Casini P et al. (2012), Hyaluronan is required for cranial neural crest...

XB-ART-45671

Dev Dyn 2012 Feb 01;2412:294-302. doi: 10.1002/dvdy.23715.

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Hyaluronan is required for cranial neural crest cells migration and craniofacial development.

Casini P , Nardi I , Ori M .

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Hyaluronan is a crucial glycosaminoglycan of the vertebrate embryonic extracellular matrix able to influence cell behaviour, both by assembling the pericellular matrices and by activating signal transducing receptors such as CD44.We showed that the hyaluronan synthases, Has1 and Has2, and CD44 display a dynamic expression pattern during cranial neural crest cells (NCC) development. By knocking down Has1 and Has2 gene functions, we revealed that hyaluronan synthesized by Has1 and Has2 is necessary for the proper development of the visceral skeleton.The data suggest that hyaluronan helps to maintain the active migratory behaviour of cranial NCC, and that its presence around pre-chondrogenic NCC is crucial for their survival. CD44 knock down also suggests that the role of hyaluronan in cranial NCC migration could be mediated, at least in part, by the activation of CD44. These findings contribute to the unveiling of the functional relation between NCC and their extracellular environment during craniofacial development.

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Species referenced: Xenopus laevis
Genes referenced: cd44 dll4 gal.2 has1 has2 mok slc12a3 snai2
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	Figure 1. Expression profile of Has1, Has2, and CD44 mRNAs during cranial neural crest development. A,A′: Lateral view (A) and a vibratome horizontal section (A′) across the broken line in A of a stage-25 embryo showing Has1 expression in NCC migrating into the four branchial arches. B,B′: Lateral view (B) and a vibratome sagittal section (B′) of a stage-33 embryo showing Has1 expression in NCC migrated into the four branchial arches. C: Has2 is expressed in the branchial region of a stage-25 embryo. C′: The vibratome horizontal section across the broken line in C showing that Has2 is present in the endodermal layer of the branchial pouches as indicated by the arrowhead for the hyoid arch. D: At stage 33 Has2 mRNA is still present in the branchial region. D′: The vibratome horizontal section across the broken line in D highlights that the expression of Has2 is switched off in the endoderm, while it appears in the neural cresterived component of the branchial arches, as indicated for the third arch by the arrow. E: Magnification of a vibratome horizontal section of a stage-41 embryo showing Has2 expression in differentiating chondroblasts. F: The lateral view of a stage-25 embryo shows CD44 expression in the four cranial NCC migratory streams. G: The lateral view of a stage-33 embryo showing CD44 expression in NCC migrated into the four branchial arches. H,I: Drawings showing a comparative expression profile of Has1, Has2, and CD44 mRNA expression. I-II-III-IV, branchial arches; e, eye; h, heart; ov, otic vesicle; s, somites; M, Meckel's cartilage; Q, quadrate cartilage; C, ceratohyal cartilage; G, gill cartilages.
	Figure 2. Hyaluronan distribution in branchial arches. A: Horizontal section of a stage-33 embryo. The biotinilated Hyaluronan Binding Complex (bHABC) staining detects hyaluronan, in red, in the extracellular matrix around post-migrated NCC. A′: The in situ hybridization with the skeletogenic NCC marker Dll4. Sections were made across the broken line drawn in the scheme of a stage-33 Xenopus tadpole, modified from Nieuwkoop and Faber (1956). I-II-III-IV, branchial arches.
	Figure 3. Cranial neural crest cells migration in Has1, Has2, and CD44 morphants. The red-gal staining identifies the injected side of each embryo. A, C, E: Dorsal view of stage-20 embryos showing the delayed onset of the cranial NCC migration process, visualized by Slug expression in Has1 (A), Has2(C), and CD44 (E) morphants. B, D, F: Lateral views of the control side of Has1 (B), Has2 (D), and CD44 (F) morphants analyzed for Dll4 expression at stage 25 to identify migrating NCC. B′, D′, F′: Injected sides of the embryos shown in B, D, and F, respectively, in which NCC migration is delayed. B″, D″, F″: Front-ventral view of embryos shown in B, D, and F, respectively; the arrows indicate the hyoid migratory streams. I-II-III-IV, branchial arches.
