Ori M et al. (2006), XHas2 activity is required during somitogenesis...

XB-ART-814

Development 2006 Feb 01;1334:631-40. doi: 10.1242/dev.02225.

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XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development.

Ori M , Nardini M , Casini P , Perris R , Nardi I .

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In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus, morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA, following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2, loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.

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Species referenced: Xenopus laevis
Genes referenced: actc1 cd44 gal.2 grem1 has1 has2 has3 myod1 pax3 slc12a3 tbxt znrd2
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	Fig. 1. XHas2 is transcriptionally activated by activin. (A,A') Ectopic XHas2 mRNA expression in activin-injected embryos detected by whole-mount in situ hybridization. The site of injection is visualized by the Red-gal staining (red arrow). A, dorsal view; A′, lateral view. (B) Comparison of the expression profile of XBrachyury and XHas2 in animal caps following activin treatment. XODC was used as positive control. RT-indicates representative samples of RNA from activin-treated animal caps, processed without reverse transcriptase and subsequently used as a negative control for XHas2, XBrachyury and XODC amplification.
	Fig. 2. Myotome alterations in XHas2-Mo injected embryos. All embryos are at stage 28-30 and are visualized by the expression of CA (A,A′) and by 12/101 immunoreactivity (B,B',C,D). Red-gal staining identifies the injected side of the embryos. (A) Bending of the embryos was observed in the injected side. (A') Example of a mild class phenotype embryo; the myotome arrangement is altered in the injected side of the embryo (arrow). (B) Lateral view of the control side of a representative case of a severe class phenotype at stage 26 (arrowheads indicate the somites). (B') Injected side of the embryo in B showing a complete disruption of somites segmentation, as indicated by the arrowheads and a strong reduction in the 12/101-positive cells (compare double-headed arrows in B and B′). Red spots on the head and trunk region (Red-gal staining) identify the site of injection. (C-D') Two representative cases of severe class phenotype embryos, longitudinally sectioned and stained with Hoechst to visualize the nuclei (C',D'). (C,D) The pattern of the 12/101 positive myocytes is altered within the somites in the injected side (arrows).
	Fig. 3. Control experiments: stage 30 embryos were analyzed for CA expression and HA distribution. (A) A representative embryo injected with a control morpholino. (B-D) Embryos injected with 15 ng of XHas2-Mo1 or with 15 ng of XHas2-Mo1 plus 800 pg XHas2 mRNA (rescue experiment). The injected side of the embryo is visualized by Red-gal staining. (B) Uninjected side of a XHas2-Mo1-injected embryo and (C) injected side of the same embryo showing the altered somites structure. (D) A representative case of a rescue experiment in which the canonical somites structure is completely recovered. (E) Ventral view of a wild-type embryo showing normal medial fusion of the two halves of the heart anlage. (F) In XHas2-Mo-injected embryos, heart formation is clearly impaired at the level of the injected side (arrow). (G) Coronal section of stage 26 XHas2-Mo-injected embryos at the trunk level and double stained with neurocan-GFP fusion protein (green) and Hoechst (blue). (H) High magnification of the XHas2-Mo-injected side region indicated by the white square in G; no HA detection is visible in the ECM surrounding the myocytes. (I) High magnification of the control side region indicated by the white square in G showing abundant HA in the ECM filling the myocytes extracellular spaces. n, notochord; nt, neural tube.
	Fig. 4. XHas2 loss-of-function phenotype during early myogenesis. Dorsal view of XHas2-Mo injected embryos at neurula stage analyzed for XmyoD (A), CA (B,B',D) and p27 (C) expression by whole-mount in situ hybridization. Notably, p27 expression appeared unaltered in primary neurons (arrow). (B′) Bisection of embryo shown in B. (D) Bisected XHas2-Mo injected embryo at stage 22 showing a reduced somite mass in the injected side. (E) Injected neurula stage embryo stained with PH3 antibody. (F) Injected embryo analyzed by a BrdU incorporation assay (light blue, X-gal staining). (G) Whole mount TUNEL staining in XHas2-Mo injected neurula stage embryo. (G′) Bisected neurula embryo showing the increased number of apoptotic cells in the presomitic mesoderm of the injected side (arrowheads). (H-I′) Lateral view at the level of trunk somitic region of XHas2-Mo injected embryos processed by TUNEL assay. (H,I) Control side of stage 24 (H) and stage 30 (I) embryos. (H′,I′) Injected side of the embryos shown in H and I. No apoptotic cells were found at these stages in both control and injected side of the embryos. (I′) In stage 30 embryos, physiological level of apoptosis was found in the telencephalon (arrow).
