XB-ART-27114Cell December 23, 1988; 55 (6): 1017-24.
A gradient of homeodomain protein in developing forelimbs of Xenopus and mouse embryos.
The expression of the homeodomain protein XIHbox 1 in developing Xenopus limbs was analyzed using specific antibodies. In the forelimb bud mesoderm XIHbox 1 shows a clear antero-posterior gradient that is strongest in the anterior and proximal region of the forelimb. Hindlimb bud mesoderm 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 bud ectoderm, 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.
PubMed ID: 2904837
Article link: Cell
Genes referenced: hoxc6
Antibodies: Hoxc6 Ab2 Hoxc6 Ab3 Hoxc6 Ab4
Article Images: [+] show captions
|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 posterior ectoderm. 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 3. Expression in the Lateral Plate Mesoderm of Early Embryos (a) Diagram showing the location of the forelimb field in the lateral mesoderm of Amblystoma maculatum tailbud-stage embryo (redrawn from Harrison, 1918). Note that the disc-shaped forelimb anlage is just posterior and ventral to the pronephros (Pn). (b) Transverse section through a stage 41 (3 day) Xenopus embryo. The left side of this slightly oblique section corresponds to the prospective forelimb field region (section immediately posterior 10 the Pn; note Pn at right side this section) and is shown at higher magnification in (d). Arrows in (d) indicate nuclei in the somatopleure (the most external layer of the lateral plate mesoderm) that express XlHbox 1. Arrowheads indicate positive mesodermal nuclei in the splanchnopleure that surrounds the endoderm (En). (c)shows a more anterior section (through the pronephric region) and (e) a more posterior section of the embryo shown in (d); note that the somatopleure nuclei do not stain with anti-XIHbox 1 antibodies in either region. In this serially sectioned tadpole the distance between sections (c) and (d), and (d) and (e). is 200 F in each case. All sections were stained with an&Xenopus antibody B (Oliver et al., 1988). The yolk platelets in the En display background staining, which is nonspecific. The central nervous system (CNS) and lung bud mesoderm (Lu) also express high levels of XlHbox 1 protein. A small proportion of myotome (My) nuclei ([d] and [e]) are stained, which we believe to be neural crest derivatives and not muscle cell nuclei. Abbreviations: D, dorsal; V, ventral: A, anterior: P, posterior: EC. ectoderm: No, notochord.|
|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.|