WNT5A/JNK and FGF/MAPK pathways regulate the cellular events shaping the vertebrate limb bud

Abstract

Background: The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown.

Results: We show that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized. Using time-lapse video microscopy, we demonstrate that cells move and divide according to this orientation. The combination of oriented cell divisions and movements drives the proximal-distal elongation of the limb bud necessary to set the stage for subsequent morphogenesis. These cellular events are regulated by the combined activities of the WNT and FGF pathways. We show that WNT5A/JNK is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb.

Conclusions: Together, these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development.

Figure 1. Characterization of Limb Bud Elongation at the Tissue and cellular Level

(A–H) Three dimensional reconstructions of Optical Projection Tomography (OPT) acquisitions at the level of the limb bud of chick embryos at stages HH18 (A, E), 20 (B, F) 21 (C, G) and 23 (D, H) showing dorsal (A, B, C, D) and lateral views (E, F, G, H). The red dots show where the measurements were made, see Movie S1. (I) Measurements of the length (in μM) of the antero-posterior (A-P, blue line), dorso-ventral (D-V, red line) and proximo-distal (P-D, green line) axes show that the limb bud elongates primarily in the P-D axis. A minimum of n=8 Limbs were analyzed for each time point, Error bars represent standard errors of the mean. (J) Transverse section of an electroporated chick embryo at stage HH18 revealing the shape of GFP-expressing cells. (K) Schematic representing the section shown in (D). A few GFP expressing cells have been outlined to show their orientation and elongated shape. (L) Transverse section of an electroporated embryo at stage 20 showing the shape of GFP expressing cells (in green). (M) Schematic representing four regions (dorsal, ventral, central and distal) of the section shown in (L). (N–Q) Enlargements of the dorsal (N), ventral (O), central (P) and distal (Q) limb bud regions of a chick embryo at stage HH21 embryos showing the shape of the GFP-expressing cells. (R–U) Quantification of the angle between the P-D axis of the limb bud and the longest axis of GFP expressing cell observed in the dorsal (R), ventral (S), central (T)_ and distal (U) regions at stage HH21. Angle of each cell longest axis is shown on a bidirectional Rosette graph which is divided into bins of 5°. The number of cells per bin is indicated on the radial axis. Quantifications were made on a total number of n=1468 cells with a minimum of n=300 cells for each area. Scale Bars in the lower right corner represent 50 μm (J, L) and 20 μm (R–U)

Figure 2. Cells of the limb exhibit oriented cell movements and oriented cell division

(A) Projection of a 15 hours time series view (corresponding to the time-lapse movie S2) of a limb explant of a GFP electroporated chick embryo. The projection is color coded; early times series are displayed in blue and late time series are progressively displayed in orange then white as the time lapse experiment progresses as indicated by the bar in the lower right corner. (see Movie S2) (B) Cell tracks from time lapse experiment presented in (A) (the colors were randomly chosen). Cells were manually tracked. (C) Schematic representing for each cell tracked in (B) the net movement (as shown by the length of each arrow) and the direction (as shown by the arrowheads) (D) Quantification of cell velocity (in μm.min⁻¹, second panel,), efficiency (i.e., ratio between the distance between t0 and tf and and the distance of covered by the whole track, third panel) and coherence (i.e., standard deviation in angle of two cell directions within a range of 50 μm, fourth panel) in three arbitrarily subdivided areas (represented by shades of green) along the P-D axis (as schematized in the first panel). (E) View of the first time series (t=0) from the experiment presented in (A). (F) Schematic representing the orientation of each cell (red arrows) that divided during the course of the time lapse experiment in (A). (See movie S2) (G) Quantification of the angle between the P-D axis of the limb bud and the axis of cell division. Angle of each cell division is shown on a unidirectional Rosette graph which is divided in bins of 5°. The relative percentage of cell division per bin is indicated on the radial axis. Quantifications were made on a total number of n=331 cell divisions, 5 embryos. (H–I) Time series at t=0 (H), t=52 min (I) and t=164 min (J) of a time lapse experiment (Movie S3) showing preferential P-D cell division at the distal end of the limb bud. The colored arrowheads points at co-electroporated cells with both a GFP (in green) and H2bRFP (in red) construct and their progeny (same arrowhead color). Scale Bars in the lower right corner represent 50 μm (B, C, E) and 20 μm (H–J)

