XB-ART-26842Development. April 1, 1989; 105 (4): 787-94.
XlHbox 8: a novel Xenopus homeo protein restricted to a narrow band of endoderm.
We report the isolation of a new homeobox gene from Xenopus laevis genomic DNA. The homeodomain sequence is highly diverged from the prototype Antennapedia sequence, and contains a unique histidine residue in the helix that binds to DNA. The homeodomain is followed by a 65 amino acid carboxyterminal domain, the longest found to date in any vertebrate homeobox gene. We have raised specific antibodies against an XlHbox 8-beta-gal fusion protein to determine the spatial and temporal expression of this gene. The nuclear protein first appears in a narrow band of the endoderm at stage 33 and develops into expression within the epithelial cells of the pancreatic anlagen and duodenum. Expression within the pancreatic epithelium persists into the adult frog. This unprecedented restriction to an anteroposterior band of the endoderm suggests that vertebrate homeobox genes might be involved in specifying positional information not only in the neuroectoderm and mesoderm, but also in the endoderm. Our data suggest that XlHbox 8 may therefore represent the first member of a new class of position-dependent transcription factors affecting endodermal differentiation.
PubMed ID: 2574662
Article link: Development.
Grant support: HD 21502-03 NICHD NIH HHS
Genes referenced: actl6a gal.2 ncoa6 nr3c1 pdx1 tbx2 wdpcp
Antibodies referenced: Pdx1 Ab2
Article Images: [+] show captions
|Fig. 1. XlHbox 8 encodes a novel type of homeodomain protein. (A) Construction of XlHbox 8 fusion protein. The top line shows the genomic EcoRl fragment (E-E) that carries the 3'-most two-thirds of the homeobox and downstream sequences. The far left end shows the rest of the homeobox to indicate the position of the EcoRl site within the homeobox. The middle line shows the Alul/Sau3A\ fragment (A-S) used in constructing the /3-gal fusion protein in pTRB 2 (bottom line). The proteincoding region is indicated by an open box, the homeobox by a shaded portion within this. The translation termination codon is asterisked. (B) Nucleotide and amino acid sequence of XlHbox 8. The sequence between the EcoRl and the Haelll site was determined on both strands (see Methods). Some additional downstream sequence is shown to indicate the Sau3AI site used in constructing the fusion protein. A Pstl site used in determining orientation in pTRB 2 is also shown. The homeodomain is indicated in bold type and a sequence of eight amino acids also found in the 'potentiator' region of human glucocorticoid receptor is underlined. (C) Comparison of XlHbox 8 with other homeodomain sequences. The relevant region of the prototype Antp homeodomain (amino acid position within the 60 residues is indicated above the sequences) is compared first with XlHbox 2 (Wright et al. 1987) and then with XlHbox 8. The position of the helix-turn-DNA-recognition helix motif is indicated (De Robertis et al. 1985; Scott et al. L988). The corresponding part of five other major classes of homeodomain are also presented.|
|Fig. 2. Spatial distribution of XlHbox 8 transcripts. (A) Stage 38 Xenopus embryos were dissected into five parts (1-5) and RNA extracted from each set of pooled fragments for analysis by RNAse protection as shown below (for full description see Methods). (B) RNAse protection of XlHbox 8 probe by total RNA from whole tadpoles or fragments thereof. Lane assignments are: P, probe incubated alone without RNAse; —, no RNA control; K, 10 j.ig adult kidney RNA control; 1-5, total RNA from 15 fragments of each type indicated in panel A; W, total RNA from 10 whole tadpoles. Probe length is indicated; fulllength protection is at 139 nucleotides (nt). The other major bands (about 8-10 nt shorter) probably represent artefactual cutting within the RNA duplex (see Wright et al. 1987), but could be due to transcripts from other copies of the XlHbox 8 gene within the tetraploid X. laevis genome with minor allelic variations (see Fritz et al. 1988). The film was deliberately over-exposed as a stringent test of the restriction of XlHbox 8 RNA to fragment 4. (C) RNAse protection of cytoskeletal actin probe by total RNA from the same samples. Lane assignments are exactly as for panel B, except that total RNA from only 9 fragments (lanes 1-5) or 6 whole tadpoles (lane W) was analysed. Probe length is indicated and the position of the cognate cytoskeletal actin band is shown. The film was exposed for approx. 