Fig. 1. (A) Wild-type Q lineage and cell migration. (Top) Q neuroblast lineage. QL and QR generate identical cell lineages to produce three neurons: the two Q.pax cells are filled circles whereas the Q.ap cell is an unfilled circle. `X' indicates cell death. (Middle) Left and right Q cells (QL in blue, QR in pink) are located in approximately the same position at hatching. Anterior is towards the left and ventral is downwards. Shortly after hatching but before division, the QL cell migrates a short distance posteriorly, while the QR cell migrates a short distance anteriorly. (Bottom) Final positions of Q descendants. QL.pax cells (filled blue circles) are located near the position of QL at hatching, whereas the QL.ap (unfilled blue circle) cell migrates into the tail. QR.pax cells (filled pink circles) migrate to the anterior body, whereas the QR.ap cell (unfilled pink circle) migrates into the head. (B) Wild-type vulva development. (Top) A schematic view of a late L1 larva with 11 of the 12 ventral Pn.p cells shown. Some of the Pn.p cells fuse with the hypodermal syncytium, hyp7, as indicated by dashes in the third row of the top panel. The unfused cells are indicated by ovals, and these are the vulval precursor cells (VPCs). The oval around P3.p is dashed because this cell remains unfused in about 50% of wild-type animals. Anterior is towards the left and ventral is downwards. (Middle) In the L3 stage, an inductive signal (green arrows) from the anchor cell (AC, also shown in the top panel), lateral signaling (blue arrow) among the VPCs and an inhibitory signal (red T) from hyp7 impose a 3°-3°-2°-1°-2°-3° pattern of cell fates. The three fates can be distinguished by the expression pattern of several markers and by cell lineage pattern. The 1° fate is characterized by three rounds of cell division, with the last divisions being all transverse (T). The 2° fate is characterized by three rounds of cell division, with the Pn.pxx cells dividing longitudinally (L), dividing transversely (T) or not dividing (N). The 3° fate is characterized by a single round of division followed by fusion with hyp7, as indicated by the dashed Pn.px circles. The descendants of P5.p-P7.p form the vulva. (Bottom) In the L4 stage, the vulval cells migrate towards the center of the animal to form seven vulval rings and begin the process of intratoroidal fusion. Vulval eversion at the end of the vulva formation process is not shown.

View larger version (28K): [in a new window]   Fig. 5. MIG-13 requires ceh-20 and unc-62, but not lin-39, to promote anterior migration. The left column shows the distribution of QR.pax cells in mig-13(mu225), lin-39(n1760), ceh-20(mu290) and unc-62(mu232) single mutants. Expression of mig-13 in all neurons under the unc-119 promoter (Punc-119::mig-13) rescues this migration defect in mig-13(-) and lin-39(-), but not in ceh-20(-) or unc-62(-) animals. In an otherwise wild-type background, Punc-119::mig-13 does not alter the distribution of these cells. The vertical gray line indicates the birthplace of the QR cell. The pink arrow indicates the total anterior distance traversed by QR and its descendants giving rise to QR.pax in wild-type animals. n=100 cells for each strain. The right column shows the distribution of the right BDU cell (BDUR) in these same strains at the end of L1. The embryonic migration of BDU starts from the region between V1 and V2 (indicated by the vertical gray line) and ends just anterior to V1 in wild-type animals. The full anterior migration of this cell is blocked in 8% of wild type, 92% of mig-13(mu225), 26% of lin-39(n1760), 82% of ceh-20(mu290) and 96% of unc-62(mu232) animals. Expression of mig-13 in all neurons restores full anterior migration in 88% and 86% of mig-13(mu225) and lin-39(n1760) animals, respectively, but in only 46% and 38% of ceh-20(mu290) and unc-62(mu232) animals, respectively. In an otherwise wild-type background, Punc-119::mig-13 does not alter the distribution of BDUR. Similar distributions were observed for the left BDU cell in these strains (data not shown). The black arrow indicates the anterior distance that BDU migrates in wild-type animals. n=50 cells for each strain.

View larger version (26K): [in a new window]   Fig. 2. Cell migration defects in ceh-20(-) and unc-62(-) mutants. The final positions of the QL.ap (left column) and QR.ap (right column) cells are graphed using the V cell daughters as reference points along the x axis, with left being anterior. The QL.ap cells in ceh-20(mu290) and unc-62(mu232) can be displaced anterior to the wild-type (WT) and mig-13(mu31) positions, but not as far anteriorly as in mab-5(e2088) animals. The QR.ap positions in ceh-20(mu290) and unc-62(mu232), as in mig-13(mu31) and lin-39(n1760) (although to a lesser degree in the latter), are significantly posterior to those found in wild type. The vertical gray line indicates the birthplace of the Q cells. The blue arrow indicates the total posterior distance that QL, QL.a and QL.ap migrate in wild-type animals. The pink arrow indicates the total anterior distance that QR, QR.a and QR.ap migrate in wild-type animals. n100 cells for each strain.

