The vertebrate limb bud arises from lateral plate mesoderm and its overlying ectoderm. Despite progress regarding the genetic
requirements for limb development, morphogenetic mechanisms that generate early outgrowth remain relatively undefined. We
show by live imaging and lineage tracing in different vertebrate models that the lateral plate contributes mesoderm to the early
limb bud through directional cell movement. The direction of cell motion, longitudinal cell axes and bias in cell division planes lie
largely parallel to one another along the rostrocaudal (head-tail) axis in lateral plate mesoderm. Transition of these parameters
from a rostrocaudal to a mediolateral (outward from the body wall) orientation accompanies early limb bud outgrowth.
Furthermore, we provide evidence that Wnt5a acts as a chemoattractant in the emerging limb bud where it contributes to the
establishment of cell polarity that is likely to underlie the oriented cell behaviours.
RESEARCH ARTICLE2551Development 137,2551-2558(2010) doi:10.1242/dev.046987◎ 2010. Published by The Company of Biologists Ltd RESEARCHARTICLE2552Development 137 (15) Accepted 18 May 2010 Oriented cell motility and division underlie early limb budmorphogenesis Laurie A. Wyngaarden1, Kevin M. Vogeli, Brian G. Ciruna13, Mathew Wells4, Anna-Katerina Hadjantonakis5and Sevan Hopyan1.6,* SUMMARY The vertebrate limb bud arises from lateral plate mesoderm and its overlying ectoderm. Despite progress regarding the geneticrequirements for limb development, morphogenetic mechanisms that generate early outgrowth remain relatively undefined. Weshow by live imaging and lineage tracing in different vertebrate models that the lateral plate contributes mesoderm to the earlylimb bud through directional cell movement. The direction of cell motion, longitudinal cell axes and bias in cell division planes lielargely parallel to one another along the rostrocaudal (head-tail) axis in lateral plate mesoderm. Transition of these parametersfrom a rostrocaudal to a mediolateral (outward from the body wall) orientation accompanies early limb bud outgrowth.Furthermore, we provide evidence that Wnt5a acts as a chemoattractant in the emerging limb bud where it contributes to theestablishment of cell polarity that is likely to underlie the oriented cell behaviours. KEY WORDS: Wnt5a, Cell migration, Cell polarity, Limb bud, Morphogenesis, Oriented cell division, Mouse, Zebrafish, Chick INTRODUCTION The earliest morphological sign of vertebrate limb formation is anapparent thickening of the lateral plate mesoderm. Forelimbinitiation occurs at a stage of development when roughly half of thesomites have been laid down, while the rostrocaudal axis of theembryo, including the lateral plate, continues to elongate. Initially,the limb ridge is elongated and grossly symmetrical in therostrocaudal axis (which corresponds to the anteroposterior axis ofthe limb). During the first day of outward (proximal to distal)growth, the limb bud acquires a stereotypical shape that is broaderposteriorly than anteriorly and is relatively narrow in thedorsoventral axis. Some of the key molecular requirements for limbinitiation and early outgrowth have been identified, and include T-box transcription factors (Agarwal et al., 2003; Ahn et al., 2002;Minguillon et al., 2005; Naiche and Papaioannou, 2003) andsignalling by fibroblast growth factor 10 (Fgf10) (Min et al., 1998;Sekine et al., 1999). By contrast, the cellular dynamics and tissuemechanisms underlying formation of the initial limb ridge andremodelling of the early limb bud are not well understood. Several cellular mechanisms that might underlie limb initiationhave been proposed. Previous analysis in the chick demonstratedthat the limb-forming region of the lateral plate mesodermmaintains a high proliferative rate, while that of the trunk regiondiminishes during early limb outgrowth (Searls and Janners, 1971).However, at least for the established limb bud, differentialproliferation cannot fully explain outgrowth.Computer simulationof limb bud growth, based solely on the regional distribution of cellproliferation found in the bud (Fernandez-Terán et al., 2006), fails ( l Program in Developmental and Stem Cell Biology, The Hosp i tal for S ick Chi l dren, T oronto, M5G 1X8, Canada. D epartments of Anatomy and of Biochemistry andBiophysics, University of Californ i a, San Francis c o, CA 94158,USA. 3Department ofMolecular Genetics, University of Toront o , M5 S 1A8, Canada. 