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First published online 17 August 2005
doi: 10.1242/dev.01967

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The HMX homeodomain protein MLS-2 regulates cleavage orientation, cell proliferation and cell fate specification in the C. elegans postembryonic mesoderm

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

View larger version (24K): [in a new window]   Fig. 1. The C. elegans hermaphrodite postembryonic M lineage. Times indicated are hours post-hatching at 25°C. (A) The M lineage with all the differentiated cell types (modified from Sulston and Hovitz, 1977). The corresponding stages referred to in the paper are indicated on the right. (B) A schematic lateral view of the M lineage through larval development. D, dorsal; V, ventral; L, left; R, right; A, anterior; P, posterior.

View larger version (90K): [in a new window]   Fig. 2. The M lineage phenotypes of cc615 and tm252 mutants. All images are lateral views of animals with anterior to the left and dorsal up; asterisk indicates position of the vulva. The number of M-derived precursors or products in each panel indicates the total number of corresponding cell types in the particular mutant. (A-I) The M lineage of wild-type (A,D,G), cc615 (B,E,H) and tm252 (C,F,I) hermaphrodites as visualized by hlh-8::gfp. (A-C) Cleavage orientation of M showing the positions of the two M daughters. (D-F) The M lineage undifferentiated products at the late L1 stage. (D) 16 M-derived cells are present in wild-type animals, with eight visible in the focal plane shown. (E) A cc615 mutant animal with 12 M-derived cells, 11 of them are visible in the focal plane shown because of defects in cleavage orientations. (F) A tm252 mutant animal with 29 M-derived cells, 14 of them visible in the focal plane shown. (G-I) Positions of SMs at late L2 stage. (G) Two SMs (one out of focus) in wild-type animals at the future vulval region. (H,I) Multiple SMs (one focal plane shown) in cc615 (H) and tm252 (I) mutants, with some SMs exhibiting migration defects. (J-L) Differentiated M lineage products in adult wild-type (J), cc615 (K) and tm252 (L) hermaphrodites. Arrow, VM1 visualized by egl-15::gfp; open arrowhead and double-line arrow, embryonically-derived CCs and M-derived CCs, respectively, as visualized by intrinsic CC::gfp. (J) A wild-type animal with two dorsal M-derived CCs and four VM1s. (K) A cc615 animal with no M-derived CCs and extra VM1s (some do not migrate properly). (L) A tm252 animal with extra VM1s and CCs.

View larger version (14K): [in a new window]   Fig. 3. The early M lineage in wild-type and mls-2(cc615) animals. (A-D) The M lineage of wild type (A) and sample animals of cc615 mutants (B-D). The M lineage was followed from L1 to L4 stage in 15 wild-type and 32 cc615 mutant animals. Orientations of the first two divisions are indicated to show randomized cleavage orientations in cc615 mutants. All cc615 mutants exhibited reduced proliferation after the 8-M stage (B-D). We consistently observed loss of both M-derived CCs and supernumerary SM-like cells in cc615 mutants. Cells that may adopt the BWM fate or fail to express any M lineage markers are unmarked.

