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doi: 10.1242/10.1242/dev.00471

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Multiple levels of transcriptional and post-transcriptional regulation are required to define the domain of Hoxb4 expression

Tim Brend^1,2, Jonathan Gilthorpe^2,*, Dennis Summerbell^1,2 and Peter W. J. Rigby^1,2,

¹ Section of Gene Function and Regulation, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK
² Division of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
^* Present address: Department of Developmental Neurobiology, King's College London, Guy's Campus, London Bridge, London SE1 1UL, UK

View larger version (19K): [in a new window]   Fig. 1. (A) Details of the Hoxb4 locus are shown along with an indication of its position within the Hoxb cluster. The first transcription start site (P1) (Gutman et al., 1994) is indicated with an arrow. Black and white boxes represent coding and untranslated sequences, respectively. The hatched region within exon 2 indicates the position of the homeobox. The positions and specificities of known regulatory regions (A, B, C) are indicated above the locus (Whiting et al., 1991). The ambiguous function of region B is indicated by a question mark. B, BglII; Hb, hindbrain; H; HindIII; N, NcoI; P, PstI; r, rhombomere; S, SalI; SC, spinal cord; so, somite. (B) Constructs used in this study. Fragments of the Hoxb4 locus are represented as in A. hsp and pA indicate the hsp68 promoter and SV40 polyadenylation signal, respectively. Exp denotes the total number of transgenic F0 embryos and lines showing a consistent pattern of X-gal staining for each construct. Asterisk indicates that analysis of construct CHZ has been reported previously (Gilthorpe et al., 2002) and the data are given here for comparison.

View larger version (48K): [in a new window]   Fig. 2. Regression of the anterior boundaries of expression of construct CHZ. (A) By 8.5 dpc, anterior limits of expression were established at the hindbrain/spinal cord boundary in the neural tube, and at so6/7 in the paraxial mesoderm. The latter corresponds to the boundary of Hoxb4 expression. (B) Expression was noticeably weaker at the anterior boundaries by 8.75 dpc. (C-E) Boundaries continued to shift posteriorly until 9.5 dpc, coming to rest alongside so6/7 in the neural tube and at so13/14 in the paraxial mesoderm. Black arrowheads indicate the position of so7. ov, otic vesicle; fl, forelimb bud.

View larger version (65K): [in a new window]   Fig. 3. Multiple regulatory elements are required to determine the anterior boundary of expression in the paraxial mesoderm. (A,B) Whole-mount immunostaining of a 10.5 dpc embryo, using an anti-Hoxb4 antibody. Hoxb4 protein was expressed up to the r6/7 boundary in the neural tube and up to so7 in the paraxial mesoderm. So7 is easily identified at this stage as it lies alongside the second dorsal root ganglion (drg2). Drg2 is the most anterior drg visible at this stage as drg1 has degenerated. (C-J) X-gal staining of 10.5 dpc transgenic embryos. (C,D) Construct CHZ was expressed only up to so14 at this stage. (E,F) Construct Cb4Z had an anterior boundary at so8, indicating that the Hoxb4 promoter can maintain much of the somitic expression established by region C but not the correct anterior boundary. (G,H) Construct b4ZC had identical boundaries of expression to Cb4Z, demonstrating that the position of region C relative to the promoter does not affect the expression pattern. (I,J) Construct CBb4Z did recapitulate the somitic boundary of Hoxb4 expression, identifying a requirement for region B in the regulation of Hoxb4 in the paraxial mesoderm.

View larger version (61K): [in a new window]   Fig. 4. Region B is required to maintain the anterior somitic boundary after 9.0 dpc. (A-F) X-gal staining of transgenic embryos. (A,B) Expression patterns for construct Cb4Z (A) and CBb4Z (B) were identical at 8.5 dpc. Both had an anterior boundary at so6/7 in the paraxial mesoderm. (C,D) Anterior boundaries of expression remained identical at 9.0 dpc. (E,F) By 9.5 dpc, the somitic boundary of Cb4Z expression shifted one segment posteriorly (E), whereas that of construct CBb4Z remains at so6/7 (F). Black arrowheads indicate the position of so7. (G-J) X-gal stained transgenic embryos counterstained with acid fuchsin. (G,H) At 9.0 dpc, two somites could be detected between the anterior boundaries of X-gal staining in the paraxial mesoderm and neural tube (black arrows) for constructs Cb4Z and CBb4Z. (I,J) By 9.5 dpc, the gap had extended to three somites with construct Cb4Z (I) but remained at two with CBb4Z (J), confirming the timing of the posterior shift of the expression boundary.