4Department of P hysical and Environmental Sciences, University o f Toront o , M1 C 1A4, Canada.Developmental Biology Program, Sl o an-Kettering Institute, New York, NY 10065,USA. 6Division of O rthopaedic Surgery, T h e Hospital for Sick Children, University of Toronto, Toronto , M5G 1L5, Canada. ) ( *Author for correspondence (s ev an. h o p yan@ si ckk i ds .c a) ) to predict the generation of an appropriately shaped elongatinglimb bud, but rather predicts the generation of a relatively sphericalmorphology (Boehm et al., 2010). Therefore, anisotropic ororiented cell behaviours might contribute to limb bud outgrowthand the generation of its stereotypical shape. Evidence for the presence of cell movement in the limb bud isfound in both mouse and zebrafish models. In mature mouse limbbuds at embryonic day (E) 11.0, four-dimensional time-lapseimaging revealed outward, rotatory movement of the surfaceectoderm (Boot et al., 2008), although these studies were not atcellular resolution and neither mesoderm nor early stages wereassessed. Lateral plate mesoderm cells lacking tbx5 in zebrafish(Ahn et al., 2002) fail to enter the pectoral fin bud. Furthermore,Fgfrl mutant cells in mouse embryo chimaeras fail to populate thelimb, in contrast to wild-type (WT) cells, which do (Ciruna et al.,1997; Saxton et al., 2000). It has also been shown that Fgf4, whichis secreted by the apical ectodermal ridge (AER), can act as achemoattractant (Li and Muneoka, 1999). An intriguing alternativemechanism is that Fgf might function to increase the liquid-likecohesiveness of mesoderm in the limb field (Damon et al., 2008;Heintzelman et al., 1978). This might cause the limb field to phaseseparate from the adjacent lateral plate mesoderm. In isolation, thisproperty would cause the limb field mesoderm to be engulfed bythe lateral plate. However, the lateral plate exhibits a unique active-rebound response that promotes limb bulging (Damon et al., 2008).Micromass culture data suggest that differential adhesiveness is animportant mechanism that underlies the segregation of cells in themature limb bud into proximodistalddomains (Barna andNiswander,2007). Another signalling molecule that might contribute to cellmovement during limb outgrowth is Wnt5a. The Wnt5a gene isexpressed in the elongating tail bud and in the early ventral limbbud ectoderm, then shortly thereafter in the distal limb budectoderm and mesoderm, among other areas of outgrowth (Gavinet al., 1990; Yamaguchi et al., 1999). Mouse embryos lackingWnt5a exhibit shortened rostrocaudal body axes and limbs(Yamaguchi et al., 1999). Wnt5a is able to cause directional cellmovement in vitro by reorienting the cytoskeleton in response to a chemokine gradient (Witze et al.,2008). It is conceivable that asimilar mechanism might contribute to limb bud outgrowth inaddition to the known positive effect of Wnt5a on mesodermproliferation (Yamaguchi et al., 1999). By contrast, it has been suggested that cell movement is a featureof limb formation only in lower vertebrates, and not in mouse orchick (Rallis et al., 2003). However, a direct survey of individualcell behaviours during early limb outgrowth in the mouse or chickhas not previously been undertaken. The possibility that orientatedcell division occurs during limb bud outgrowth has been addressed,although not systematically tested (Hornbruch and Wolpert, 1970).Here we utilisee genetic, live-imaging and lineage-tracingtechniques to directly survey the movements, shapes and divisionplanes of mesodermal cells in mouse, chick and zebrafish embryosto define the morphogenetic mechanisms that generate the earlylimb bud and address whether equivalent cell behaviours drive thisevent across vertebrates. Our studies reveal the directionalmovement of mesoderm into the early limb bud, as well as spatiallydistinct biases in cell shape and cell division plane between thelateral plate and limb bud across species. A transition of theselargely parallel parameters accompanies, and is likely to contributeto, early outgrowth of the bud. Cell polarity, which is partiallyconferred by Wnt5a, is likely to underlie these oriented cellbehaviours. MATERIALS AND METHODS Embryo culture CAG::H2B-EGFP (Hadjantonakis and Papaioannou, 2004) and CAG::myr-Venus (Rhee et al., 2006) transgenic mouse lines were used, and crossedwith Wnt5a mutants (Yamaguchi et al., 1999). E9.25-9.5 embryos[corresponding to late Theiler stage 14 (18-20 somites) to stage 15 (21-25somites); Bard et al., 1998] were dissected and decapitated in DMEMcontaining 10% fetal calf serum. For live imaging, embryos weresubmerged just below the surface of optimised media (see Results)containing 25% DMEM and 75% rat serum. Cheese cloth or fragments of1% agarose were used to position the lateral plate mesoderm and early limbbud directly against a coverslip at the bottom of a metallic confocal well,such that the entire depth of the tissue under study could be visualised.Time-lapse imaging experiments were performed for periods of up to 3hours in a humidified chamber at 37°℃ in 5% CO2. The