XB-ART-19495Nucleic Acids Res July 11, 1995; 23 (13): 2555-62.
Xenopus sonic hedgehog as a potential morphogen during embryogenesis and thyroid hormone-dependent metamorphosis.
The hedgehog family of proteins have been implicated as important signaling molecules in establishing cell positional information and tissue patterning. Here we present the cloning and characterization of a hedgehog homologue from Xenopus laevis similar to the sonic class of vertebrate hedgehog genes. We isolated Xenopus hedgehog (Xhh) from a subtractive hybridization screen designed to identify genes induced by thyroid hormone during metamorphosis of the X.laevis gastrointestinal tract. In the intestine, Xhh mRNA expression was up-regulated at the climax of metamorphosis (stage 62) when intestinal epithelium underwent morphogenesis. Treatment of pre-metamorphic tadpoles with exogenous thyroid hormone (TH) resulted in a similar pattern of Xhh induction. Furthermore, TH induction was resistant to inhibitors of protein synthesis suggesting that Xhh is a direct thyroid hormone response gene. The expression and TH regulation of Xhh was not limited to the intestine, but was also observed in the limb and a mixture of pancreas and stomach. Throughout development, Xhh mRNA was present at varying levels with the earliest expression being detected at neurula stage. The highest levels of Xhh were observed between stages 33 and 40 shortly before tadpole feeding begins. Whole mount in situ hybridization analysis of Xhh expression in pre-hatching, stage 32 tadpoles demonstrated staining in the notochord and floor plate similar to that observed for other vertebrate hedgehog genes. Together, these data suggest a putative role for Xhh in organ development during both amphibian embryogenesis and metamorphosis.
PubMed ID: 7630736
PMC ID: PMC307066
Article link: Nucleic Acids Res
Genes referenced: dhh ihh rpl8 shh thra
Article Images: [+] show captions
|Figure 1. Schematic of intestinal changes in X.laevis during metamorphosis. The larval (tadpole) intestine is comprised of a single layer of fully differentiated epithelial cells (55,56). There is a single fold in the intestine called the typhlosole. During metamorphosis extensive remodeling occurs to create the adult frog intestine (9,10). The first stage of this process is the death of larval epithelial cells followed by adult epithelial cell proliferation and differentiation. In addition, there is an increase in the connective and muscle tissue. By the end of metamorphosis, the adult frog intestine consists of multiple epithelial folds, highly resembling the intestines of other vertebrates. bb, brush border of EP. EP, epithelial cells; CT, connective tissue.|
|Figure 2. Comparison of Xenopus, mouse and chicken hedgehog sequences. Sequence comparison at the amino acid level is displayed for Xenopus hedgehog (Xhh), mouse sonic hedgehog (M-Shh) (17), chicken sonic hedgehog (C-Shh) (20), mouse Indian hedgehog (M-Lhh) (17) and mouse Desert hedgehog (M-Dhh) (17). Blanks in the sequence indicate identical amino acids as compared with Xhh. Gaps represented by dots were introduced to allow for the best alignment. Xhh is 71% identical to M-Shh, 73% to C-Shh, 60% to M-Ihh and 54% to M-Dhh. The termination codons are marked by asterisks (*). The predicted site of signal peptide cleavage is marked with an arrow (57). The triple repeat amino acid sequence unique to Xhh is underlined.|
|Figure 3. In vitro translation of Xhh cDNA. Full length Xhh cDNA (XHH) or full length Xhh cDNA fused to a T7 gene 10 tag (T7-XHH) were translated in reticulocyte lysate using radiolabeled methionine. In vitro translation of reticulocyte lysate alone is indicated by (-). Protein products were displayed by SDS-PAGE and visualized by autoradiography. Protein markers are indicated on the left. Note that the shorter polypeptide was most likely derived from the C-terminus as it had the same size from the intact and tagged Xhh clones.|
|Figure 4. Xhh is activated during intestinal metamorphosis. Intestinal RNA was isolated from the stages indicated and analyzed by RNA blot hybridization using Xhh cDNA as a probe. The filter was also hybridized with rpL8 which served as a loading control. 28S and 1 8S ribosomal markers are indicated on the left.|
|Figure 5. Xhh expression is also activated during TH induced intestinal metamorphosis. Stage 56 tadpoles were treated with 5 nM T3 for various times as indicated in days. RNA was isolated from the intestine for Northern blot analysis using either Xhh or rpL8 as a probe. 28S and 18S ribosomal markers are indicated on the left. It is interesting to note that in the absence ofexogenous TH it normally takes 8 days for stage 56 tadpoles to reach stage 60 when Xhh mRNA levels are upregulated (Fig. 4), 11 days to reach the peak levels of Xhh mRNA (stage 62), and 3 weeks to reach stage 66 when Xhh expression is repressed again.|
|Figure 6. Xhh is a directTH response gene. (A) Kinetics ofT3 induction. Stage 52-54 tadpoles were treated with 5 nM T3 for various times as indicated in hours. RNA was isolated from intestine for RNA blot hybridization using full length Xhh orrpL8 as a probe. (B)TH activation ofthe Xhh gene is independent of new protein synthesis. Protein synthesis inhibitors cyclohexamide and anisomycin (CHX) were added to stage 52-54 tadpoles 1 h before the addition of 50 nM T3. The treatment was continued for another 12 h before RNA was isolated from intestine for RNA blot analysis using Xhh or rpL8 as a probe. Ribosomal markers, 28S and 18S, are indicated on the left.|
|Figure 7. Tissue specific induction ofXhh by TH. Tadpoles at stage 52-54 were treated in the presence (+) or absence (-) of T3 for up to 24 h. After treatment, poly(A)+ RNA was isolated from brain, hindlimb (limb), tail and intestine; total RNA was isolated from a mixtre of pancreas and stomach (panc/stomach). RNA was copied into cDNA using reverse transcriptase, restricted to small fragments, ligated to a PCR-linker, and amplified by PCR. Southern blot hybridization was performed on these cDNAs using Xhh as the probe. Filters were also hybridized with rpL8 (see Materials and Methods) to assay for variation in loading and PCR amplification.|
|Figure 8. Xhh is highly expressed during embryogenesis. Total RNA was isolated from whole animal at various stages throughout development as indicated. This RNA was used for Northern blot hybridization with Xhh cDNA as the probe. 28S and 18S ribosomal markers are indicated on the right. An equal amount of RNA was present in each lane as confirmed by staining the membrane with methylene blue (43). Note that high levels ofXhh mRNA were present in the animal from neurula stages (stages 16/17) to the onset of tadpole feeding (stages 44/45).|
|Figure 9. In situ hybridization localized Xhh mRNA in the notochord and floor plate. In situ hybridization was performed on stage 32 X.1aevis embryos with a digoxygenin labeled Xhh cRNA and the expression of Xhh mRNA was analyzed after cross-sectioning the hybridized embryos. The most intense hybridization was detected in the notochord (N) and the neural floor plate (FP). The background signal below the notochord is likely due to non-specific binding by the yolk proteins present in early embryos.|