2h to allow the band in fragment 5 to show properly - for this reason the intensities of the bands in lanes K, 1 and W are well outside the linear range of the film. From other exposures, the band intensities are in good agreement with the recovery of total RNA as judged by agarose gel electrophoresis. Markers are single-strand sequence ladders from an M13 subclone of XlHbox 2 (Wright et al. 1987).|
|Fig. 3. Immunolocalization of XlHbox 8 protein. Xenopus embryos staged according to Nieuwkoop & Faber (1967) were serially sectioned and stained with anti-XlHbox 8 antibody. (A) Frontal section of a stage 33 embryo. A narrow band of expression of the XlHbox 8 protein (bracketed) is faintly visible in the endoderm slightly posterior to the pharynx. (B) Sagittal section through the left half of a stage 38 embryo. In this section the anterior border where expression of the XlHbox 8 antigen starts is clearly visible. The dorsal and ventral pancreatic rudiments are positive for the antigen as indicated. The stained endodermal tube in the centre will later mature into duodenum (note the cavity at its anterior tip). The liver is entirely unstained. (C) Sagittal section through a stage 41 embryo. Note the band of expression in the endoderm (dark stain) while the remainder of the embryo is unstained. (D) Transverse section (slightly oblique angle) through a stage 41 embryo at low magnification. Only endodermal cells are stained; neuroectoderm and mesoderm are unstained. (E) Same section as in D but at higher magnification. Note XlHbox 8 expression in the gut epithelium as well as in the pancreatic excretory duct. All nuclei in the endodermal band are stained; the lower right corner appears unstained because of the slightly oblique plane of sectioning. (F) The same section as in E counterstained with the fluorescent DNA stain Hoechst 33258. (G) Section through a stage 49 embryo. All nuclei in the epithelium of the duodenum and pancreatic ducts are stained, but only a small percentage of cells within the pancreas proper contain XlHbox 8 protein at this stage. No XlHbox 8 protein is expressed in liver, stomach, or small intestine. (H) Same section shown in G at higher magnification. (I) Section through an adult Xenopus pancreas. All the nuclei in the pancreatic duct system are stained with anti-XlHbox 8 antibody. The nuclei of cells in the acini are weakly stained. Abbreviations; ph, pharynx; en, endoderm; p, pancreas; dp, dorsal pancreatic anlage; vp, ventral pancreatic anlage; 1, liver; pd, pancreatic excretory duct; s, stomach; in, intestine; ens, central nervous system; pn, pronephros.|
|Fig. 4. Spatial expression pattern of XlHbox 8 protein visualized by antibody whole-mount staining. (A) A side view of a stage 40 tadpole immunostained with XlHbox 8 primary antibody and peroxidase-coupled anti-rabbit second antibody, visualized with diaminobenzidine/hydrogen peroxide, and cleared in benzyl benzoate/benzyl alcohol. Samples were processed exactly as described by M. Klymowsky (personal communication). (B) Diagrammatic interpretation of panel A. The regions labelled dp and vp are the dorsal and ventral pancreatic rudiment, respectively; duo represents the region of the duodenum. The epithelial nuclei of these regions together constitute the only specific XlHbox 8 staining in the embryo. The punctate appearance of the lower left edge of the embryo is a photographic artefact caused by the extreme curvature of the tadpole combined with nonunitorm illumination by the microscope condenser. This whole-mount embryo staining allows us to conclude that the endoderm is the only place where XlHbox 8 antigen is found. Photomicrograph was taken in a BioRad confocal microscope using an image-enhancement program.|