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. 2010 Mar 25:10:32.
doi: 10.1186/1471-213X-10-32.

Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo

Poongodi Geetha-Loganathan  1 , Suresh Nimmagadda, Bodo Christ, Ruijin Huang, Martin Scaal
Affiliations

Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo

Poongodi Geetha-Loganathan et al. BMC Dev Biol. .

Abstract

Background: Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis.

Results: In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II.

Conclusion: Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm.

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Figures

Figure 1
Figure 1
In situ hybridizations of chicken wing buds with probes against the chondrogenic markers Sox9 (A, B) and Coll2a (C, D), after removal of the dorsal ectoderm at HH-stage 20-21 and 24 or 48 h reincubation. On the left side is the control limb, the right limb has been manipulated. Arrowheads indicate the site of ectoderm removal. A Expression of Sox9 after 24 h. Expression is stronger on the operated side. a Transverse section of the control limb showing low Sox9 expression. a' Transverse section of the operated limb showing strong Sox9 expression, which is extending ectopically towards the dorsal surface of the limb. B Expression of Sox9 after 48 h. Expression is stronger on the operated side. b Transverse section of the control limb showing normal Sox9 expression. b' Transverse section of the operated limb showing enhanced Sox9 expression, which is extending ectopically towards the dorsal surface of the limb. C Expression of ColIIA after 24 h. Expression is stronger on the operated side. Dislocated fragments of gold foil are still visible. c Transverse section of the control limb showing low ColIIA expression. c' Transverse section of the operated limb showing strong ColIIA expression, which is extending ectopically towards the dorsal surface of the limb. D Expression of ColIIA after 48 h. Expression is stronger on the operated side. d Transverse section of the control limb showing normal ColIIA expression. d' Transverse section of the operated limb showing enhanced ColIIA expression, which is extending ectopically towards the dorsal surface of the limb. Scale bar 500 μm.
Figure 2
Figure 2
Alcian-blue staining of the cartilaginous skeleton of embryonic chicken wings. A-C Control wings. A1-C1 Wings after injection of Wnt6-CHO cells at HH-stage 20-21 and 4 days reincubation. Skeletal elements are shorter in length compared to the control wings. A1 to C1 illustrate increasingly severe defects depending on the quantity and spreading of injected cells, A1 being the mildest, C1 being the severest phenotype. A1 only the humerus is affected. B1 stylopod and zeugopod are affected, C1 the entire limb is affected. Scale bar 500 μm.
Figure 3
Figure 3
In situ hybridization of chicken wing buds with probes against chondrogenic markers Sox9 (A, B) and Coll2a (C, D), after injection of Wnt6-CHO cells at HH-stage 20-21 and 24 or 48 h reincubation. On the left side is the control limb, the right limb has been manipulated. Dotted line indicates the site of injection. A Expression of Sox9 after 24 h. Expression is severely reduced on the operated side. a Transverse section of the control limb showing normal Sox9 expression. a' Transverse section of the operated limb showing weak Sox9 expression. B Expression of Sox9 after 48 h. Expression is reduced on the operated side. b Transverse section of the control limb showing normal Sox9 expression. b' Transverse section of the operated limb showing reduced Sox9 expression. C Expression of ColIIA after 24 h. Expression is locally reduced on the operated side. c Transverse section of the control limb showing normal ColIIA expression. c' Transverse section of the operated limb showing weak ColIIA expression. D Expression of ColIIA after 48 h. Expression is reduced on the operated side. d Transverse section of the control limb showing normal ColIIA expression. d' Transverse section of the operated limb showing reduced ColIIA expression. Scale bar 500 μm.
Figure 4
Figure 4
Comparison of the expression domains of Sox9 and Wnt6 in the limb. Slightly oblique transverse sections of wing buds at HH-stage 24. A Sox9 expression is limited to the chondrogenic region, which is located in the center of the limb bud mesenchyme. B Wnt6 is expressed in the entire circumference of the limb bud ectoderm. Scale bar 500 μm
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References

    1. Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. J Cell Biochem. 2006;97:33–44. doi: 10.1002/jcb.20652. - DOI - PubMed
    1. Foster JW, Dominguez-Steglich MA, Guioli S, Kowk G, Weller PA, Stevanovic M, Weissenbach J, Mansour S, Young ID, Goodfellow PN. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature. 1994;372:525–30. doi: 10.1038/372525a0. - DOI - PubMed
    1. Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, Pasantes J, Bricarelli FD, Keutel J, Hustert E. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell. 1994;79:1111–20. doi: 10.1016/0092-8674(94)90041-8. - DOI - PubMed
    1. Marshall OJ, Harley VR. Molecular mechanisms of SOX9 action. Mol Genet Metab. 2000;71:455–62. doi: 10.1006/mgme.2000.3081. - DOI - PubMed
    1. Tickle C. Making digit patterns in the vertebrate limb. Nat Rev Mol Cell Biol. 2006;7:45–53. doi: 10.1038/nrm1830. - DOI - PubMed

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