presence ofpyknotic nuclei disqualified live-imaging experiments from analysis. Two transgenic zebrafish lines, h2af/z:gfp (Pauls et al., 2001) andBactin:hras-egfp (Cooper et al., 2005), wereused. Embryos were culturedusing Mesab (tricaine, Sigma) anaesthetic in egg water at 28°C for up to 3hours in air. Some zebrafish embryos were cultured in egg water in thepresence of 4 uM latrunculin A or its carrier 0.1% DMSO. Image acquisition Laser-scanning confocal data were acquired using a Zeiss LSM 510 METAmicroscope system and a LiveCell culture chamber (Neue Biosciences).GFP and Venus fluorophores were excited using a 488 nm argon laser. Forthree-dimensional time-lapse experiments, a high frequency of mitoses anda lack of pyknotic nuclei in every acquired z-stack, as visualised directlywith the aid of the CAG::H2B-EGFP and h2afz:gfp reporters, confirmedgood tissue health (Plusa et al., 2008). During the stages investigated in thisstudy, there is normally little to no apoptosis in the mouse limb bud(Fernandez-Teran et al., 2006). Therefore, the presence of any pyknoticnuclei disqualified data sets from analysis. Confocal images were acquiredas z-stacks of xy images taken at 2-5 um z intervals. We focused on the two-dimensional coronal plane that is composed ofthe proximodistal (defined here as the x-axis) and rostrocaudal (oranteroposterior, defined as the y-axis) axes and did not record data in thedorsoventral (z) axis, as resolution along this axis is poor with currentinstrumentation and optimal acquisition for maintaining sample viability. Lineage tracing Chick embryos at Hamburger and Hamilton stage (HH) 15-17 wereexposed through a window in the shell. Hand-pulled glass needles wereused to deliver small spots of DiI to the lateral plate mesoderm. Embryoswere photographed using a Leica MZ16 F stereomicroscope to documentthe position of the dye, and then cultured in ovo at 39℃ until HH 20 priorto being rephotographed. Zebrafish embryos were injected at the one-cell stage with DMNB-FITCfor ubiquitous distribution and allowed to develop in the dark until 16hours post-fertilisation (hpf). Cells in the lateral plate were then labelledthrough photo-activation of the FITC with a 365 nm laser focused througha 20× objective on a compound microscope. Embryos were allowed todevelop until 28 hpf and were then immunostained against FITC to amplifythe signal. Chemotaxis assays Acrylic beads were soaked for 1 hour in Wnt5a (1 mg/ml), Fgf8 (1 mg/ml),sonic hedgehog (Shh) (1 mg/ml), retinoic acid (RA) (10ug/ml) (Helms etal., 1994), or carrier (PBS; DMSO for RA). HH 16 chick embryos wereaccessed in ovo through a window. The ectoderm overlying the lateral platemesoderm was incised just enough to allow implantation of an acrylic beadinto the mesoderm. Small spots of DiI were placed within 200 um rostraland caudal to the bead for optimal sensitivity based on the findings of aprevious chemotaxis assay (Li and Muneoka, 1999). Distance wasmeasured directly from captured images using the scale bar. Embryos werephotographed using a Leica MZ16 F stereomicroscope to document theposition of the dye relative to the bead and then cultured in ovo at 39°℃until HH 20, prior to being rephotographed. Maintenance of separationbetween the bead and the DiI was taken to imply a lack of chemoattraction,as was seen with all carrier-soaked beads. Approximation of previouslyseparated DiI to the bead in the form of a contiguous streak was taken toimply chemoattraction of mesoderm to the bead. Image processing Particle image velocimetry (PIV; DaVis, LaVision, Gottingen, Germany)was used to determine bulk tissue motion based on the trajectory offluorescent nuclei over successive time-lapse frames in two dimensions. Cell polarity Paraffin-embedded sections were stained against GM130 (Golga2), a Golgimatrix protein, and counterstained with the nuclear marker DAPI. Theangle, from 0-359°, at which the Golgi lay with respect to the centre of thenucleus was measured for individual cells using the rostrocaudal embryoaxis as the 0/180° line of reference. Confocal microscopy was used toobtain multiple z-stacks through the entire specimen under investigation.The nucleus-Golgi angles were measured for all cells while avoidingduplication by selecting z-stacks separated by one cell diameter.Measurements were grouped into twelve segments of 30°and wererepresented on a polar plot. Orientation of cell division Time-lapse movies of CAG::H2B-EGFPand h2af/z:gfp transgenic mouseand zebrafish limb buds imaged in the coronal plane were inspected frame-by-frame using Volocity software (Improvision). Confocal z-stacks,together encompassing the entire depth of the tissue under study, wereassessed individually. Care was taken not to record duplicate mitoses onadjacent