Lai CJ et al. (1995), Patterning of the neural ectoderm of Xenopus la...

XB-ART-19414

Development 1995 Aug 01;1218:2349-60.

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Patterning of the neural ectoderm of Xenopus laevis by the amino-terminal product of hedgehog autoproteolytic cleavage.

Lai CJ , Ekker SC , Beachy PA , Moon RT .

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The patterns of embryonic expression and the activities of Xenopus members of the hedgehog gene family are suggestive of role in neural induction and patterning. We report that these hedgehog polypeptides undergo autoproteolytic cleavage. Injection into embryos of mRNAs encoding Xenopus banded-hedgehog (X-bhh) or the amino-terminal domain (N) demonstrates that the direct inductive activities of X-bhh are encoded by N. In addition, both N and X-bhh pattern neural tissue by elevating expression of anterior neural genes. Unexpectedly, an internal deletion of X-bhh (delta N-C) was found to block the activity of X-bhh and N in explants and to reduce dorsoanterior structures in embryos. As elevated hedgehog activity increases the expression of anterior neural genes, and as delta N-C reduces dorsoanterior structures, these complementary data support a role for hedgehog in neural induction and anteroposterior patterning.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actl6a ag1 dhh gsc hesx1 ihh nog nrp1 otx2 prl.1 shh tbxt
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	Fig. 1. Autoproteolytic cleavage of Xenopus hh proteins in vitro and in embryos. (A) Percentage of amino acid identity between the predicted N and C domains of the Xenopus hh gene family members (Ekker et al., 1995). (B) Depiction of constructs encoding full-length X-bhh, N, DN-C, and UHA. The polypeptides predicted to be formed in vivo after translation and cleavage of the signal sequence, and the autoproteolytic cleavage of the full-length polypeptide, are shown to the right, and are described in Materials and methods. SS denotes the signal sequence, U refers to the unprocessed polypeptide after cleavage of the signal sequence, N depicts the amino-terminal region after signal sequence cleavage and after autoproteolysis (at the site indicated by the downward arrowhead), and C denotes the carboxyterminal domain after autoproteolysis. The filled box in C denotes a histidine at position 270, and the box with a check denotes mutation of histidine 270 to an alanine. (C) Processing of X-bhh (lane 1), Xshh (lane 2), and X-chh (lane 3) upon translation in vitro. Each lane contains three hh-associated protein products (indicated by arrowheads), of which the two smaller products arise by autoproteolytic cleavage of the larger unprocessed form (see text and D below). The two X-bhh cleavage products are of similar size, and appear as a doublet in lane 1 (see D below). (D) Detailed analysis of X-bhh processing in vitro. The X-bhh open reading frame was mutated to yield UHA, N, and DN-C constructs as diagrammed in B. Cartoons clarifying the region of X-bhh present in the translation product are shown to the left of lane 4. Lanes 4 and 8: translation of wild-type X-bhh. Lane 5: translation of UHA. Lane 6: translation of N. The protein product comigrates with a fragment generated by autoproteolysis of X-bhh (compare lane 6 with lanes 4 and 8). Lane 7: translation of DN-C whose primary translation product undergoes autoproteolysis (refer to cartoon). The lower of the two bands within the doublet comigrates with a fragment generated by autoproteolysis of X-bhh (compare lanes 7 and 8 with reference to the cartoon), and the band migrating near the 6´103 Mr marker is the small N-terminal fragment remaining after autoproteolysis (refer to cartoon). (E) Processing of X-bhh in embryos. X-bhh or UHA were co-injected with [35S]methionine into embryos and the resulting extracts were immunoprecipitated with an antibody to the carboxy region of X-bhh (see Materials and methods). The upper gel is useful solely for showing the presence of full-length X-bhh denoted by an arrowhead. The lower gel was overexposed to resolve lower molecular mass species arising by processing of X-bhh. Lane 9 (both gels): proteins generated from in vitro translation of DN-C. Lane 10 (both gels): in vitro translation of X-bhh. Lane 11 (both gels): Immunoprecipitation of embryo extracts with a C-terminal antibody after injection of UHA demonstrates the presence of full-length X-bhh polypeptide (arrowhead, upper gel), but no bands co-migrating with C-terminal polypeptides (lower gel). Lane 12 (both gels): Immunoprecipitation of embryo extracts after injection of X-bhh RNA demonstrates the presence of full length X-bhh in the upper gel. In lane 12 of the lower gel, two lower molecular mass bands (arrowheads) are noted, which are absent from the UHA-injected embryos (lane 11), and absent from uninjected embryos (not shown). The lower of these two bands comigrates with C generated by in vitro translation of X-bhh (lane 10) or DN-C (lane 9). The approximately 30´103 Mr band in lane 12 (arrowhead) is presumed to be a modification of the C protein, possibly glycosylation at a predicted N-linked glycosylation site (Ekker et al., 1995). Unmarked bands are not hh-derived as determined by immunoprecipitation of labeled embryos not injected with X-bhh RNAs (not shown).
