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The expression of the homeodomain protein XIHbox 1 in developing Xenopus limbs was analyzed using specific antibodies. In the forelimb budmesoderm XIHbox 1 shows a clear antero-posterior gradient that is strongest in the anterior and proximal region of the forelimb. Hindlimb budmesoderm is devoid of XIHbox 1, indicating an early molecular difference between arm and leg. The innermost ectodermal cell layer is positive throughout the forelimb and hindlimb budectoderm, but no other areas of the skin. Similar results are obtained in developing mouse limbs, suggesting that XIHbox 1 participates in forelimb development in a variety of tetrapods. In early tadpoles analyzed at stages preceding limb bud formation, the lateral plate mesoderm is positive in the region corresponding to the earliest "field" of forelimb information, but not in the hindlimb field. These results suggest a molecular link between morphogenetic fields, gradients, and homeobox genes in vertebrate development.
Figure 1. A gradient of XlHbox 1 protein in X. laevis Forelimb Buds
(a-f) Serial transverse sections through a stage 51 tadpole (3 week; all stages according to Nieuwkoop and Faber, 1987). Section (a) is the most
anterior and section(f) the most posterior. Note the anteroposterior gradient of stained nuclei in the mesoderm. XlHbox 1 protein was immunolocalized
with antibody B (Oliver et al., 1988) which is directed against amino acids 2-148 of the long XlHbox 1 protein and therefore detects both the long
and the short versions of this protein that have been described by Cho et al. (1988). (g-k) Serial parasagittal sections through a stage 52 (4 week)
tadpole. (g) is the most distal and (k) the most proximal. Note the mesodermal gradient of expression (best seen in panel [i]) and the stronger staining
over the posteriorectoderm. lmmunolocalizations were with antibody C (Oliver et al., 1988) which is specific for amino acids 2-73 of the long protein
and therefore does not react with the short XlHbox 1 protein (Cho et al., 1988; Oliver et al., 1988). Transcription of XlHbox 1 in embryos is driven
by two prdmoters, resulting in the production of either a short or a long protein, which differ by 82 amino acids at the amino terminus (Cho et al.,
1988). All the results reported here for limb bud mesoderm and ectoderm, both in frogs and mice, can be explained by expression of the long XlHbox
1 protein exclusively. This is because the immunostaining with antiserum C (Oliver et al., 1988) which is specific for the first 73 amino acids of the
long protein’([g-k]) is the same as that obtained with antibody B ([a-f]), which does not distinguish between the two types of protein. Similar results
were obtained by using the “nonoverlapping†antibody A (see Experimental Procedures). (I) Schematic representation of the plane and direction
of sectioning in the preceding panels in stylized limb buds. Note that at these stages Xenopus forelimb buds are contained inside a cavity called
the atrium (Nieuwkoop and Faber, 1987). Abbreviations: A, anterior; P posterior; D, dorsal; V, ventral; Di, distal; Pr, proximal.
Figure 2. Expression of XlHbox 1 in Xenopus Hindlimb Buds
Transverse section through the hindlimb buds of a stage 50 (2 week)
Xenopus tadpole immunostained with antibody B. Note that the mesoderm is not stained above background, while the posterior (medial) region of the ectoderm (EC) shows strong expression. Arrow indicates
ectodermal staining. Abbreviations: HL, hindlrmb; Re, rectum; D, dorsal;
V, ventral; En, endoderm.
Figure 4. XlHbox 1 Protein Expression in the Forelrmb and Hindlrmb Bud of Day 10 Mouse Embryos
Serial transverse sections were immunostained with an antibody raised against the last 106 amino acids of the human homolog of the XlHbox 1
gene. (a-d) are from the forelimb, and (e-h) are from the hindlimb of the same embryo. (a) and (e) are most anterior, and (d) and (h) are the most
posterior sections. The arrowheads indicate the AER. The curvature of this embryo was exceptionally favorable, permitting the sectioning of all four
limb buds in an approxtmately transverse plane. Note that the mesodermal gradient is present in the forelrmb but absent from the hindlimb. Identical
results were obtained in mouse limb buds with antibodies derived from nonoverlapping Xenopus fusion proteins (antibodies A, B, and C; Oliver et
al., 1988). All antibodies reacted with similar embryonic regions in mouse and Xenopus embryos, regardless of whether the fusion protein was of
human or frog origin. The strongest staining of mouse sections was obtained wtth the antibody derived from the human antigen. Abbreviations:
D, dorsal; V, ventral; DI, distal; Pr, proximal; Ant, anterior; Post, posterior.
