Wyngaarden LA , Vogeli KM , Ciruna BG , Wells M , Hadjantonakis AK , Hopyan S .

R01-DK084391 NIDDK NIH HHS , R01-HD052115 NICHD NIH HHS , R01 DK084391 NIDDK NIH HHS , R01 HD052115 NICHD NIH HHS

	Fig. 1. Mesoderm trajectories during mouse early limb outgrowth. For vector velocity fields (VVFs), longer arrow length and red end of the spectrum correlate with higher relative velocity within a given experiment. (A) VVF of lateral plate mesoderm adjacent to somites 8-12 of a CAG::H2B-EGFP transgenic embryo at late Theiler stage 14 (E9.0-9.25, 18-20 somites), just prior to limb outgrowth. Rostral-to-caudal tissue movement relative to a stationary somite boundary (asterisk) is evident. Inset depicts field of view. (B) Dorsal view of early forelimb buds of a Theiler stage 15 (E9.25-9.5, 21-25 somites) wild-type (WT) embryo. The inset and arrow depict the direction of view; the orange line indicates the margin of the right-hand side lateral plate mesoderm. The hindlimb bud is beyond the field of view. The boxed regions indicate the field of view in C-E. (C) VVF of the anterior margin of a early WT limb bud at Theiler stage 15. Mesoderm moves obliquely from lateral plate mesoderm (lpm) toward the limb bud (lb). (D,E) Separate z-stacks of a Theiler stage 15 WT forelimb bud. Within the early limb bud, mesoderm moves in a posterodistal direction. Tissue near the posterior margin rotates in a proximal direction. (F) Schematic depiction of regional tissue vectors during early limb outgrowth. (G) Lateral plate mesoderm of Wnt5a–/–;CAG::H2B-EGFPTg/+ at Theiler stage 14 lacks the coordinated movement seen in the WT embryo (A). Asterisk overlies somite. (H) The long-body axis and lateral plate mesoderm of a Wnt5a–/–;CAG::H2B-EGFPTg/+ embryo at Theiler stage 15 are shortened as compared with the WT embryo (B). The orange outline delineates the truncated lateral plate mesoderm and the hindlimb bud (*), which is in abnormally close proximity to the forelimb bud. (H′) Expression ofTbx4 by in situ hybridisation confirms that the bulge close to the forelimb is indeed the hindlimb. The forelimb (boxed region) is shown in I. (I) Tissue movement into the early Wnt5a–/–;CAG::H2B-EGFPTg/+ limb bud is evident, albeit at a lower velocity, compared with WT embryos (see text).
	Fig. 2. Regional lineage of chick lateral plate mesoderm during early wing bud outgrowth. Merged bright-field and fluorescent images. (A,A′) Chick embryo labelled with spots of DiI (orange) at HH 16 (26-28 somites) at the start of wing bud outgrowth and cultured in ovo. The rostral-most spots on the left side are adjacent to somites 17/18. (B,B′) In the same embryo at HH 20, DiI-labelled tissue has become displaced into the wing bud. Streaks of DiI suggest caudal-lateral oblique movement of mesoderm into the anterior wing bud from a rostral position. (C-D′) Lineage tracing in another embryo with images taken before (C,C′) and after (D,D′) culture in ovo. DiI spots adjacent to somites 19/20 at HH 16/17 (C,C′) were displaced in a linear fashion by HH 20, heading directly lateral (D,D′). These findings are consistent with the movements observed in the mouse embryo, as shown in Fig. 1. (E-H) Pre- and post-culture images showing that chemoattraction of DiI-labelled mesoderm is not apparent toward a bead (arrowheads) soaked in PBS (E,F), but is apparent toward a bead soaked in Wnt5a protein in PBS (G,H).
	Fig. 3. Lineage and movement of mesoderm during early pectoral fin development in zebrafish. (A) Fluorescein uncaged in the lateral plate mesoderm (arrow) at 16 hpf (18 somites). (B) By 28 hpf (30 somites), in the same embryo as in A, focal fluorescence is found in the early fin bud (arrow). (C) In tbx5 morpholino (MO) knockdown embryos, a fin bud fails to form and labelled tissue becomes scattered throughout the mesoderm (arrow). (D) Summary of lateral plate lineage-tracing experiments. Mesoderm adjacent to somites 1 to 4, but not 5, contributes to the pectoral fin bud. (E) Since the pectoral fin bud arises adjacent to somites 2 and 3, we infer that lateral plate mesoderm condenses during fin initiation. (F,F′) Field of view (boxed) and position of a maturing pectoral fin bud at 44 hpf (arrowhead), positioned as in G-I. (G) VVF of an h2af/z:gfp transgenic embryo at 44 hpf. (H) Schematic representation of tissue movements in the maturing (35-45 hpf) fin bud. The movements are comparable to those of the mouse limb bud, taking into account the different orientation of the two buds with respect to the embryo body. (I) VVF of a 44 hpf embryo treated with 4 μM latrunculin A. Tissue motion is halted (but not with 0.1% DMSO carrier) as demonstrated by the lack of arrows under PIV analysis.
