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Dev Dyn
1998 Jul 01;2123:352-63. doi: 10.1002/(SICI)1097-0177(199807)212:3<352::AID-AJA3>3.0.CO;2-G.
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Hedgehog family member is expressed throughout regenerating and developing limbs.
Stark DR
,
Gates PB
,
Brockes JP
,
Ferretti P
.
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Members of the hedgehog family have been shown to play a key role in many developmental processes, including limb patterning and chondrogenesis. We have therefore investigated whether members of this family are also expressed during regeneration of the adult urodele limb and are regulated by retinoic acid (RA), since this derivative induces proximodistal duplications in regenerating limbs, and has been shown to regulate sonic hedgehog (shh) in the developing limbs of birds and mammals. We report here that a newt homologue of Xenopus banded hedgehog, called N-bhh, is uniformly expressed by mesenchymal blastemal cells from the initial stages of regeneration and is up-regulated by RA. In addition, we show that N-bhh is uniformly expressed in the early limb bud of the newt embryo. Since bhh has not been detected in developing limbs of higher vertebrates, its expression in developing and regenerating newt limbs may be related to the regenerative capability of urodeles.
Fig. 4. Localisation of N-bhh transcripts in the limb during regeneration and development by whole-mount in situ hybridization using the p740 probe. The alkaline phosphatase-conjugated anti-digoxigenin antibody was detected with BCIP/NBT substrate (blue). A: Vibratome section of a 15-day blastema showing N-bhh expression in the blastema (Bl). The inset shows the proximal region of a blastema and stump. Note that the wound epidermis (we) and stump tissue, in particular muscle (m), are largely negative; some reactivity, however, is observed around the subepidermal glands (arrowheads). B: Microtome section of a 15-day blastema with wound epidermis (we) negative and blastema (Bl) positive C: Microtome section of a stage 32 embryo showing that whereas some structures such as the limb bud (lb), balancers (b), which are adhesive organs functionally similar to the cement gland of anurans, and periderm (pe) express N-bhh, brain (br) and notochord (n) are clearly negative. The inset shows the tail of a whole mount tail stage embryo (arrowheads). D: Microtome section of a stage 32 embryo at high magnification showing staining of the limb bud (lb). Reactivity is also observed in the periderm (pe). Scale bars=50μm(A,D),and25μm(C);A and B are at the same magnification.
Fig. 5. Localisation of N-bhh transcripts in the newt embryo by whole mount in situ hybridisation with the p740 probe using AP-red as a substrate instead of BCIP/NBT. The reaction product of this substrate appears as a brown deposit on sections. Sections were counterstained with Mayer haematoxylin (blue). A: Lateral section of developing limb from a 3-digit stage embryo showing the absence of N-bhh transcripts in the developing cartilage (c). B: Oblique section through the intestine of the same embryo as A. N-bhh transcripts (brown) were detected in the epithelium of the intestine but not in the mesenchyme (m). Scale bars = 300 μm (A) and 75 μm (B).