View larger version (92K): [in a new window]   Fig. 5. Downregulation of transgene expression in later development. (A-C) At 11.0 dpc, the anterior boundaries and overall expression patterns of constructs CHZ (A), Cb4Z (B) and CBb4Z (C) were identical to those seen at earlier stages of development. (D-F) By 11.5 dpc, downregulation of expression had commenced in the posterior mesoderm with all three constructs (white brackets). (G-I) Downregulation was complete by 12.5 dpc. Mesodermal expression was absent from embryos carrying construct CHZ (G) and was restricted to cervical regions for constructs Cb4Z (H) and CBb4Z (I). (J-L) At 13.5 dpc, neural expression of CHZ was also downregulated (J), whereas neural and mesodermal expression was maintained by constructs Cb4Z (K) and CBb4Z (L). White arrowheads indicate drg2. Black arrowheads and arrows indicate the anterior and posterior boundaries of mesodermal expression, respectively.

View larger version (81K): [in a new window]   Fig. 6. Post-transcriptional regulation of Hoxb4. (A-D) In situ hybridisation for Hoxb4. Transcripts were present in so7-13 (black arrowheads) but not in more posterior somites. By contrast, Hoxb4 was expressed throughout the neural tube posterior to the r6/7 boundary (white arrowhead). (D) Strong staining was seen in the tailbud (white arrow). (E-H) Whole-mount immunostaining for Hoxb4. Protein was detected in so7-13 and in the posterior hindbrain (E,F), but was absent from posterior neural tube and somites (G). (H) Protein was not expressed in the tailbud (black arrow). (I-J) Transverse section of embryos subjected to in situ hybridisation for Hoxb4 (I,J) or immunostaining for Hoxb4 (K,L). (I) Transcripts were widely distributed at forelimb level with relatively high levels in the dorsal neural tube (white arrowhead) and dermomyotome (white arrows). (J) At posterior levels transcripts were expressed throughout the neural tube but not in the adjacent somites. (K) At forelimb level, Hoxb4 protein was not detected in the neural tube but was expressed in adjacent tissue with a high level in the dermomyotome (white arrows). (L) In the posterior embryo, protein was detectable in neither somites nor neural tube, but was seen in the notochord (black arrow) and the mesonephric ducts (black arrowheads). (M-O) In situ hybridisation of a lacZ probe to a transgenic embryo carrying construct CBb4Z. The pattern was identical to that seen with X-gal staining (Fig. 3I,J), with strong expression in all somites posterior to so7 (black arrowhead) and in the tailbud (white arrow). (P) X-gal staining of a 10.5 dpc transgenic embryo carrying construct b4ZCBpA. Strong staining was seen only in so7-13 (black arrowheads). (Q) Structure of construct b4ZCBpA [construct 6 (Whiting et al., 1991)]. This schematic follows the format used in Fig. 1.

View larger version (13K): [in a new window]   Fig. 7. Regulatory organisation of paralogous group 4 Hox genes in the mouse. (A) A revised version of Fig. 1A incorporating the data presented in this study. The spatial and temporal specificities of regulatory regions are indicated below the Hoxb4 locus. Red and yellow boxes indicate neural and paraxial mesodermal specificity, respectively. Early and late regulatory elements have been more precisely mapped within region A and are indicated accordingly (Gould et al., 1997; Gould et al., 1998). (B) Comparison of the regulatory organisation of Hoxa4, Hoxb4 and Hoxd4. Black boxes represent the coding sequences of each gene. The question mark represents the ambiguous role of the Hoxa4 intron (see Discussion). Asterisks indicate that enhancer activity may be located in either or both of the regions 3' of Hoxd4. M, maintenance. This diagram incorporates data from Behringer et al. (Behringer et al., 1993), Keegan et al. (Keegan et al., 1997), Morrison et al. (Morrison et al., 1997), Sharpe et al. (Sharpe et al., 1998), Whiting et al. (Whiting et al., 1991) and Zhang et al. (Zhang et al., 1997).