z-stacks by ensuring that they were separated by one cell depth.Two regions were analysed separately: lateral plate mesoderm medial towhere the limb bud protrudes beyond the trunk, and the limb bud lateral tothat line. Tracing paper was used to draw a line precisely representing themetaphase-to-telophase transition of every single mitosis identified. Anglesof these lines were measured from 0-180° using a compass with referenceto the rostrocaudal axis of the embryo as derived from low-magnificationimages, with 0 representing the rostral end. The planes of cell division weregrouped into six segments of 30°and were represented on a polar plot withsegments shaded symmetrically about the centre. Segment length was usedto denote the proportion of divisions within that range. This methodologyyis capable of detecting a bias in cell division along the rostrocaudal andproximodistal axes, but not the dorsoventral axis. RESULTS The trajectory of tissue movement into the mouseearly limb bud To characterise cell movements during early limb bud outgrowth, wefirst established a system facilitating the ex utero development ofintact lateral plate and early limb bud mesoderm in which thosetissues could be live imaged. Transgenic CAG::H2B-EGFP mouseembryos (Hadjantonakis and Papaioannou, 2004) were used becauselocalisation of the H2B-GFP fusion reporter to chromatin facilitatesthe visualisation of nuclei during all phases of the cell cycle, suchthat cells can be segmented and individually tracked in fourdimensions (three-dimensional time-lapse). We optimised 3-hourstatic culture conditions of decapitated E9.0-9.5 (18-25 somites)mouse (embryosbby testing variousSmedia andrat serumconcentrations. Using the quality of extracted ribosomal RNA bandsas a readout, we determined that in a 5% CO2 environmentmaintained at 37°C, 75% rat serum supplemented with DMEMprovided optimal ex utero development of static samples in achamber resting on the microscope stage (see Fig. S1A in thesupplementary material). We confirmed that short-term culture underthese conditions did not result in increased cell death as assessed byLysoTracker Red staining (see Fig. S1B in the supplementarymaterial), nor in dysmorphology of the limb bud (see Fig. S1C in thesupplementary material). Using this formulation coupled with rapiddissection and short-term (up to 3 hours) time-lapse imagingexperiments, pyknotic nuclei (a sign of cell necrosis, see Materialsand methods) were rarely observed in CAG::H2B-EGFP transgenictissues during live-image data acquisition. As forelimb and hindlimb development might be subject todistinct kinetics, we chose to focus our studies on the forelimb.During Theiler stage 144 (~E9.0-9.25,,18-20 somites) theprospective mouse forelimb field appears as a barely perceptiblethickening of lateral plate mesoderm between somites 8 and 12.Live imaging at this site revealed tissue movement in a rostral-to-caudal direction relative to somite boundaries (n=8 embryosstudied) (Fig. 1A; see Movie 1 in the supplementary material). Thisfinding explains previous fate-mapping (Chaube, 1959) and Tbx5gene expression (Agarwal et al., 2003; Ahn et al., 200; Zhao etal., 2009) studies that suggest a caudal progression of theprospective forelimb-forming region of the lateral plate mesodermrelative to the somites, prior to overt limb morphogenesis. At Theiler stage 15 (~E9.25-9.5, 21-25 somites), lateral platemesoderm was seen entering the early forelimb bud at an obliqueangle from rostral positions (Fig. 1B,C), and laterally from morecentral positions relative to the limb bud (Fig. 1D; see Movie 2 inthe supplementary material). The relative velocity of movementgradually increased from proximal to distal within the bud (Fig.1D,E). This finding suggests that velocity is additive within thebud, and that the substrate for movements is the neighbouring cellsthemselves. Movement into the limb bud from caudal positionsrelative to the bud was not observed. Rather, tissue in the posterioraspect of the limb bud demonstrated rotational movement, suchthat cells at the posterior edge moved slightly back towards theembryo (n=10) (Fig. 1E; see Movie 2 in the supplementarymaterial). This pattern of movement is partially analogous to therotational motion of later limb ectoderm (Boot et al., 2008) and topolonaise movements that occur during primitive streak formation(Cui et al., 2005; Voiculescu et al.,2007), suggesting that rotationof tissue commonly accompanies longitudinal growth duringdevelopment. In summary, these data indicate that lateral platemesoderm cells migrate into the early mouse limb bud, and do soprimarily from rostral and lateral positions (Fig.1F). Wnt5a Wnt5a7/- Wnt5a7- Fig. 1. Mesoderm trajectories during mouse early limb outgrowth. For vector velocity