	Fig. 5. XCD44 gene expression pattern. (A) XCD44 expression during Xenopus development analyzed by RT-PCR. (B) Dorsal view of a stage 20 embryo showing XCD44 mRNA localization in the presomitic mesoderm. (C) Lateral view of a stage 24 embryo: XCD44 gene expression is detected in somites, cement gland (arrowhead) and, at lower level, in the cranial NCC (arrows). (C') Bisected embryo at stage 24 showing that in the trunk region XCD44 is localized exclusively in the somites. The notochord (n) at this stage does not express XCD44 mRNA. (D) Lateral view of stage 32 embryo: XCD44 mRNA is present in somites, branchial arches (arrow) and in the otic vesicle (arrowhead). A horizontal section at the level of the otic region is shown in the inset. (E) Lateral view of a stage 37 embryo showing the localization of XCD44 transcripts in the CNS (arrow), the dorsal somite tips (black arrowhead), the notochord and migrating hypaxial muscle cells (red arrowheads). (F-H) Dorsal view of XCD44-Mo injected embryos at neurula stage, the injected side of the embryos is visualized by the Red-gal staining. The expression of the following markers was detected by whole mount in situ hybridization: (F) MyoD, (G) p27 and (H) CA. (I) An example of a XCD44-Mo injected embryo processed by TUNEL assay. The apoptotic cells are in blue; red spots indicate the site of injection.
	Fig. 6. Analysis of hypaxial cell migration in XHas2-Mo and XCD44-Mo injected embryos. (A,A′) Lateral view of control (A) and injected (A′) side of an XHas2-Mo injected embryo (mild phenotype) at stage 37 showing the relative position of differentiating hypaxial muscle cells, as highlighted by 12/101 antibody staining (arrows). (B,B') Transversally sectioned XHas2-Mo injected embryo hybridized with CA and coloured with Hoechst to visualize the nuclei, in which it is evident that the somite structure is altered and the ventrolateral migration of hypaxial muscle cells is impaired (red arrow). (C) Lateral view of the control side of a XHas2-Mo-injected embryo analyzed for XPax3 expression by in situ hybridization at stage 37 highlighting hypaxial muscle cells migrating ventrally (arrow). (C′) Injected side of the embryo shown in C, in which hypaxial muscle cells are blocked at the ventral aspect of somites (red arrow). (D,D′) Stage 37 XCD44-Mo injected embryos immunostained with the 12/101 antibody. Although the trunk musculature appears unaffected by XCD44 loss, migration of hypaxial cells is greatly reduced. (D) Control side of the embryo; (D') injected side. (E-G) Ventral views of stage 43 embryos immunostained with the 12/101 antibody that show the final position of the ventral body wall musculature in uninjected (E), XCD44-Mo injected embryo (F) and XHas2-Mo injected embryo (G). The side of injection is visualized by X-gal staining in blue and indicated by the arrow. (E′-G′) Lateral views of the same embryos shown in E-G, respectively.
	Fig. 7. Stage 32 XHas2-Mo and XCD44-Mo injected embryos analyzed for XGremlin expression by whole-mount in situ hybridization. (A) Lateral view of the control side of two embryos, showing NCC migration pathways. (A′) Lateral view of the injected side, revealed by Red-gal staining, of the embryos shown in A: no defined NCC migration pathways are recognizable. (B) Higher magnification of one of the embryo showed in A. (B′) Injected side of the embryo shown in B; NCCs are still present but they do not follow their normal migration routes. (C) Longitudinal section of an XHas2-Mo injected embryo: trunk NCCs appear dispersed along the somite external aspect (injected side, red arrowheads) instead of migrating along the intersomitic boundaries (control side, yellow arrowheads). (D,D′) Double-labelling of stage 32 embryo for XGremlin (blue) and 12/101 muscle-specific antigen (orange), whereas red staining reveals the injected side of the embryo. From the comparison of the control (D) and the injected sides (D'), it seems that the impairment of trunk NCC migration parallels the reduction and developmental alterations of somites. (E,E') Stage 32 XCD44-Mo injected embryos analyzed for XGremlin expression. No differences in NCC migration are discernible between the control side (E) and the injected side (E′) of the embryo.
	cd44 (CD44 molecule (Indian blood group) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior right.
	cd44 (CD44 molecule (Indian blood group) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view dorsal up, anterior right.
	cd44 (CD44 molecule (Indian blood group) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, mid-trunk region, transverse section, dorsal up.
	cd44 (CD44 molecule (Indian blood group) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view dorsal up, anterior right. Insert: A horizontal section at the level of the otic region is shown in the inset.