Gersch RP et al. (2012), Mustn1 is essential for craniofacial chondrogen...

XB-ART-44759

Gene Expr Patterns 2012 Jan 01;123-4:145-53. doi: 10.1016/j.gep.2012.01.002.

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Mustn1 is essential for craniofacial chondrogenesis during Xenopus development.

Gersch RP , Kirmizitas A , Sobkow L , Sorrentino G , Thomsen GH , Hadjiargyrou M .

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Mustn1 is a vertebrate-specific protein that, in vitro, was showed to be essential for prechondrocyte function and thus it has the potential to regulate chondrogenesis during embryonic development. We use Xenopus laevis as a model to examine Mustn1 involvement in chondrogenesis. Previous work suggests that Mustn1 is necessary but not sufficient for chondrogenic proliferation and differentiation, as well as myogenic differentiation in vitro. Mustn1 was quantified and localized in developing Xenopus embryos using RT-PCR and whole mount in situ hybridization. Xenopus embryos were injected with either control morpholinos (Co-MO) or one designed against Mustn1 (Mustn1-MO) at the four cell stage. Embryos were scored for morphological defects and Sox9 was visualized via in situ hybridization. Finally, Mustn1-MO-injected embryos were co-injected with Mustn1-MO resistant mRNA to confirm the specificity of the observed phenotype. Mustn1 is expressed from the mid-neurula stage to the swimming tadpole stages, predominantly in anterior structures including the pharyngeal arches and associated craniofacial tissues, and the developing somites. Targeted knockdown of Mustn1 in craniofacial and dorsal axial tissues resulted in phenotypes characterized by small or absent eye(s), a shortened body axis, and tail kinks. Further, Mustn1 knockdown reduced cranial Sox9 mRNA expression and resulted in the loss of differentiated cartilaginous head structures (e.g. ceratohyal and pharyngeal arches). Reintroduction of MO-resistant Mustn1 mRNA rescued these effects. We conclude that Mustn1 is necessary for normal craniofacial cartilage development in vivo, although the exact molecular mechanism remains unknown.

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Species referenced: Xenopus laevis
Genes referenced: mustn1 sox9
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	Fig. 2. Mustn1 is differentially expressed both temporally and spatially during X. laevis development. Agarose gel results of RT-PCR show an increase and plateau of Mustn1 expression during later neurulation (A). Mustn1 expression was further elucidated via RT-PCR on Stage 20 (B) and Stage 23 (C) embryos respectively. Total RNA was isolated from these tissues and RT-PCR was used to visualize the relative expression levels of Mustn1 compared to Whole Body samples. Expression of the metabolic gene Ornithine Decarboxylase (ODC) was used to normalize for RNA recovery, cDNA synthesis and gel loading. Mustn1 expression was visualized in wild-type embryos and analyzed via anti-sense in situ hybridization during development. Dorsal view of representative antisense stained St. 20 embryo (D) and sense control (H) respectively. Lateral view of an antisense stained embryo at St. 25 (E), and St. 39 (F). Lateral view of a sense stained embryo at St. 23 (I), and St. 39 (J). Panel G shows a magnified view of the Mustn1-antisense stained stage 39 tadpole head shown in panel F. In panels A, B and C, WB denotes RNA derived from the whole body of the embryo while H denotes tissues dissected from Head, D denotes Dorsal tissues, V denotes Ventral tissues, and P denotes Posterior regions as indicated in Nieuwkoop and Faber diagrams Nieuwkoop and Faber, 1967 with dissection planes indicated by dotted lines. In panels D–F, Black arrows denote paraxial mesoderm and somites, and in D–F, and G the black arrowhead denote craniofacial structures (M, mandibular arch; H, hyoid arch; B, branchial arch), the white arrowhead denotes heart (He in panel G), and the white arrow indicates the otic vesicle. All scale bars = 1 mm.
	Fig. 3. Phenotype of injected embryos during embryogenesis. Embryos were injected with morpholinos (35 ng/embryo) dorsal to the midline at the 4-cell stage. Lateral views comparing Co-MO-injected (A), Mustn1-MO-injected (B), and Rescue-injected (C) embryos at stage 37–38. The Mustn1-MO-injected embryos have smaller eyes, truncated body axes and/or tail kinks. When the same analysis was completed at stage 40; Co-MO-Injected tadpoles appear normal (D), while Mustn1-MO-injected embryos show severe defects – the eyes shrink or disappear and the body axis length is also reduced along with curvature of the tail (E). Rescue-injected embryos show reduced severity of these defects (F). All micrographs were taken at the same magnification.
	Fig. 4. Quantification of phenotypes in injected embryos during embryogenesis. Quantification of eye phenotypes in injected embryos, scored at stage 40 (A). Labels correspond to each phenotypic category, for Co-MO-injected, Mustn1-MO-injected, and Rescue-injected embryos. Embryos were categorized as having two normal eyes, two small eyes, one eye, or no eyes. Analysis of body axis length (B); embryos were categorized as having either a normal or shortened body axis. Analysis of Tail Kink phenotype (C); embryos were categorized as having a normal, mild, or severe tail curvature (see Experimental Design for details).
	Fig. 5. Effects of Mustn1 depletion on Sox9. Lateral views of embryos subjected to in situ whole mount hybridization to detect Sox9 expression. These embryos were from among those scored for phenotypes in Fig. 3 and Fig. 4. High magnification micrographs were taken of lateral views of the head illustrating Sox9 expression at stage 40 in a Co-MO-injected embryo (A), a Mustn1-MO-injected embryo (B) and a Rescue-injected embryo (C). qPCR analysis of Sox9 expression in dorsal tissue dissections of Co-MO-, Mustn1-MO-, and Rescue-injected embryos assessed at stages 25, 34, 39 (graph). No significant difference in Mustn1 mRNA expression was found between Co-MO-injected and Rescue-injected groups at any developmental stage, but a significant difference between Co-MO-injected and Mustn1-MO-injected embryos was detected at stages 34 and 39. All bars represent average values of pooled mRNA (n = 5 embryos). Error bars indicate standard deviation of PCR runs (n = 3). Significance was determined by Mann–Whitney test vs. Co-MO-injected levels, ∗p < .05. Black arrows indicate staining in cranial cartilage, white arrows indicate pharyngeal arch staining, and black arrowheads denote staining of the anterior structures such as mandibular and nasal processes. Scale bar = 250 μm.
	Fig. 6. Effect of Mustn1 knockdown on cartilage development. Ventral views of unilaterally injected embryos (stage 45) following Alcian blue staining. Co-MO-injected embryo, with a dotted line indicating the midline (A). The same embryo shown in A after the removal of adherent tissue (B). The same analysis of a representative Mustn1-MO-injected embryo shows alteration of midline and loss of symmetrical cartilaginous structures on the contralateral, injected side (C and D, respectively). Black arrows indicate the ceratohyal cartilage; and white arrows denote the gill (branchial) arches. Scale bar = 0.5 mm
	mustn1 (musculoskeletal, embryonic nuclear protein 1) gene expression in Xenopus laevis embryos, NF stage 20, as assayed by in situ hybridization, dorsal view, anterior down.
	mustn1 (musculoskeletal, embryonic nuclear protein 1) gene expression in Xenopus laevis embryos, NF stage 25, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.
	mustn1 (musculoskeletal, embryonic nuclear protein 1) gene expression in Xenopus laevis embryos, NF stage 39, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.

References [+] :

Alfandari, Mechanism of Xenopus cranial neural crest cell migration. 2010, Pubmed, Xenbase