	Figure 4. Increased apoptosis in the branchial region of stage-30 Has1 and Has2 morphants. A, D, G: Lateral views of the control side of Has1 (A), Has2 (D), and CD44 (G) morphants analyzed at stage 30 for Dll4 expression. A′, D′, G′: Injected sides of embryos in A, D, and G showing a significant reduction of post-migrated NCC streams size. B, B′, E, E′, H, H′: Lateral views of the control and injected side of stage-28 Has1, Has2, and CD44 morphants processed for TUNEL assay to highlight apoptotic cell death. C, F, I: Vibratome horizontal sections at the branchial level of Has1, Has2, and CD44 down-regulated embryos processed for TUNEL assay. Increased apoptotic signal in the branchial arches of the injected side is indicated by the arrows (C, F). L: Quantitative evaluation of apoptosis by measurement of the number of TUNEL signal-positive cells within the pharyngeal region of CD44, Has1, and Has2 down-regulated embryos. In the histogram, the average numbers of TUNEL-positive cells in Has1-Mo- and Has2-Mo-injected sides are, respectively, approximately 5-fold and 4-fold higher compared with the wt side. Statistically, no significant difference in the number of TUNEL-positive cells was found between the wt-side and CD44-Mo-injected side. Vibratome sections of the pharyngeal region of three embryos for each down regulation have been analysed. The comparisons were performed using the Student's t-test and the results presented as means SEM. P < 0.05 was considered to be statistically significant (*). I-II-III-IV, branchial arches; e, eye.
	Figure 5. Skeletal alterations in Has1, Has2, and CD44 morphants. Stage-49 flat-mounted embryos processed for Alcian blue staining. All specimens are shown in a ventral view with the anterior to the top. A, C, E: Visceral skeleton of Has1 (A), Has2 (C), and CD44 (E) morphants showing a mild alteration of the skeletal elements derived from the four NCC migratory streams: the Meckel's, palatoquadrate, ceratohyal, and gill cartilages appear reduced in size in the injected side (arrows). B, D: Visceral skeleton of Has1 (B) and Has2 (D) morphants showing a severe alteration: in the injected side the gill cartilages are strongly reduced (arrows), while the other skeletal elements failed to develop. F: Scheme of a wild type visceral skeleton structure, modified from Sadaghiani and Thiebaud (1987). G: The percentages of mild and severe affected skeletons in the injected embryos. M, Meckel's cartilage; Q, palatoquadrate cartilage; C, ceratohyal cartilage; G, gill cartilages; Bh, basihyal cartilage.
	Supp. Fig. S1. Molecular and morphological analysis of embryos injected with Has1, Has2, and CD44 control morpholinos. The injected side of each embryo is identified by the red-gal staining. A': Cranial NCC migration occurred normally in embryos injected with Has1-MoK, Has2-MoK, and CD44-MoK as revealed by the gene expression of Slug and Dll4. G, G', I, I', M, M': In embryos injected with Has1-MoK, Has2-MoK, and CD44-MoK TUNEL-positive cells are detected within the brain and the eye but never in the branchial arches region. H, L, N: The NCC-derived visceral skeleton developed correctly in Has1-MoK, Has2-MoK, and CD44-MoK embryos. e, eye; cg, cement gland.
	Supp. Fig. S2. Control experiments: hyaluronan distribution in branchial arches of Has1 and Has2 stage-33 morphants. A''', B''': Cryostat horizontal sections at the branchial level of Has1 and Has2 morphants, showing the Hoechst staining to visualize nuclei (A, B), the GFP fluorescence to identify the injected side (A', B'), the biotynilated Hyaluronan Binding Complex (bHABC) staining to detect hyaluronan (A'', B''), and the merge (A''', B'''). In the injected side of morphants, a decreased amount of hyaluronan is present in the branchial extracellular spaces with respect to the uninjected counterpart. I-II-III-IV, branchial arches.