Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position

Abstract

Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the antero-posterior axis remains largely unknown. Hox genes have long been suspected to control limb position; however, supporting evidences are mostly correlative and their role in this process is unclear. Here, we show that limb position is determined early in development through the action of Hox genes. Dynamic lineage analysis revealed that, during gastrulation, the forelimb, interlimb, and hindlimb fields are progressively generated and concomitantly patterned by the collinear activation of Hox genes in a two-step process. First, the sequential activation of Hoxb genes controls the relative position of their own collinear domains of expression in the forming lateral plate mesoderm, as demonstrated by functional perturbations during gastrulation. Then, within these collinear domains, we show that Hoxb4 anteriorly and Hox9 genes posteriorly, respectively, activate and repress the expression of the forelimb initiation gene Tbx5 and instruct the definitive position of the forelimb. Furthermore, by comparing the dynamics of Hoxb genes activation during zebra finch, chicken, and ostrich gastrulation, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether, our results that characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb positioning in avians show a direct and early role for Hox genes in this process.

Keywords: Hox genes; Tbx5; birds; chicken; collinearity; gastrulation; lateral plate mesoderm; limb; patterning.

Graphical abstract

Figure 1

The Forelimb Position Is Already Determined by the End of Gastrulation (A) LPM rotation procedure in a stage 11 chicken embryo. FL, Forelimb; IL, Interlimb. (B–D) Fgf10 (B and C) and Tbx5 (D) expression 48 hr after LPM rotation, showing complete (B; n = 3/23 embryos, red brackets) or partial (C and D; n = 12/23 embryos, red arrowhead) displacement of the forelimb bud. (E) Chicken embryo grafted with memGFP transgenic quail somatopleure (green) showing a posterior displacement of the forelimb upon rotation (n = 3/3 embryos). (F–I) Transverse sections of quail-chick chimera at the forelimb (F and G) and interlimb (H and I) levels, stained with phalloidin (red), GFP antibody (green), and DAPI (blue). (G) and (I) are higher magnification of (F) and (H), respectively. NT, neural tube; S, somite; IM, intermediate mesoderm; Som, somatopleure. Scale bars are 500 μm in (B)–(E) and 100 μm in (F)–(I).

Figure 2

Progressive Formation of the LPM and Concomitant Patterning by Hox Genes (A) Time series showing LPM formation from stage 5 to stage 11. LPM precursors are electroporated with H2b-RFP (red) and GFP (green). White brackets outline the presumptive LPM in the PS; white asterisks, Hensen’s node; PS, primitive streak; prLPM, presumptive LPM; HF, head folds. See also Video S1. (B) Position of electroporated cells in the LPM (y axis) as a function of their timing of ingression (x axis); n = 57 tracked cells, 8 embryos (see Video S2). (C) Timeline of Hoxb4 (red), Hoxb7 (light blue), and Hoxb9 (blue) activation in the presumptive LPM with regard to LPM formation (black arrows). For detailed expression data, see Figure S2. (D) Schematic summarizing anterior (red) and posterior (blue) Hox genes expression in the LPM. (E–G) Expression of Hoxb4 (E), Hoxb7 (F), and Hoxb9 (G) at stage 13. (E’)–(G’) are higher magnifications of (E)–(G), showing Hox genes posterior (Hoxb4; E’) or anterior (Hoxb7 and Hoxb9; F’ and G’, respectively) border of expression in the LPM (dashed black line). Red asterisks mark the somite 20. (H–L) Stage 13 embryos, electroporated at stage 4 with GFP (H), Hoxb4/GFP (I), Hoxb7/GFP (J), Hoxb9/GFP (K), or Hoxb4 dn-GFP (L). Yellow brackets highlight different distribution of electroporated cells in the LPM. (M and M’) Hoxb4 endogenous expression (M’) in a Hoxb4 dn-GFP electroporated embryo (M). Note the decrease in Hoxb4 expression on the electroporated side (red brackets, n = 13/21 embryos) compared to the control side (black dashed line). (N) Distribution of electroporated cells along the A-P axis for GFP-only (gray, 16 embryos, 6,994 cells), Hoxb4/GFP (red, 12 embryos, 3,203 cells), Hoxb7/GFP (green, 11 embryos, 2,359 cells), Hoxb9/GFP (blue, 15 embryos, 3,922 cells), and Hoxb4 dn-GFP (dashed red, 22 embryos, 20,528 cells) electroporated embryos. Distribution is presented as mean ± SEM. Scale bars are 100 μm. See also Figures S1, S2, and S3A and Videos S1, S2, and S3.

Figure 3

Hox Genes Determine the Position of the Early Tbx5-Positive Forelimb Field (A and B) Stage 15 embryos electroporated at stage 4 with GFP (A; n = 4 embryos) or Hoxb4 dn-GFP (B; n = 9 embryos). Note that intense signal in the anterior somite and neural tube in (B) is non-specific auto-fluorescence. (A’ and B’) Tbx5 expression in corresponding embryos is shown. Note the decrease in Tbx5 expression on the electroporated side of Hoxb4 dn-GFP electroporated embryo (B’; red brackets; n = 7/9 embryos). (C–F) Embryos electroporated with GFP alone (C; n = 20 embryos) or in combination with Hoxb4 (D; n = 16 embryos), Hoxb4+Hoxc9 dn (E; n = 19 embryos), or Hoxc9 dn (F; n = 15 embryos) in the interlimb domain at stage 14 and re-incubated for 24 hr. Note that variation in the position of the electroporated domain is due to technical variation. (C’–F’) Tbx5 expression in corresponding embryos is shown. Ectopic Tbx5 expression in the interlimb region is denoted by red arrowheads compared to normal endogenous expression denoted by green arrowheads (E’; n = 9/19 embryos). Scale bars are 100 μm. See also Figure S3.