	Fig. 2. Inductive activities of X-bhh-derived polypeptides in animal cap explants. Animal caps from embryos injected with various RNAs were cultured until sibling embryos had reached stage 25, at which time samples were processed for RT-PCR for the markers shown. Minus (-) lanes are controls omitting reverse transcriptase in the first strand synthesis, and plus (+) lanes contain reverse transcriptase. Lanes 1, 2: stage 25 embryos as positive controls. Lanes 3, 4: animal caps from uninjected embryos as negative controls. Lanes 5, 6: animal caps from embryos injected with Xbhh RNA. Lanes 7, 8: animal caps from embryos injected with N. Lanes 9, 10: animal caps from embryos injected with UHA. Lanes 11, 12: animal caps from embryos injected with a frame-shifted version of X-shh (X-shhfs) as a negative control. Lanes 13, 14: Animal caps from embryos injected with noggin RNA for comparison of induced neural markers. Note the high-level induction by active constructs (X-bhh, N, and UHA) of the cement gland marker XAG-1 and the relatively lower level induction of the anterior neural markers XANF-2 and Otx-A, without induction of the general neural marker, N-CAM.
	Fig. 3. X-bhh modifies the anteroposterior pattern of neural gene expression in explants under the influence of endogenous neural inducers. (A) Isolation of dorsal explants from injected embryos for the preparation of Keller sandwiches (Keller and Danilchik, 1988; Doniach et al., 1992; redrawn from Doniach, 1993). (B) Keller sandwiches were made from uninjected (lanes 1 and 2) and X-bhh-injected (lanes 3 and 4) embryos, total RNA was isolated when control embryos reached stage 20, and RT-PCR was used to analyze the expression of XAG-1 and neural markers. XAG-1 is a cement gland marker, XANF-2 is an anterior pituitary marker, Otx-A is a forebrain marker, En-2 demarcates the midbrain-hindbrain boundary, Krox-20 marks rhombomeres 3 and 5 of the hindbrain, and XlHbox-6 is a spinal cord marker. N-CAM is a general neural marker whose expression is not restricted along the anteroposterior axis. The EF-1a control demonstrates that a comparable amount of RNA was assayed in each set. Note that expression of XAG-1 and anterior neural markers is stimulated by X-bhh treatment, whereas expression of posterior neural markers is suppressed.
	Fig. 4. X-bhh and derived polypeptides modify the anteroposterior pattern of neural gene expression in activin-treated animal caps. Embryos were injected with X-bhh or prolactin RNA and animal cap explants were isolated from blastulae and incubated in the presence or absence of activin. (A) Explants were processed when the sibling embryos reached stage 11, and total RNA was assayed by RT-PCR for the mesodermal markers brachyury (Xbra), goosecoid, and Xwnt-8 and the control EF-1a. Lanes designated plus and minus refer to the presence or absence of reverse transcriptase in the first strand cDNA synthesis. Lanes 1, 2, 5, 6: control animal caps from uninjected embryos. Lanes 3, 4, 7, 8: animal caps from embryos injected with X-bhh. Lanes 9, 10: animal caps from embryos injected with bovine prolactin RNA. Animal caps in lanes 5- 10 were treated with activin A. Note that no mesodermal markers are induced by X-bhh. (B) A second group of explants from the same experiment and in the same order as in A, were cultured until tailbud (stage 25) and assayed for the expression of anteriorposterior neural markers. The actin acted as a control for induction of mesoderm. Expression of anterior neural markers was enhanced by combined treatment with activin and X-bhh relative to either treatment alone; note also the reduction in expression of posterior neural markers by X-bhh in activin-treated explants. (C) In independent experiments, embryos were injected with N or DN-C, and some animal cap explants were treated with activin before culturing until sibling embryos reached tailbud stage. Lanes 1, 2: control animal caps from uninjected embryos. Lanes 3, 4: control animal caps from uninjected embryos, treated with activin. Lanes 5, 6: animal caps from embryos injected with N and treated with activin. Lanes 7, 8: animal caps from embryos injected with DN-C and treated with activin. Whereas N displays activities in activin-treated explants similar to those of X-bhh (see B) DN-C produces the opposite effect, decreasing anterior and increasing posterior neural marker expression.
	Fig. 5. DN-C interferes with X-bhh and N activity in animal cap explants. Embryos were injected with various RNAs, animal cap explants were cultured until sibling embryos reached tailbud (stage 25), at which time RT-PCR was used to analyze the expression of the cement gland marker XAG-1 and the control RNA, EF-1a. Lanes 1, 2: control animal caps from uninjected embryos. Lanes 3, 4: animal caps from embryos injected with both X-bhh and prolactin RNAs. Lanes 5, 6: animal caps from embryos injected with both X-bhh and DN-C. Lanes 7, 8: animal caps from embryos injected with both N and prolactin RNAs. Lanes 9, 10: animal caps from embryos injected with both N and DN-C. The N and X-bhh experiments were conducted independently and thus absolute levels in lanes 3-6 should not be compared to those in lanes 7-10. Note that the induction of XAG-1 expression by X-bhh or N is reduced by co-injection of DN-C.
	Fig. 6. Distinct effects of expression of N and DN-C in Xenopus embryos. Uninjected embryos (WT), and embryos injected with N, or DN-C were photographed at tadpole stage (A) or analyzed by in situ hybridization (B, C). (A) Cement glands (arrow) and other anterior structures are enlarged in N-injected embryos. In contrast, embryos injected with DN-C display a smaller cement gland, reduced anterior structures, and enhanced posterior structures. (B) In situ hybridization was performed with a pan neural RNP marker. Arrows indicate the otic vesicle. The neural tissue anterior to the otic vesicle is enlarged in N-injected embryos, and reduced in DN-Cinjected embryos as compared to control embryos. (C) In situ hybridization was performed with the forebrain marker Otx-A. The pattern of Otx-A expression (arrows) is expanded in N-injected embryos while expression in DN-C-injected embryos is similar, but reduced in comparison to that of control embryos.