fields (VVFs), longer arrow length andred end of the spectrum correlate with higher relative velocity within agiven experiment. (A) VVF of lateral plate mesoderm adjacent tosomites 8-12 of a CAG::H2B-EGFP transgenic embryo at late Theilerstage 14 (E9.0-9.25, 18-20 somites), just prior to limb outgrowth.Rostral-to-caudal tissue movement relative to a stationary somiteboundary (asterisk) is evident. Inset depicts field of view. (B) Dorsal viewof early forelimb buds of a Theiler stage 15(E9.25-9.5,21-25somites)wild-type (WT) embryo. The inset and arrow depict the direction ofview; the orange line indicates the margin of the right-hand side lateralplate mesoderm. The hindlimb bud is beyond the field of view. Theboxed regions indicate the field of view in C-E. (C) VVF of the anteriormargin of a early WT limb bud at Theiler stage 15. Mesoderm movesobliquely from lateral plate mesoderm (lpm) toward the limb bud (lb).(D,E) Separate z-stacks of a Theiler stage 15 WT forelimb bud. Withinthe early limb bud, mesoderm moves in a posterodistal direction. Tissuenear the posterior margin rotates in a proximal direction. (F) Schematicdepiction of regional tissue vectors during early limb outgrowth.(G) Lateral plate mesoderm of Wnt5a-;CAG::H2B-EGFPT9/+ at Theilerstage 14 lacks the coordinated movement seen in the WT embryo (A).Asterisk overlies somite. (H) The long-body axis and lateral platemesoderm of a Wnt5a-;CAG::H2B-EGFP19/+ embryo at Theiler stage15 are shortened as compared with the WT embryo (B). The orangeoutline delineates the truncated lateral plate mesoderm and thehindlimb bud (*), which is in abnormally close proximity to the forelimbbud. (H’)Expression ofTbx4 by in situ hybridisation confirms that thebulge close to the forelimb is indeed the hindlimb. The forelimb (boxedregion) is shown in l. (I) Tissue movement into the earlyWnt5a--;CAG::H2B-EGFPI9/+ limb bud is evident, albeit at a lowervelocity, compared with WT embryos (see text). Wnt5a has been implicated in the regulation of cell polarity anddirectional cell movement in a number of contexts, including themorphogenesis of early mesoderm (Sweetman et al., 2008; Witzeet al., 2008). Wnt5a mutant mice exhibit severe shortening of thebody axis and limbs (Yamaguchi et al., 1999). To determine whether this mutant phenotype might be due, in part, to a cellmigration defect, we used the CAG::H2B-GFP reporter to liveimage Wnt5a- embryos. At Theiler stage 14, lateral platemesoderm cells of Wnt5a’-embryos lacked the rostral-to-caudalmovement seen in WT littermates (n=5) (Fig. 1G; see Movie 3 inthe supplementary material). At Theiler stage 15, a correspondingshortening of the lateral plate mesoderm (and entire long-bodyaxis) was clearly evident (Fig.1H). This shortening is underscoredby the unusual proximity of the hindlimb bud field (as identifiedby expression of Tbx4, a specific marker of the hindlimb) to theforelimb (Fig. 1H'). Cells in the early Wnt5a forelimb budmoved with a vector comparable to that of WT littermates, albeitat a reduced velocity (Fig. 1I; see Movie 4 in the supplementarymaterial). Themeannvelocity of randomly selected cellsindividually traced frame-by-frame from z-stacks midway throughthe dorsoventral axis for 1.5 hours in WT embryos was 20.2um/hour (s.d.=2.6), compared with 10.8 um/hour (s.d.=2.3) in themutants, (P<0.0001, n=20 cells traced in three embryos each).Therefore, these data suggest that Wnt5a is necessary to maintainthe normal velocity of mesoderm cell movements. Tissue movement in chick To cross-validate our directional movement findings from themouse embryo, we studied tissue displacements in the chickembryo by lineage tracing. The chick wing bud arises from lateralplate mesoderm adjacent to somites 18-22 at HH 16 (26-28 somitestotal) (Chaube, 1959). HH 15-17 chick embryos were labelled withDiI and cultured in ovo at 39°C until HH 20 (40-43 somites).Lateral plate mesoderm labelled in the rostral portion of the wingbud-forming region, adjacent to somites 17 and 18, left a trail ofdye entering the early wing bud in a caudal and lateral direction(n=10) (Fig. 2A,B). Mesoderm that was labelled more centrally andinferiorly, adjacent to somites 19-21, resulted in a more directlylateral trail of DiI entering the bud (n=6) (Fig. 2C,D). Tissue caudalto somite 21 was not found to enter the wing bud, but ratherbecame displaced just caudal to the bud (n=7; data not shown). Thelinear displacement of dye that we observed, as opposed to thedisplacement of intact spots, could result from a number ofmechanisms, including directed cell migration and oriented celldivision. Nonetheless, the pattern of mesoderm movement into theearly wing bud, however, was similar to that of the mouse limbbud. Wnt5a as a chemoattractant To identify candidate molecules