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First published online 15 September 2004
doi: 10.1242/dev.01367

Development 131, 5009-5019 (2004)
Published by The Company of Biologists 2004
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An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Christelle Adolphe1, Monica Narang1,*, Tammy Ellis1, Carol Wicking1, Pritinder Kaur2 and Brandon Wainwright1,{dagger}

1 Institute for Molecular Bioscience, and Special Research Centre for Functional and Applied Genomics, University of Queensland, and the Cooperative Research Centre for the Discovery of Genes for Common Human Diseases, Victoria, Australia
2 Epithelial Stem Cell Biology Laboratory, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia



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  		Fig. 1. Range of developmental phenotypes resulting from ectopic hedgehog signalling activity. Shh, Dhh and Ihh overexpression gave rise to Regular transgenic embryos that present with skin indistinguishable from wild-type epidermis (A), and mild limb phenotypes such as a post-axial nubbin (B, arrowhead) and/or digit duplication (C, arrowhead; D, alcian blue staining of digit duplication). A subset of Shh and Dhh transgenics presented with a Wrinkled skin phenotype which presented with grossly wrinkled skin (E,F) and blisters over regions of exposed brain (G, arrow) and spinal cord (G, star), pre-axial polydactyly (H-K), complete inter-digital soft tissue webbing (H, arrows), excessive soft-tissue (J, arrow) and calcification at the distal tip (K, arrowhead). Shh and Dhh overexpression also gave rise to Translucent embryos [displayed severe developmental defects (L,M)] and Translucent limbs often lacked proximal distal growth, presenting as limb buds (N,O, arrowheads).

 


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  		Fig. 3. Histological analysis of hedgehog-induced skin lesions. (A) Wild-type skin is opaque in colour and (D) presents with regularly spaced skin folds and hair follicles. Wrinkled transgenic skin on the other hand presents with excessive skin folds (B) and is characterised by epidermal hyperplasia (E). (C) Translucent transgenic skin is macroscopically thin and fragile (arrowheads) with regions of opaque blisters (arrows) and (F) presents as an epidermal bilayer that lacks any HF or sebaceous structures. (G-K) Basal cell lesions were evident from E17.5 in both Wrinkled (G-I) and Translucent epidermis (J,K). Lesions displayed clefting (G,I,K, arrows) and palisading cells, characteristic of human BCCs. (L-M) Gross morphology of wild-type (L) and Wrinkled (M,N) skin grafts two months post-graft. (M) Grafts from Wrinkled embryonic skin displayed thick tumour masses and ulceration was often evident (arrow). (P) Histological analysis of Wrinkled graft skin identified epidermal hyperplasia, sebaceous hyperplasia, and (O,T,Q-S) basaloid growths such as basaloid follicular harmatomas (BFHs) (T) and regions resembling micronodular (O,R, arrows) and sclerosing BCCs (Q,S).

 


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  		Fig. 2. Shh and Dhh overexpression results in epidermal basal cell anomalies. (A,D) Wild-type skin is characterised by a single-cell layer of basal cells. (B,E) Wrinkled transgenic skin on the other hand presents with an increase in the number of basal cell layers, (H,N) the epidermis exists in a hyperproliferative state and (K) exhibits a delay in terminal differentiation. (C,F) Translucent skin lacks a substantial amount of basal cells and (L) presents as a bilayer of differentiated cells. (O) Few proliferative cells are observed within the basal layer of Translucent skin (arrows), with proliferation mainly restricted to basaloid lesion invaginating into the dermis (unbroken lines).

 


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  		Fig. 4. Hedgehog pathway activity is perturbed in embryonic Wrinkled and Translucent skin phenotypes. (A) Shh expression is evident in both the basal and suprabasal layers of E18.5 Wrinkled epidermis. (B) Although Ptc1 expression is evident in stratified epithelia of Wrinkled skin, endogenous Ptc1 expression is completely ablated within the basal cell compartment (arrow). (D,E) High levels of Shh (D) and Ptc1 (E) expression are present within the PMD cells and basaloid lesions of E18.5 Translucent epidermis. We also observed high levels of Gli1 within the basaloid lesions of Wrinkled (D) and Translucent (J) skin.

 


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  		Fig. 5. Analysis of p63 expression in E18.5 Shh mouse models. (A) Within wild-type epidermis, p63 is expressed within a subset of epidermal basal cells. (B) Loss of Shh activity (Shh null embryos) does not alter the expression of p63 within the basal cell compartment. (C) Wrinkled epidermis displays a marked increase in the number of epidermal cells expressing p63 whereas (D) Translucent skin displays a complete ablation of p63 expression.

 


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  		Fig. 6. Shh pathway activity can increase keratinocyte growth potential. (A) E18.5 wild-type epidermis contains an average of 41 keratinocyte colonies per mouse whereas Wrinkled epidermis contains over 150 keratinocyte colonies per mouse. (B,C) A typical representation of wild-type colony density (B) and Wrinkled colony density (C). Translucent keratinocytes lacked colony-forming efficiency.

 


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  		Fig. 7. The level of hedgehog expression increases with the severity of the skin phenotype. (A-C) RNA in situ analysis of transgene expression showed an increase in transgene expression from Regular (A), to Wrinkled (B) and to Taut (C). (D) Detection of transgene expression within Translucent skin was hindered by the decrease in basal cells. (E) Genomic PCR analyses indicate that the relative level of transgene copy number is Translucent > Wrinkled > Regular. This suggests that differences between transgenic phenotypes may be related to the level of transgene copy number, and an increase in embryonic phenotype severity correlates with a significant increase in copy number.

 


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  		Fig. 8. Ihh overexpression perturbs epidermal hedgehog signalling activity. In response to high levels of Ihh transgene expression (A) and ectopic Ihh protein within the basal cells of mouse skin (B), we observed high levels of Shh (C) and Ptc1 (D) expression in both the basal and suprabasal layers of E18.5 Ihh transgenic epidermis.

 

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© The Company of Biologists Ltd 2004