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First published online 11 February 2009
doi: 10.1242/dev.029363

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MIG-32 and SPAT-3A are PRC1 homologs that control neuronal migration in Caenorhabditis elegans

Departments of Pediatrics and Molecular Biology, Division of Pediatric Hematology-Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

View larger version (38K): [in this window] [in a new window]   Fig. 1. MIG-32 and SPAT-3A are RING-domain proteins closely related to Polycomb-group family members. Alignment and comparison of MIG-32 and SPAT-3A with related proteins from other species. (A) Amino acids highlighted in blue indicate the RING domain. (B) MIG-32 compared with related human and Drosophila proteins. The green box indicates the RING domain; the black box indicates a C-terminal region that participates in complex formation with RING1B (Li et al., 2006). Percentage amino acid sequence identities (shown as a number within the compared regions) are indicated in pair-wise comparisons between MIG-32 and the other proteins. (C) Amino acid sequence alignments of the RING domain of SPAT-3A and dRING1 with related proteins. There is little or no additional sequence similarity between SPAT-3A and RING1B/RING2 homologs from these other species. (D) Pair-wise comparisons of the amino acid identities between the RING domains of MIG-32 and SPAT-3A from C. elegans with BMI-1 and RING1B from humans.

View larger version (48K): [in this window] [in a new window]   Fig. 2. mig-32 is required for ubiquitylation of histone H2A. (A) Western blot analysis of acid-extracted histones from wild-type, mig-32(n4275) and spat-3(gk22) mutant C. elegans. The filter shown in the left panel was probed with anti-ubiquitin antibody. Arrowheads indicate ubiquitylated H2A and H2B. In the right panel, this blot was stripped and probed with anti-H2A antibody. Arrowheads indicate H2A and ubiquitylated H2A. (B) spat-3 mutants have greatly reduced levels of ubiquitylated H2A. A western blot using anti-ubiquitin antibody detects a trace amount of ubiquitylated H2A in most preparations of acid-extracted histones from spat-3(gk22) mutants, consistent with this allele being strong loss-of-function, but not null. A spat-3(RNAi) construct was fed to wild-type animals in two experiments [spat-3(RNAi-1) and spat-3(RNAi-2)]. The RNAi construct reduces, but does not eliminate, ubiquitylated H2A levels. Loading was not equal in all lanes of this blot.

View larger version (70K): [in this window] [in a new window]   Fig. 3. mig-32 is broadly expressed, localized to nuclei, and concentrated in nucleoli. Differential interference contrast (A,C,E) and epifluorescence (B,D,F) images of transgenic C. elegans. (A,B) Mixed-stage embryos. (C,D) L1-stage larva showing the lateral hypodermal cells. Arrowheads highlight nucleoli within hypodermal nuclei. (E,F) L4-stage male tail. Arrowhead highlights a nucleolus within a neuronal nucleus. (G,H) COS-7 cell transfected with FLAG-epitope-tagged C. elegans MIG-32, stained with (G) DAPI to visualize DNA and (H) anti-FLAG antibody. Arrowheads highlight nucleoli in the COS cell nucleus. In C-F, anterior is to the left and ventral down. Scale bars: 10 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 4. mig-32 acts parallel to most known pathways that position Ray 1, with the exception of the PRC2 pathway. (A) Ventral views of the tails of male wild-type and mig-32 mutants and a lateral view of a mig-2; mig-32 mutant male. The nine bilateral rays are numbered. Arrows indicate Ray 1 pairs in the wild-type and mig-32 mutants; an arrowhead indicates crumpled Rays 2 and 3 in the mig-32 mutant. (B) The percentage of animals with anterior Ray 1 in wild-type and mutant animals. The severity of the Ray 1 migration defect is categorized as Class I, in which the ray is located anterior to and outside of the normal position of the cuticular fan, or as Class II, in which the ray is anterior but present within the fan (Fujii et al., 2002; Ginzburg et al., 2002). (C) The percentage of animals with anterior Ray 1 in wild-type and mutant animals of the indicated genotypes, scored as in B.

View larger version (52K): [in this window] [in a new window]   Fig. 5. mig-32 mutants have defects in neuronal migration and process extension. Images of wild-type and mig-32 mutant animals carrying gfp reporters. (A) An otherwise wild-type animal expressing Pnlp-1gfp in the HSN neurons. HSN cell bodies are indicated with a large white arrowhead. The position of the vulva is indicated. Small white arrowheads highlight the axons of the two HSN neurons, which proceed along the ventral body wall to the head. (B)A mig-32 mutant expressing Pnlp-1gfp. Large white arrowheads indicate the HSN cell bodies and small arrowheads highlight the HSN axons. (C) Ventral view of a wild-type animal expressing Punc-47gfp in the VD neurons. Small white arrowheads indicate lateral commissures. (D) Ventral view of a mig-32 mutant expressing Punc-47gfp in the VD neurons. Small white arrowheads indicate lateral commissures. Large arrows indicate three commissures on the wrong side. (E) A wild-type animal expressing Psra-6gfp in the PVQL and PVQR neurons. A small white arrowhead indicates the normal separation of the PVQ axons into the right and left sides of the ventral nerve cord. (F)A mig-32 mutant expressing Psra-6gfp in the PVQ neurons. Arrows indicate inappropriate crossing by the PVQ neuronal axons. The scale bar in B applies to A-D, which show adult animals. E and F show L1-stage larvae. Anterior is to the left in all images; ventral is down in A and B, up in C and D, and slightly rotated in E and F.

View larger version (23K): [in this window] [in a new window]   Fig. 6. mig-32 and spat-3 act similarly in HSN migration. (A) The embryonic migration path (red arrow) and final positions of the HSN neurons in wild-type and mutant transgenic animals. The Pnlp-1gfp reporter, which is expressed in the HSNs (Li et al., 1999), was used to identify the final positions of the HSNs (see Materials and methods). The shaded boxes indicate the percentage of animals with HSNs in specific regions. The percentage of HSN axons that failed to reach the head is shown. n, number of HSNs assayed. (B) The migration path (red arrow) and final positions of the ALM neurons. The Pmec-7gfp reporter, which is expressed in the ALMs (Hamelin et al., 1992), identified their final positions.

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