that might function to drawmesoderm into the early limb field, we performed chemotaxisassays in chick embryos. We tested Wnt5a, Fgf8, sonic hedgehog(Shh) and retinoic acid (RA). Wnt5a is expressed in the earlyectoderm and distal mesenchyme of the nascent limb bud (Gavinet al.,1990), whereas Fgf8 is expressed by the AER (Crossley andMartin, 1995). Shh is expressed in the posterior limb budmesoderm (Riddle et al., 1993), and RA is expressed in lateral platemesoderm prior to, and during, limb initiation, and is necessary forforelimb induction (Zhao et al.,2009). Acrylic beads were soakedin protein or carrier for 1 hour prior to implantation into HH 16chick embryo lateral plate mesoderm. Spots of DiI were placedrostral and caudal within 200 um of the bead for optimal sensitivityof the assay (Li and Muneoka, 1999), and the position of thesespots relative to the bead was documented. Following overnightincubation in ovo, the position and configuration of the DiI relativeto the bead was again documented. Beads soaked in Wnt5a, but notPBS, frequently became surrounded by DiI that was contiguous Fig. 2. Regional lineage of chick lateral plate mesoderm duringearly wing bud outgrowth. Merged bright-field and fluorescentimages.(A,A') Chick embryo labelled with spots of Dil (orange) at HH16 (26-28 somites) at the start of wing bud outgrowth and cultured inovo. The rostral-most spots on the left side are adjacent to somites17/18. (B,B') In the same embryo at HH 20, Dil-labelled tissue hasbecome displaced into the wing bud. Streaks of Dil suggest caudal-lateral oblique movement of mesoderm into the anterior wing budfrom a rostral position. (C-D') Lineage tracing in another embryo withimages taken before (C,C') and after (D,D') culture in ovo. Dil spotsadjacent to somites 19/20 at HH 16/17 (C,C') were displaced in a linearfashion by HH 20, heading directly lateral (D,D'). These findings areconsistent with the movements observed in the mouse embryo, asshown in Fig. 1. (E-H) Pre- and post-culture images showing thatchemoattraction of Dil-labelled mesoderm is not apparent toward abead (arrowheads) soaked in PBS (E,F), but is apparent toward a beadsoaked in Wnt5a protein in PBS (G,H). with the previously distinct spot of dye (P=0.04, Fisher’s exact test)(Table 1, Fig. 2E-H). Neither carrier alone, nor any of the otherproteins tested, exhibited evidence of chemotaxis. These datasuggest that Wnt5a acts as a chemoattractant of mesoderm. Ourobservations are consistent with similar findings in the developingmouse palate (He et al., 2008). Tissue movement in zebrafish We extended our tissue movement analyses to zebrafish (Daniorerio) to establish whether the early mechanisms of budding areconserved despite the divergent structural features of the maturelimb and fin. The zebrafish pectoral fifinbbud becomesmorphologically distinct from lateral plate mesoderm adjacent tosomites 2 and 3 at 24 hpf (26 somites). To localise the region of thelateral plate mesoderm that gives rise to the pectoral fin bud, weperformed lineage tracing. Ubiquitously distributed, but non-emitting fluorescein (DMNB-FITC), was uncaged in lateral platemesoderm adjacent to specific somites using a focused laser at 18hpf (18 somites), prior to fin initiation (Fig. 3A). Embryosdeveloped until 28 hpf (30 somites) and were then immunostained Table 1. Wnt5a acts as a chemoattractant of mesoderm No effect PBS 5 Fgf8 in PBS Wnt5a in PBS Shh in PBS DMSO Retinoic acid in DMSO Shown are the number of beads exhibiting evidence of chemoattraction inchemotaxis assays performed in chick embryos. Fig. 3. Lineage and movement of mesoderm during early pectoralfin development in zebrafish. (A) Fluorescein uncaged in the lateralplate mesoderm (arrow) at 16 hpf (18 somites). (B) By 28 hpf (30somites), in the same embryo as in A, focal fluorescence is found in theearly fin bud (arrow). (C) In tbx5 morpholino (MO) knockdown embryos,afin bud fails to form and labelled tissue becomes scattered throughoutthe mesoderm (arrow). (D) Summary of lateral plate lineage-tracingexperiments. Mesoderm adjacent to somites 1 to 4, but not 5,contributes to the pectoral fin bud. (E) Since the pectoral fin bud arisesadjacent to somites 2 and 3, we infer that lateral plate mesodermcondenses during fin initiation. (F,F) Field of view (boxed) and position ofa 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) Schematicrepresentation of tissue movements in the maturing (35-45 hpf) fin bud.The movements are comparable to those of the mouse limb bud, takinginto account the different orientation of the two buds with respect to theembryo body. (I) VVF of a 44 hpf embryo treated with 4 uM latrunculin A.Tissue motion is halted (but not with 0.1% DMSO carrier) asdemonstrated by the lack of arrows under