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Genes Dev
1989 May 01;35:641-50. doi: 10.1101/gad.3.5.641.
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Complementary homeo protein gradients in developing limb buds.
Oliver G
,
Sidell N
,
Fiske W
,
Heinzmann C
,
Mohandas T
,
Sparkes RS
,
De Robertis EM
.
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A new human homeo box-containing gene designated Hox-5.2 was cloned and mapped to human chromosome 2. This homeo box is related in sequence to Abdominal-B, a Drosophila homeotic gene that specifies identity of posterior segments. An antibody probe was made using a human Hox-5.2 fusion protein and was found to stain posterior regions of mouse, chicken, and Xenopus embryos. Unexpectedly, when the distribution of Hox-5.2 antigen was compared with that of X1Hbox 1 antigen, a non-overlapping and mutually exclusive pattern was detected (e.g., in developing limb buds, intestine, and somites). Regions expressing Hox-5.2 do not express X1Hbox 1 protein, and vice versa. Hox-5.2 antigen is detected strongly in developing fore- and hindlimb buds, where it forms a gradient of nuclear protein throughout most of the mesenchyme. This gradient is maximal in distal and posterior regions. Hox-5.2 expression is activated in Xenopus limb regeneration blastemas, as expected for any gene involved in pattern formation. As described previously, a gradient of X1Hbox 1 protein can be detected in the forelimb. The latter gradient has the opposite polarity to that of Hox-5.2. i.e., maximal in anterior and proximalmesoderm. These two opposing gradients (and possibly others) could be involved in determining positional values in developing limb buds.
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Hox-5.2 antigen in mouse embryos is expressed posteriorly and exhibits a pattem complementary to that of X1Hbox 1. (A)
Figure 3. Day-10.5 mouse embryo sectioned parasagittally and stained with an antibody raised against the last 106 amino acids of the human homolog of the X1Hbox 1 gene (Oliver et al. 1988a). The protein is detected in anterior regions of the spinal cord beginning in the hindbrain (HB), in somites, and in mesoderm of the digestive tract. (B)The adjacent mouse section was immunostained with anti- Hox-5.2 antibody (raised against the last 73 amino acids of the protein). Nuclear staining is detected over the same tissue derivatives as X1Hbox 1, but its distribution is in more posterior regions of the embryo (e.g., this protein is absent in hindbrain and only the posteriordigestive tract is stained). The mutually exclusive expression pattern of both proteins is better seen in the two following panels. (CNS) Central nervous system. (C) Transverse section through an intestinal loop of a mouse embryo stained with X1Hbox 1 antibody. Staining is detected in the mesoderm of an anterior intestinal loop. (D) Adjacent section immunostained with anti-Hox-5.2 antibody; note that only the posterior intestinal loop is positive. These mutually exclusive staining patterns strongly support the specificity of the antibodies used in this analysis.
Figure 4. lmmunolocalization of Hox-5.2 antigens in developing hindlimb buds. (A) Day-10.5 mouse embryohindlimb buds (HL) sectioned transversely and stained with the X1Hbox I antibody raised against the human homolog (0liver et al. 1988b).As described previously, staining is detected only in the surrounding ectoderm (indicated by arrows). (B) Staining of an adjacent section with anti-Hox-5.2 antibody. Mesodermal cells exhibit a distal (Dist) to proximal (Prox)gradient of nuclear protein. {C}Transverse section at the level of the hindlimb buds of a Xenopus stage-50 tadpole (2-week)stained with Xenopus antibody B (Cho et al. 1988; 0liver et al. 1988a). As previously reported (0liver et al. 1988b), no mesodermal staining is detected in hindlimbs, while the medial region of the ectoderm shows expression (arrows).(D) An adjacent section was stained with anti-Hox-5.2 antibody and, as in mouse, the staining is observed in the mesoderm in a distal to proximal gradient. (CNS) Central nervous system.
Figure 5. Anti-Hox-5.2 antibody staining in developing day-10.5 mouse forelimbs. (A) Longitudinal {frontal} section at the level of the forelimb (FL)bud stained with the human X1Hbox 1 antibody. Staining is detected in the anterior {Ant)mesoderm and in the surrounding ectoderm. {Post}Posterior; {Prox)proximal; (Dist) distal. (B) Staining of an adjacent section with anti-Hox-5.2 antibody detects this antigen in a complementary pattern to that of X1Hbox 1, i.e., negative in the ectoderm and position in those mesodermal cells that do not show staining for X1Hbox 1.
Figure 6. Section through a stage-25 chicken forelimb showing a clear nuclear gradient of Hox-5.2 antigen. In this oblique section Hox-5.2 gradient is maximal in the distal (and posterior) region, and minimal in the proximal (and anterior) region of the limb bud. The arrow indicates the apical ectodermic ridge, a thickening of the ectoderm involved in limb growth.
Figure 7. Activation of Hox-5.2 protein during limb regeneration. (A) Normal hindlimb bud from a stage-54 Xenopus tadpole (oblique section). Very weak staining is detected with Hox-5.2 antibody. (B) Contralateral hindlimb bud amputated distally 8 days previously (stage 52). Note that Hox-5.2 antigen is strongly expressed in the regeneration blastema (arrows).
