QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 24, 2005
doi: 10.1242/10.1242/dev.01787

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rallis, C. Articles by Logan, M. P. O. Search for Related Content PubMed PubMed Citation Articles by Rallis, C. Articles by Logan, M. P. O.

Tbx3 can alter limb position along the rostrocaudal axis of the developing embryo

Division of Developmental Biology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK

View larger version (62K): [in a new window]   Fig. 1. Normal expression of Tbx3 in the chick (A-F). (A) At stage13, Tbx3 is expressed in the presumptive forelimb (FL) and hindlimb (HL) levels (arrowheads). (B) At stage 16, Tbx3 expression is more robust in the posterior of the limb (arrowheads). (C) Stage16 section at the forelimb level. Tbx3 is expressed in the lateral plate mesoderm and somite. (D) Higher magnification of section in C. Tbx3 is present in the mesoderm but not ectoderm of the wing and in the ventrolateral lip of the dermomyotome. (E) Stage 19, Tbx3 is located in the posterior limb mesenchyme (arrowheads). (F) Stage 25, Tbx3 is present in the limbs in two stripes in the anterior and posterior limb mesenchyme (arrowheads). (G) Schematic diagrams of Tbx3 constructs. (H) Bar chart representing the relative luciferase activity obtained in luciferase assays with Tbx3 constructs. S, somite; LP, lateral plate mesoderm; V, ventral lip of dermomyotome; M, mesoderm; E, ectoderm.

View larger version (80K): [in a new window]   Fig. 2. Misexpression of Tbx3 and Tbx3EN causes a rostral shift in limb position but the shifted limb is patterned normally. Dorsal views of embryos; injected side is on the right, control side is on the left. Rostral is towards the top. (A) Whole-mount in situ hybridization with MyoD marking somites and Shh marking the ZPA. There is a one somite-level shift in limb position (compare level of left and right bar). (B) Whole-mount in situ hybridization with Pax3 marking migrating myoblasts from the lateral lip of dermomyotome into the limb mesenchyme. Pax3 is not detected in the lateral lip of the dermomyotome in somites at levels that normally contribute to limb musculature (black arrowhead), as these cells have migrated into the limb. Following a limb shift on the injected side, these Pax3-expresssing cells are still present in somites at the same axial level (red arrowhead). (C) Whole-mount immunohistochemistry for the neurofilament-associated antigen (3A10). Axons from axial levels that normally innervate the limb fail to send projections to the shifted limb (right, black arrowheads). At more rostral levels, following mislocation of the limb, axons that normally do not innervate the limb on the injected side, project into the limb mesenchyme (red arrowheads). (D-I) Genes expressed in regions of the limb ectoderm or mesenchyme. Fgf8 (D), Bmp2 (E), Hoxc5 (F), Tbx5 (G), Tbx15 (H) and Tbx2 (I) are all expressed in their normal pattern in the shifted limb. (J-O) Skeletal preparations of an embryo injected with Tbx3 in the wing (J-L) or in the leg (M-O). Although the wing and the leg are shifted rostrally (compare bar levels between left and right side in J and M), no alterations are observed in the vertebral column. Arrow in J indicates scapula, arrow in M indicates ischium. (K) Injected wing; (L) contralateral control wing. There is no alteration in the pattern of the limb elements. (N) Injected leg; (O) uninjected leg. No change is observed in digit patterning following Tbx3 misexpression. Chick stages: (A) 22, (B) 23, (C) 24, (D-I) 21, (J-O) 27.

View larger version (83K): [in a new window]   Fig. 3. Early effects of Tbx3 misexpression at the forelimb level. In all panels, injected side is rightwards and control side is leftwards. Rostral is towards the top. (A) Levels of Tbx3 transcripts are unaffected at stage 13. (B) By stage 16, Tbx3 transcripts are detected throughout the limb field on the injected side. (C,D) There is no alteration in the rostral border (red arrowheads) of Hoxb8 (C) or Hoxb9 (D) following misexpression of Tbx3. (E) Wnt2b is expressed normally in the early forelimb-forming territory. The limb-forming region extends between somites 15 and 20 (numbered). (F) Tbx5 expression is expanded rostrally. The extent of rostral expansion is indicated with a broken yellow line. Red arrowheads indicate equivalent rostrocaudal levels. (G) Rostral expansion of Hoxc5 expression (indicated with a broken yellow line). Red arrowheads show equivalent rostrocaudal levels. (H). DiI labelling of the prospective limb-forming region and adjacent somites (for axial reference). In the uninjected limb, cells that normally contribute to the posterior limb stay in register with somites at the same axial level (left, arrowhead). On the injected side (right), cells at the same level instead contribute to the inter-limb flank but remain in register with adjacent somites (arrowhead). Somites 17 and 20 are numbered.