PIV analysis. against FITC. We found that cells labelled in the lateral plateadjacent to somites 1-4, but not those adjacent to somite 5,contributed to the mature pectoral fin bud (Fig. 3B,D). Forvalidation of our lineage-tracing method, we performed the sameexperiment in the presence of morpholinos against tbx5 that areknown to produce deficient pectoral fins. As expected (Ahn et al.,2002), mesoderm in these embryos that was labelled adjacent tosomites 1-4 became scattered, rather than focused, in the limb bud(Fig. 3C). Our lineage-tracing observations suggest that lateralplate mesoderm condenses during formation of the pectoral fin(Fig. 3E). In contrast to the mouse embryo, in which primarilyrostral lateral plate tissue migrates into the limb bud, zebrafishlateral plate tissue migrates from both rostral and caudal positionsto enter the early bud. To assess cell movements in the more mature pectoral fin bud,but prior to cartilaginous condensation, we live imaged h2afz:gfp(Pauls et al., 2001) transgenic zebrafish embryos. With the embryoin the sagittal plane, views of the broad two-dimensionalproximodistal-anteroposterior surface of the pectoral fin bud,comparable to those of the mouse forelimb bud, were achieved(Fig.3F,F). Limited cell movements were seen between 28 and 40 hpf (see Movie 5 in the supplementary material). From 40 to 45hpf, fin mesoderm cells could be seen to move in a rotatoryfashion, from proximal-anterior to distal-posterior (n=16) (Fig. 3G;see Movie 6 in the supplementary material). This movement wasanalogous to those in the mouse limb bud at Theiler stage 15 (Fig.3H), taking into account the different orientation of the limb andfin buds. Tissue motion was abolished in the presence of 4 uMlatrunculin A, but not 0.1% DMSO carrier (n=4) (Fig. 3I; seeMovie 7 in the supplementary material), indicating that actin-basedmechanisms are required for cell movement. Regional differences in cell shape and polarity Having shown that the movement of cells is regionally distinct in theforelimb-forming region of each organism, we sought to establishwhether mesoderm cells in these regions exhibit a distinct shape andorientation. We performed live imaging of CAG::myr-Venustransgenic mice (Rhee et al., 2006) and Bactin:hras-egfp (Cooper etal., 2005) transgenic zebrafish. These transgenic reporters illuminatethe cell perimeter, avoiding potential artefactual changes in cellmorphology that might be associated with tissue fixation. Multiplez-stacks encompassing the entire depth of the relevant tissue weretaken in the coronal plane. By imaging multiple planes andidentifying the distinct morphological characteristics of the single-cell layer of ectoderm (Fig. 4), we ensured that only mesoderm wasanalysed. Regional differences in mesoderm cell shape wereidentified in both mouse and zebrafish. Mouse lateral platemesoderm cells at Theiler stage 14 (n=11) and those just medial tothe early limb bud at Theiler stage 15 (n=9) displayed elongatedshapes that ran parallel to the rostrocaudal axis of the embryo body(Fig. 4A,C). However, in Wnt5amutants, elongated cell shapeswere not apparent at either stage (n=7 and 4, respectively) (Fig.4B,D). WT (n=9) and Wnt5amutant (n=9) limb bud cells lateralto the trunk at Theiler stage 15 exhibited isotropic shapes without alongitudinal bias (Fig. 4A-F). Little to no intervening space betweencells was evident by live imaging (Fig. 4A-F). To test whether mesoderm cells exhibit polarity that correspondsto their direction of movement, we determined the location of theGolgi apparatus relative to the nucleus. The Golgi apparatus iscommonly located near the leading edge of a motile cell (Nabi,1999). At Theiler stage 14 (20 somites), we identified a caudal andlateral bias in the position of the Golgi in WT mesoderm cells(n=3) (Fig. 4G,I). However, in Wnt5amutant mesoderm, thisbias was not apparent (n=3, P<0.05, x’test) (Fig. 4H,J). Thesefindings correlate with the direction of mesoderm movement inWT embryos as well as with the diminished movements seen inWnt5amutants, and suggest that cell polarity is required fordirected cell movements. In zebrafish, lateral plate cells at the base of the pectoral fin budalso exhibited a longitudinal bias parallel to the rostrocaudal axisof the embryo (n=6) (Fig. 4K). Most of the cells within the fin budwere isotropic between 35 and 45 hpf, as in the mouse bud, butcells at the proximal-anterior portion of the 40-45 hpf fin buddisplayed a distinctive mediolateral longitudinal orientation,parallel to the proximodistal axis of the fin bud and perpendicularto the rostrocaudal embryo axis (n=10) (Fig. 4L,M). Silencing ofthe Bactin:hras-egfp transgene resulted in mosaic reporterexpression and facilitated the visualisation of individual cellmorphologies. We observed actin-rich protrusions pointingposterodistally from the distal end of the elongated cells (Fig.4K,M). These protrusions were likely to be filopodia, localised tothe leading edge of cells and extending in the direction ofmovement. Fig. 4. Regional differences in cell shape during early limb budoutgrowth.