Figure 4

Hox Genes Regulate the Definitive Forelimb Position (A–C) Embryos electroporated with GFP in combination with Hoxb4+Hoxc9 dn in the interlimb domain at stage 14 and re-incubated for 48 hr. The unilateral expansion of the forelimb is denoted by red arrowheads compared to contralateral side denoted by green arrowhead (n = 13/22 embryos). (A’)–(C’) shows Fgf10 (A’), Tbx5 (B’), and Shh (C’) expression in corresponding embryos. White arrowhead in (A’) points at ectopic Fgf10 expression within the electroporated region. (D–G) Embryos electroporated with GFP alone (D and F; n = 7 embryos) or in combination with Hoxb4+Hoxc9 dn (E and G; n = 22 embryos) in the interlimb domain at stage 14 and re-incubated for 4 days. (F and G) Alcian Blue staining in GFP (F; n = 5 embryos) or Hoxb4-Hoxc9 dn-GFP (G; n = 9 embryos) electroporated embryos is shown. Note the posterior shift of the forelimb on the right electroporated side of Hoxb4-Hoxc9 dn-GFP electroporated embryos (E and G; n = 11/22 embryos) highlighted with red dashed line (E) and red arrow (G). Stars, somites; dashed lines, forelimb outline; arrows, vertebral level of the forelimb. Scale bars are 200 μm in (A)–(C) and (A’)–(C’) and 500 μm in (D)–(G). See also Figure S4.

Figure 5

Variations in Wing Position Correlate with Changes in Tbx5, Hoxb4, and Hoxb9 Expression Domains in the LPM (A–C) Alcian blue-Alizarin red staining of chicken (A; E20), zebra finch (B; E13), and ostrich (C; E37). Red arrowheads point at the wing position level (15^th, 13^th, and 18^th vertebrae in chicken, zebra finch, and ostrich embryos, respectively); red dots mark each cervical vertebra. (D–F) Tbx5 expression in stage 18 chicken (D), zebra finch (E), and ostrich embryos (F). The position of Tbx5 posterior border of expression is indicated in somite number. (E’) and (F’) are higher magnification of (E) and (F), respectively. (G–I) Hoxb4 expression in chicken (G; 20-somite stage), zebra finch (H; 20-somite stage), and ostrich embryos (I; 34-somite stage). The position of Hoxb4 posterior border of expression is indicated in somite number. (H’) and (I’) are higher magnification of (H) and (I), respectively. (J–L) Hoxb9 expression in chicken (J; 20-somite stage), zebra finch (K; 20-somite stage), and ostrich embryos (L; 36-somite stage). The position of Hoxb9 anterior border of expression is indicated in somite number. (K’) and (L’) are higher magnification of (K) and (L), respectively. Dashed black lines show variation in posterior-anterior border of expression in zebra finch (E’, H’, and K’) and ostrich embryos (F’, I’, and L’) compared to chicken embryo (represented by red asterisks). Scale bars are 3 mm in (A)–(C) and 100 μm in (D)–(L), (E’), (F’), (H’), (I’), (K’), and (L’).

Figure 6

Relative Changes in Hox Collinear Activation Timing Underlie Variation in Limb Position between Chicken and Ostrich (A–I) Hoxb4 expression in chicken (A–D) and ostrich (E–I) embryos. (J–R) Hoxb9 expression in chicken (J–M) and ostrich (N–R) embryos. (S) Timeline of Hoxb4 (red) and Hoxb9 (blue) activation in chicken (top diagram) and ostrich (bottom diagram). Scale bars are 100 μm. Asterisks represent the Hensen’s node; black brackets outline the presumptive LPM.

Figure 7

Changes in Retinoic Acid Signaling during Gastrulation Modulate the Forelimb Field Position (A–C) Cyp26a1 expression in zebra finch (A), chicken (B), and ostrich (C) embryos. Red arrowheads highlight the onset of Cyp26a1 expression in the presumptive LPM (black brackets); black asterisks, Hensen’s node; H, head. (D–F) Hoxb4 expression in stage 13 control (D), retinoic-acid-treated (E), and AGN193109-treated (F) embryos. (G) Position of Hoxb4 posterior border of expression in control (n = 14 embryos), retinoic-acid-treated (n = 15 embryos), and AGN193109-treated embryos (n = 22 embryos). (H–J) Hoxb9 expression in stage 13 control (H), retinoic-acid-treated (I), and AGN193109-treated (J) embryos. (K) Position of Hoxb9 anterior border of expression in control (n = 15 embryos), retinoic-acid-treated (n = 18 embryos), and AGN193109-treated embryos (n = 23 embryos). (L–N) Tbx5 expression in stage 18 control (L), retinoic-acid-treated (M), and AGN193109-treated (N) embryos. (O) Position of Tbx5 posterior border of expression in control (n = 12 embryos), retinoic-acid-treated (n = 15 embryos), and AGN193109-treated embryos (n = 13 embryos). Red lines show changes in posterior-anterior border of expression in treated embryos compared to control embryos (dashed black lines). Scale bars are 100 μm. (G, K, and O) Each dot represents one embryo; error bars represent mean ± SEM. Statistical analysis, ANOVA with Fisher least significant difference (LSD) post hoc test; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.