Figure 3. Wn5a regulates limb bud elongation and cell orientation in the mouse

(A) Wnt5a expression detected by in situ hybridization showing higher expression in the distal mesenchyme of the early limb bud. (B) Wild Type (WT) (green) and Wnt5a^−/− mutant limb buds (red) reconstructed and virtually dissected from Optical Projection Tomography acquisitions of E10.5 Mouse embryo. Wnt5a^−/− limbs exhibit elongation defects and appear roundish as compared to control. (See Movie S4) (C) Measurements (in μM) of the A-P (left bars), D-V (central bars) and P-D (right bars) axes lengths in WT (green bars) and Wnt5a^−/− (red bars) embryos. (D–E) Transverse sections of WT (D) and Wnt5a^−/− (E) mouse embryos at E10.5 stained with Phalloidin (in white). (F) Outline of the WT (in green) and Wnt5a^−/− (in red) limb buds shown in (D) and (E), respectively, showing the effect of the loss of Wnt5a on the relative proportions of the limb bud (i.e., A-P axis, white arrowheads and P-D axis (white arrow)). (G, J) Transverse sections of WT GFP X^+/− (G) and Wnt5a^−/−; XGFP^+/− (J) mouse embryos at E9.25 showing the shape of GFP expressing cells. (H, K) Schematics representing outlines of GFP expressing cells from sections shown in (G) and (J). (I, L) Schematics showing the net movement (arrows lengths) and the direction (arrowheads) of cells during time lapse experiments performed in WT GFP X^+/− (I) and Wnt5a^−/−; XGFP^+/− (L) mouse embryos at E9.25 (Movie S5). (M) Quantification of cell velocity (first panel), efficiency, (second panel) and coherence (third panel) in the proximal, central and distal regions of WT mouse limb bud (represented by shades of green, as schematized in the first panel of Figure 2D) and in the most distal and dorsal/ventral parts of Wnt5a ^−/− mouse limb buds ( dark red and light red respectively). (N–O) Transverse section of chick limb buds electroporated with a GFP construct (in green) and implanted with control (O) or cWnt5a-expressing (N) DF1 cells stained with DiI (in red). (P, R) First time series (t=0) from time lapse experiments (Movie S6) showing control DF1 cells (R) or cWnt5a-expressing cells (P, in red) implanted in limb buds previously electroporated with a GFP construct (in green). (Q, S) Schematics showing the net movement (arrows lengths) and direction (arrowheads) of GFP-expressing cells from (P) and (R). GFP-expressing cells move toward the source of Wnt5a in (Q), and normally, towards the ectoderm in (S). The position of control DF1 cells (S) and Wnt5a-expressing cells (Q) is indicated in red. (see Movie S6). (T) Quantification of cell velocity (first panel), efficiency, (second panel) and coherence (third panel) of GFP labeled cells surrounding implanted DF1 cells (in green) or Wnt5a expressing cells (in red). Scale Bars in the lower right corner represent 50 μm.

Figure 4. Wnt5a/JNK regulates orientation of cell division

(A, G) First time series (t=0) of time lapse experiments (Movie S7) on limb buds of WT/H2bGFP (A) or Wnt5a^−/−; H2bGFP (G) mouse embryos at E9.5. (B, H) Schematics representing the orientation of cell division (red arrows) from time lapse experiments in (A) and (G), respectively. (C–E, I–K) Enlargements of the dorsal (C, I), distal (D, J) and ventral (E, K) areas showing regional differences in the orientation of cell divisions. (F, L, P) Quantifications of the angle between the P-D axis and the axis of cell divisions identified from the time lapse experiments performed in WT H2bGFP (F) and Wnt5a^−/−; H2bGFP (L) mouse embryos at E9.5, and chick limb buds treated with SP600125 (P), respectively as shown in (A, G and M). The rosette graph is divided in bins of 5°, the relative proportion in percentage of cell division per bin is indicated on the radial axis. (M) First time series (t=0) from a time lapse experiment (see Movie S7) of chick limb bud explants electroporated with GFP (in green) and H2bRFP (in red) constructs and cultured in presence of the JNK inhibitor SP600125. (N) Schematic showing the net movement (arrows lengths) and the direction (arrowheads) of cells shown in (M). (O) Schematic representation of the direction of cell division (red arrows) shown in (M) (Q) Quantification of cell velocity ( first panel), efficiency, (second panel) and coherence (third panel) in the proximal, central and distal regions (represented by shades of orange, as schematized in the first panel of Figure 2D) of chick limb treated with SP600125.

Figure 5. FGF/MAPK signalling promotes cell movement

(A) Transverse section showing FGF8 expression detected by in situ hybridization in the distal ectoderm of a chick limb bud. (B) Transverse section of a limb bud stained with a phosphorylated-ERK/MAPK antibody. (C) Intensity profile of pERK (green line) and non specific red autofluorescence (red line) along the Proximal-Distal axis of the limb as shown in (B) correlated with the velocity of cells from the proximal, central and distal regions of the limb bud ( Columns) as shown in Figure 2D. (D) Transverse sections of chick embryos limb buds co-electroporated with constitutively active Mek (CA-MEK) and GFP constructs. (E) High magnification showing lamellipodia of electroporated, GFP expressing cells (white arrowheads). (F) Transverse sections of chick limb buds electroporated with both dominant negative MEK1 and GFP constructs. (G) High magnification showing filopodia protruding from cells electroporated with dominant negative MEK1 and GFP (white arrows). (H, K, N) First time series (t=0) of time lapse experiments (Movie S8) on chick limb buds electroporated with Constitutive active MEK1/GFP (K), GFP only (H, N) but cultured in presence of the MEK1 inhibitor U1026 (H), or GFP only and cultured with a bead soaked in FGF8 (N) (see movie S8). (I, L, O) Cell tracks from a time lapse experiment shown in (H), (K) and (N), respectively. (J, M, P) Schematics representing the net movement (arrows lengths) and direction (arrowheads) of cells tracked (I), (L), and (O), respectively. (Q) Left graph: quantifications of the velocity (in μm.min⁻¹, left panel) of cells electroporated with GFP-only (left bars), constitutive active MEK1 (central left bars), in presence of FGF8 beads (central right bars, yellow) or U0126 (right bars).within the proximal, central and distal regions (shades of green, see Fig. 2D) of the limb bud. Centre graph: Quantification of the efficiency of proximal cells of the limb electroporated with GFP only or in presence of a FGF8 bead (yellow). Right graph: quantification of the coherence of the movement of cells located within the proximal region of the limb bud electroporated with GFP (in green) or exposed to FGF8 beads (in red). Scale Bars in the lower right corner represent 50 μm.