View larger version (58K): [in a new window]   Fig. 4. Effects of Tbx3 and Tbx3EN misexpression on dHand and Gli3 expression. (A-C) Schematics of the limb-forming region. (A) At stage 16, dHand is present throughout the limb forming region. (B) At stage 17, high levels of dHand expression are observed in the posterior limb mesenchyme owing to repression by Gli3, which is expressed at higher levels in the anterior. (C) At stage 18, Gli3 is expressed in the anterior limb mesenchyme, while dHand in the posterior is required for the establishment of Shh expression in the ZPA. In medial limb mesenchyme, dHand and Gli3 expression domains overlap. (D) At stage 17, dHand is restricted in the posterior limb mesenchyme in the control side (left, arrowhead shows rostral border of expression). Following Tbx3 misexpression, dHand is expressed throughout the limb-forming region (right, arrowhead). At stage 19 (E) and stage 21 (F), when the alteration in limb position is apparent, normal expression of dHand is observed within the limb mesenchyme of the shifted limb. (G) At stage 17, following Tbx3 misexpression, Gli3 is downregulated in the injected side (right arrowhead) compared with the control side (left arrowhead). A rostral shift in the graded Gli3 expression domain is observed (compare left and right arrowhead levels). At stage 19 (H) and at later stages (stage 21) (I), Gli3 is expressed normally within the mesenchyme of the shifted limb. Shh expressed in cells of the ZPA in the posterior of the limb is detected in more rostral locations in the shifted limb by stage 19 (J) and also at stage 21 (K). Hoxd12, a downstream target of Shh, is similarly expressed in more rostral locations in the Tbx3-injected limb by stage 19 (L) and also at stage 21 (M).

View larger version (50K): [in a new window]   Fig. 5. Misexpression of Tbx3VP16 causes a caudal shift in axial limb position. (A) MyoD expression marks the somites and Shh marks the ZPA. There is a two-somite level caudal shift (marked by bars) in the injected limb (right). (B) dHand expression at stage 17 following Tbx3VP16 misexpression. dHand is restricted to the posterior limb mesenchyme in the control side (left, arrowhead shows rostral expression border), while in the injected side, dHand expression is displaced caudally (right, arrowhead). (C) Gli3 expression at stage 17 following Tbx3VP16 misexpression. Gli3 is upregulated in the injected side (right) compared with the control side (left). The graded domain of Gli3 expression is displaced caudally (compare left and right arrowheads).

View larger version (85K): [in a new window]   Fig. 6. Misexpression of a Gli3ZnF-VP16 activator form of Gli3 can influence limb positioning. (A) Schematic of full-length Gli3 protein (Gli3) and a Gli3ZnF-VP16 activator form. Full-length Gli3 protein contains an N-terminal repression domain (RD), a zinc finger DNA-binding domain (ZnF), and an activation domain in the C-terminus (Act. Domain). The Gli3 activator form contains the ZnF fused with two VP16 activation domains. (B-E) Dorsal views of the embryo; injected limb is on the right. (B) Whole-mount in situ hybridization with MyoD indicates the somites and Shh marks the ZPA. There is a one-somite level shift in limb position (compare level of left and right bars) following misexpression of Gli3ZnF-VP16 in the limb. (C) The anterior border of the Tbx3 (C) and dHand (D) expression domains in the forelimb region at stage 17 have been displaced rostrally (arrowheads) following Gli3ZnF-VP16 misexpression. (E) Gli3 expression is downregulated following misexpression of Gli3ZnF-VP16 (arrowhead).

View larger version (26K): [in a new window]   Fig. 7. Models for the interactions between dHand, Gli3 and Tbx3, in the limb-forming region at stages prior to Shh expression, that refine limb position along the rostrocaudal axis of the embryo. (A) At stage 16, Tbx3 is expressed throughout the limb-forming region but expression is more robust in the future posterior limb mesenchyme. dHand is expressed throughout the limb-forming region. (B) At stage 17, Gli3 is expressed throughout the limb mesenchyme with higher levels in the anterior. dHand expression is restricted to the posterior. (C) Ectopic expression of Tbx3 in more anterior regions leads to a repression of Gli3, resulting in a de-repression of dHand and expansion of its expression domain. (D) A model illustrating how the genetic antagonism between dHand and Gli3 that positions the future ZPA may be mediated through Tbx3. – and + symbols indicate, respectively, the transcriptional repressor and activator function of each protein. In addition, in the posterior limb, Tbx3 and dHand cooperate to induce Shh expression and thereby specify the position of the ZPA in the posterior limb mesenchyme. A, anterior; P, posterior.