	Fig. 4. Regional differences in cell shape during early limb bud outgrowth. (A-F) Theiler stage 15 (21-25 somites) CAG::myr-Venus transgenic mouse embryos imaged live using a confocal microscope. Rostral positions are towards the top of the images. Somites, which contain cells of distinctive shape for comparison, are indicated by asterisks (A,B). WT lateral plate mesoderm (lpm) cells adjacent to the early limb bud are oriented longitudinally, parallel to the rostrocaudal axis of the embryo. This is observed as longitudinal streaking adjacent to the somites in A, and anteromedial to the early limb bud in C. Cells within the early WT limb bud (lb) are isotropic (A,C,E). By contrast, cells in Wnt5a–/– littermates (B,D,F) are isotropic in both the lateral plate and the limb bud. This finding correlates with the shortening and relative lack of tissue movement observed in Wnt5a–/– lateral plate mesoderm. (G,H) GM130 (Golga2) stain (green) highlights the location of the Golgi relative to nuclei (DAPI, blue). In Theiler stage 14 embryos (20 somites), Golgi are commonly found caudal and lateral to corresponding nuclei in the WT forelimb mesoderm field (arrows), but not in Wnt5a–/– mutants. Since Golgi are found at the leading edge of motile cells, these data correlate well with the direction of tissue motion in WT embryos, and the lack of movement of Wnt5a–/– mesoderm demonstrated in Fig. 1. (I,J) Polar plots summarising Golgi angle in relation to the nuclear centre, with embryonic reference marks as shown. (K-M) Separate z-stacks of a live βactin:hras-egfp transgenic zebrafish fin bud at 42 hpf. Lpm cells at the base of the fin bud (blue arrowhead, K) are elongated, parallel to the rostrocaudal axis of the embryo. Most cells in the fin bud are isotropic. However, cells in the proximal-anterior region of the fin bud are elongated, with an anteroproximal-to-posterodistal long axis, parallel to their direction of movement (red arrowhead, L). Some of these cells exhibit protrusions at their posterodistal tip (white arrowheads, K,M). ecto, ectoderm; lpm, lateral plate mesoderm; lb, limb bud; fb, fin bud.
	Fig. 5. Evidence for regionally localised oriented cell division during early limb development in mouse and zebrafish. (A) Method of measuring cell division angle. h2af/z:gfp transgenic zebrafish chromatin undergoing mitosis (arrowheads) is visualised on successive frames. The angle of a line joining the daughter chromatin centres at telophase is measured with reference to the longitudinal axis of the lateral plate mesoderm (fourth frame down). The level at which the limb bud protruded beyond the lateral plate was selected as the boundary between these two regions on every individual z-stack (long white line, fourth frame down). Every mitotic angle visualised on all z-stacks, together encompassing the entire limb bud, was measured over the whole duration of each time-lapse experiment. (B-J) Polar plot representations of cell division planes in which the rostrocaudal axis is defined at 0/180°, and the lateral and future distal axis of the limb bud is defined at 90°. The proportion of cell divisions in each 30° segment is represented by the length of the segment. (B) During mouse Theiler stage 14 (18 somites), lateral plate mesoderm adjacent to somites 8-12 exhibits a preferential plane of cell division that is parallel to the rostrocaudal embryo axis. At Theiler stage 15 (21-25 somites), although the same preferential plane of division is found in the lateral plate (C), cells in the early limb bud exhibit a different orientation of division that is perpendicular to the rostrocaudal axis, but parallel to the direction of bud outgrowth (D). In Wnt5a–/– mutants, orientation of cell division in the lateral plate is less apparent (E), although some orientation in the limb bud is evident (F). This finding correlates with the severe shortening and lack of tissue movement in the lateral plate mesoderm of Wnt5a–/– mutants, whereas budding of the limbs takes place with evident cell movement, as seen in Fig. 1. In zebrafish at 35 hpf, the orientation of cell division is similar to that of the mouse embryo (G), although more pronounced in the limb bud (H). By 44 hpf, orientation of cell division in the lateral plate is lost (I), while it persists in the fin bud (J).

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