View larger version (71K): [in a new window]   Fig. 4. mls-2 encodes a homeodomain protein of the HMX family. (A) mls-2 gene structure and constructs. The upper diagram represents the six exons of the mls-2 genomic sequence, with the molecular lesions in cc615 and tm252 indicated. Shaded areas represent the homeodomain. The lower diagram depicts various fusion constructs used in this work (see Materials and methods). Both pYJ59 and pYJ62 were able to rescue the mls-2(cc615) mutant phenotypes. (B) A Clustal W sequence alignment of MLS-2, other HMX (Nkx5) family homeodomain proteins and the C. elegans Nkx2.5 homolog CEH-22. Only residues within the homeodomain (residues 1-60) and two additional domains (in dashed boxes) immediately after the homeodomain were aligned. The proteins share no similarity outside this compared region. Identical amino acids are in white letters against black background. Similar amino acids are in black letters against gray background. The alignment is shown for CeMLS-2 (this work), C. briggsae CBG14538(WormBase), SOHo1 (chicken) (Deitcher et al., 1994), SpHmx (sea urchin) (Martinez and Davidson, 1997), TgHbox5 (sea urchin) (Wang et al., 1990), GH6 (chicken) (Stadler et al., 1994), DHmx (Drosophila) (Wang et al., 2000), murine Hmx3 (Nkx-5.1) (Bober et al., 1994), murine Hmx2 (Nkx-5.2) (Bober et al., 1994), human HMX2 (Wang et al., 2000), murine Hmx1 (Yoshiura et al., 1998), human HMX1 (H6) (Stadler et al., 1992) and CEH-22 (Okkema and Fire, 1994). (C) A phylogenetic tree of the HMX family generated using the neighbor-joining method of Clustal W. The tree is rooted with CEH-22 in TreeView version 1.6.6 (Page, 1996). Only the homeodomain and the two additional conserved domains immediately after the homeodomain (as shown in B) were used in the analysis.

View larger version (84K): [in a new window]   Fig. 5. Expression pattern of mls-2 in wild-type, cc615 and tm252 hermaphrodites. (A,B,E,F,M) Wild-type embryos (A,B) and L1 larvae (E,F,M) stained with anti-MLS-2 antibody (A,E,M) or DAPI (B,F). (C,G,N) Expression pattern of a functional GFP::MLS-2 fusion in wild-type embryos (C) and early L1 larvae (G,N). (D,H) Corresponding DIC images for panels C and G. Nuclear localized MLS-2 protein was seen in a subset of cells during embryogenesis (A,C), in six head neurons (arrowhead in E; the neuronal signal was out of focus in G), in the M mesoblast (arrows in E and G) and in the eight M descendants (M,N; only four cells visible in this focal plane). No MLS-2 protein was detected after the 8-M stage (n>50). Note that the other signals shown in G are due to gut autofluorescence. (I-L) cc615 (I,J) and tm252 (K,L) L1 larvae stained with anti-MLS-2 antibody (I,K) or DAPI (J,L). (I) No MLS-2 staining was detected in cc615 mutants (n>50). (K) The MLS-2(gf) protein in tm252 mutants was detected in the 16 M descendants (eight visible in this focal plane) and the head neurons (data not shown). (O) GFP signal from a mls-2p::gfp transgene in an animal with 13 M descendants (six GFP-expressing cells visible in the focal plane shown). The GFP signal is primarily cytoplasmic. (P-U) Transgenic animals carrying an integrated hlh-8p::lacZ reporter were double-stained with both anti-MLS-2 (P,S) and anti-ß-galactosidase (Q,T) antibodies. (R,U) Merged images. MLS-2 protein was detected in the M mesoblast (P-R), but not in the SMs (S-U). (V) Summary of mls-2 expression pattern in the M lineage. The wild-type M lineage with overlay of mls-2 expression highlighted in red. Solid red lines indicate mls-2 expression detected by both GFP::MLS-2 and anti-MLS-2 antibodies; dashed red lines indicate mls-2 expression detected only by a transcriptional mls-2p::gfp reporter.

View larger version (69K): [in a new window]   Fig. 6. mls-2 is specifically required for the M lineage expression of hlh-1. (A,B) A lin-39(n1760) mab-5(e1239) mutant animal (A) and a hlh-1(cc561ts) mutant animal (B) stained with anti-MLS-2 antibodies, showing mls-2 expression in M (arrow) and in six head neurons (arrowhead). (C) M lineage expression (arrow) of mab-5::gfp in a cc615 mutant animal. (D-F) A mls-2(cc615) ayIs6(hlh-8::gfp) mutant animal at the 4-M stage stained with anti-HLH-1 antibodies (red in D,E) and DAPI (F). Note that HLH-1 was present in the embryonic BWMs (white arrows in D-F), but not in the four M granddaughter cells (blue arrows in E-F).