View larger version (15K): [in a new window]   Fig. 3. ceh-20 and unc-62 can function independently of Hox genes lin-39 and mab-5 when positioning cells along the AP axis. The histograms show the distributions of QR.pax cells in wild-type animals as well as in ceh-20(mu290), unc-62(mu232) and lin-39(n1760) single mutants. In the double mutants ceh-20(mu290) lin-39(n1760) and unc-62(mu232); lin-39(n1760), the anteriorwards migrations of QR descendants are shortened. In unc-62(mu232); lin-39(n1760) double mutants, some cells even actively migrated towards the posterior. In the double mutants ceh-20(mu290) mab-5(e2088) and unc-62(mu232); mab-5(e2088), QR descendants did not migrate to the wild-type positions achieved in mab-5(e2088) single mutants. The vertical gray line indicates the birthplace of the QR cell. The pink arrow indicates the total anterior distance traversed by QR and its descendants giving rise to QR.pax in wild-type animals. n=100 cells for each strain.

View larger version (31K): [in a new window]   Fig. 4. ceh-20 and unc-62 affect cell positioning along the AP axis. (A) In the double mutant mig-13(mu31); egl-20(n585), QR.pax cells were displaced further posteriorly than in mig-13(mu31) or egl-20(n585) single mutants. QR descendants actively migrated towards the posterior in many of these double mutants animals. (B,C) QR.pax cells could be displaced to a location posterior to the birthplace of QR in double mutants ceh-20(mu290); egl-20(mu320) and unc-62(mu232); egl-20(mu320), but not in double mutants ceh-20(mu290); mig-13(mu31) or unc-62(mu232); mig-13(mu31). The penetrance of mig-13(mu31) phenotypes is equal to that of the molecular null, mu225 (Sym et al., 1999). egl-20(mu320) is likely a null allele (Ch'ng et al., 2003). Double mutants of ceh-20(mu290) or unc-62(mu232) with a strong allele of egl-20, n585, also displayed QR descendants migrating posteriorly instead of anteriorly (histogram not shown). In ceh-20(mu290); egl-20(n585) or unc-62(mu232); egl-20(n585) double mutants, 24% or 13%, respectively, of QR.pax cells were located in the posterior body between V5.a and V6.a. Distributions of QR.pax cells in ceh-20(mu290), unc-62(mu232), egl-20(mu320) and mig-13(mu31) single mutants are also shown. There is no significant difference in the QR.pax distributions between ceh-20(mu290) and ceh-20(mu290); mig-13(mu31) (P>0.05). (D) QR.pax cells were displaced further posteriorly in lin-39(n1760); egl-20(mu320) and lin-39(mu26); mig-13(mu31) double mutants than in lin-39(n1760), egl-20(mu320) or mig-13(mu31) single mutants. However, QR descendants did not actively migrate towards the posterior in these double mutants. The mutation in lin-39(mu26) is predicted to eliminate DNA-binding activity, whereas lin-39(n1760) is a null allele lacking the homeodomain (Clark et al., 1993; Wang et al., 1993). lin-39(mu26) and lin-39(n1760) single mutants have nearly identical QR.pax distributions. The vertical gray line indicates the birthplace of the QR cell. The pink arrow indicates the total anterior distance traversed by QR and its descendants giving rise to QR.pax in wild-type animals. The green arrows denote active posterior migration of cells giving rise to QR.pax. n=100 cells for each strain.

View larger version (67K): [in a new window]   Fig. 6. The Muv phenotype in ceh-20(mu290) animals. Nomarski photomicrographs of L4 larvae. (A) Wild-type animal showing a normal vulval invagination formed by P(5-7).p (arrowhead). (B) ceh-20(mu290) animal showing separate invaginations formed by P5.p and P6.p while P7.px (not shown) failed to divide further. P4.p also formed a small invagination. (C) ceh-20(mu290) animal showing an invagination at P2.p in addition to the invagination formed by P(5-7).p (not shown). (D) ceh-20(RNAi) animal showing P4.p forming a separate invagination adjacent to the invagination formed by P(5-7).p. (E) ceh-20(mu290) lin-39(n1760) animal showing separate invaginations formed by P5.p and P6.p. P7.p did not divide in this animal. (F) unc-62(mu232) animal showing the incomplete migration of some P7.p descendants towards P6.p descendants, resulting in the asymmetric invagination. Animals in all panels are oriented with anterior towards the left.

View larger version (119K): [in a new window]   Fig. 7. ceh-20 is expressed in nuclei of Q, P and V cells and their descendants. (A-C) Fluorescence micrographs of wild-type larvae carrying ceh-20::gfp. This plasmid rescued the cell migration defects (QL and QR descendants, BDU) and the multivulval defect of ceh-20(mu290) animals. Dorsal is upwards and anterior is towards the left. (A,B) At hatching, ceh-20 is expressed in the nuclei of ectodermal such as the Q cells, P cells, V cells and hyp7. The mesodermal precursor, M, also expresses ceh-20 robustly. (C) Six to eight hours after hatching, V cell daughters, hyp7 cells and P cells continue to express ceh-20. In this animal, the right side is shown. The descendants of QR have migrated anteriorly and are in the process of dividing (not visible by fluorescence). (D) In this L2 animal, P(1-11).p cells express ceh-20, although only P(3-8).p are shown. Ventral cord neurons between the Pn.p cells also express ceh-20::gfp.

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