LeBlanc-Straceski JM et al. (2009), Developmental expression of Xenopus myosin 1d a...

XB-ART-39615

Dev Growth Differ 2009 May 01;514:443-51. doi: 10.1111/j.1440-169X.2009.01107.x.

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Developmental expression of Xenopus myosin 1d and identification of a myo1d tail homology that overlaps TH1.

LeBlanc-Straceski JM , Sokac A , Bement W , Sobrado P , Lemoine L .

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Xenopus laevis myosin 1d (XlMyo1d) is a member of the myosin I class, subclass 4. Members of this class are single headed, bind calmodulin light chains and have lipid binding domains in their tails. The rat myo1d homologue has been implicated in endosome vesicle recycling in epithelial cells. Mutations in the Drosophila myosin 1d homologue cause situs inversus in the abdomen. The XlMyo1d cDNA has been cloned and the derived amino acid sequence is 80% identical to the rat and human homologues. Sequence comparison revealed a novel isoform-specific tail homology embedded in the Tail Homology 1 (TH1) domain characteristic of myosin I isoforms. Western blot analysis using a polyclonal antibody raised against an isoform-specific peptide showed that the protein is present in eggs and levels increase at early neurula through tadpole stages. Whole mount in situ hybridization using a probe containing the 5'UTR (untranslated region) showed that XlMyo1d mRNA is expressed in neural tube, pre-somitic mesoderm, somites and all three segments of cranial neural crest cells during their migration. Sections of the in situ hybridizations revealed that during somitogenesis, XlMyo1d mRNA was localized to a stripe overlapping the nuclear region of somites during early tadpole stages.

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Species referenced: Xenopus laevis
Genes referenced: actl6a cda myo1c myo1d nucb1

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	Fig. 1. Domain structure and amino acid sequence analysis of Xenopus laevis myosin 1d (XlMyo1d). (A) A comparison of the isoform-specific region between the invariant GESGAGKT and the very highly conserved EAFGNAKT sequences. Identical amino acids are highlighted. Sequences compared are myo1d isoforms from frog, XlMyo1d (accession number: AF540952); fruit fly, DmMyo31DF (accession number: S45573); zebrafish, DrMyo1d (accession number: CAK11490); rat RnMyo1d (originally identified as myr4) (accession number: X 71997); human, HsMYO1D, (accession number: BC030602); and a myo1c isoform, HsMYO1C (accession number: AAH44891). (B) The two IQ motifs previously reported for the myo1d subfamily were identified in XlMyo1d. Sequences of several species were compared with the IQ motif consensus sequence from the myosin I family (I/V/L)QxxxRGxxx- (R/K)xx(F/I/L/V/W/Y) described previously (BÃ¤hler and Rhoads 2002). Positions of conserved amino acids are designated by asterisks in the alignment, and amino acids found in the consensus sequence are shaded. (C) The TH1 of the myosin I family was compared with representative members of the myo1d subfamily using the ClustalW sequence comparison program. The top row shows the TH1 consensus sequence obtained from the CDD (Conserved Domain Database) of National Center for Biotechnology Information (NCBI). The second row shows a myo1d tail consensus sequence, which was obtained by comparing representative sequences from fly, fish, frog, and rat. Shaded amino acids are identical to the TH1 (top row). Bold amino acids are identical to the myo1d tail consensus.
	Fig. 2. Xenopus laevis myosin 1d (XlMyo1d) expression increases during early development. Protein samples were prepared from various stages of embryogenesis and analyzed by Western blot. One half-embryo equivalents were loaded into lanes of a 7.5% polyacrylamide gel. (A) The rabbit polyclonal antibody, Ab35, identified a protein product of the predicted molecular weight, for XlMyo1d, 116 kDa. (B) An anti-tubulin antibody was incubated with the stripped blot to control for gel loading.
	Fig. 3. Xenopus laevis myosin 1d (XlMyo1d) expression is detected in somites, neural tissue and neural crest. XlMyo1d mRNA expression was detected using whole mount in situ hybridization. (Aâ€“K) Embryos hybridized to the XlMyo1d probe. (A) Stage 12. Yolk plug stage. (B) Stage 20, dorsal view. (C) Stage 20, lateral view, showing mandibular, hyoid and branchial neural crest cells, anterior to posterior. (D) Stage 20.5 lateral view. (Eâ€“G) Stage 22. (E) Side view. (F) Dorsal view. (G) Lateral view, at higher magnification. The rotation of the cells during the formation of the somite in an anterior to posterior direction is detected. (Hâ€“K) Stage 30. (H) Dorsal, oblique view. (I) Lateral view. (J) Lateral view close-up of the head region. (K) Close up of the head, dorsal view. (L) Stage 26 embryo hybridized to XlMyo1d sense probe. (M) Stage 30 embryo hybridized to the actin probe. BA/CNC, branchial arch region and cranial neural crest segments; CG, cement gland; E, eye; H, heart; NT, neural tube; OV, otic vesicle; PSM, pre-somitic mesoderm; S, somite; YP, yolk plug.
	Fig. 4. Xenopus laevis myosin 1d (XlMyo1d) expression is detected in the nuclear region of somites and pre-somitic mesoderm. Horizontal, 10 Î¼M sections were taken from embryos that had undergone whole mount in situ hybridization to the XlMyo1d and actin probes. Head is left and tail is right. Since the dorsal side of the embryo is curved, only the anterior-most and posterior-most portions of the neural tube and notochord are present in the sections shown in (A) and (B). (A) Actin probe hybridization. (Bâ€“C) XlMyo1d probe. (C) Somite structure at a higher magnification than shown in (B). Each somite measures 70 Î¼M anterior to posterior and 90 Î¼m proximal to distal. NC, notochord; NT, neural tube; Nuc, nuclei of somite; PSM, presomitic mesoderm; S, somite.
	myo1d (myosin ID) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
	myo1d (myosin ID) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.