(A-F) Theiler stage 15 (21-25 somites) CAG::myr-Venustransgenic mouse embryos imaged live using a confocal microscope.Rostral positions are towards the top of the images. Somites, whichcontain cells of distinctive shape for comparison, are indicated byasterisks (A,B). WT lateral plate mesoderm (lpm) cells adjacent to theearly limb bud are oriented longitudinally, parallel to the rostrocaudalaxis of the embryo. This is observed as longitudinal streaking adjacentto the somites in A, and anteromedial to the early limb bud in C. Cellswithin the early WT limb bud (lb) are isotropic (A,C,E). By contrast, cellsin Wnt5a--littermates (B,D,F) are isotropic in both the lateral plate andthe limb bud. This finding correlates with the shortening and relativelack of tissue movement observed in Wnt5a--lateral plate mesoderm.(G,H)GM130 (Golga2) stain (green) highlights the location of the Golgirelative to nuclei(DAPI, blue). In Theiler stage 14 embryos (20 somites),Golgi are commonly found caudal and lateral to corresponding nucleiin 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 WTembryos, and the lack of movement of Wnt5a--mesodermdemonstrated in Fig. 1. (I,J) Polar plots summarising Golgi angle inrelation to the nuclear centre, with embryonic reference marks asshown.(K-M)Separate z-stacks of a live Bactin:hras-egfp transgeniczebrafish fin bud at 42 hpf. Lpm cells at the base of the fin bud (bluearrowhead, K) are elongated, parallel to the rostrocaudal axis of theembryo.Most cells in the fin bud are isotropic. However, cells in theproximal-anterior region of the fin bud are elongated,with ananteroproximal-to-posterodistal long axis, parallel to their direction ofmovement (red arrowhead, L). Some of these cells exhibit protrusions attheir posterodistal tip (white arrowheads, K,M). ecto, ectoderm; lpm,lateral plate mesoderm; Ib, limb bud; fb, fin bud. Our data revealed parallels between cell orientation and thedirection of tissue movement. A transition of cell behaviours islikely to involve active remodelling of the cytoskeleton becausefilopodia and Golgi localise to the leading side of mesoderm cellsand latrunculin treatment impairs cell movement. The lack ofapparent intercellular matrix seen by live imaging supports theproposal that the substrate upon which motion occurs is the cellsthemselves. Cell division might therefore be capable of influencingtissue motion. Regional differences in the orientation of celldivision We sought to integrate data on cell division planes with our otherfindings. Time-lapse images of CAG::H2B-EGFP and h2afiz:gfptransgenic mouse and zebrafish buds, respectively, captured in thecoronal plane were inspected frame by frame. Analysis ofindividual z-stacks facilitated the identification of mitotic planes(see Movie 8 in the supplementary material). The angle of themetaphase-to-telophase transition of every mitosis identifiedthroughout a given specimen was measured with respect to therostrocaudal axis (Fig.5A). In both mouse and zebrafish embryos, we found evidence forspatially distinct preferences in cell division planes. In lateral platemesoderm at Theiler stage 14, the preferred plane of division wasparallel to the rostrocaudal axis of the embryo (n=2 embryos, 61mitotic angles measured; Fig. 5B). This bias was similar in lateralplate tissue medial to the early limb bud at Theiler stage 15 (n=2,66 mitoses; Fig. 5C). In the early bud itself, a bias in the plane ofcell division was identified perpendicular to the long axis of theembryo (n=2, 224 mitoses; Fig.5D). The distribution of divisionplanes was significantly different between WT lateral platemesoderm and limb bud (P=0.007,x’test). Cell division planes inWnt5a mutant lateral plate mesoderm lacked the distinct orientationbias of WT embryos (n=2,50 mitoses, P=0.05; Fig. 5E). However,in the Wnt5a mutant limb bud, there remained evidence of a biasin division plane largely perpendicular to the long axis of theembryo, similar to that of WT embryos (n=2, 59 mitoses, P=0.9;Fig.5F). Zebrafish embryos demonstrated similarities in cell divisionplanes to mouse embryos. At 35 hpf, lateral plate mesoderm cellsdivided with a bias similar to that of mice (n=2, 24 mitoses; Fig.5G), whereas fin bud cell divisions were more strikingly biasedperpendicular to the embryo long axis (n=2, 30 mitoses, P=0.02;Fig. 5H). By 44 hpf, a preferred plane of cell division was nolonger apparent in the lateral plate as compared with 35 hpfembryos (n=2, 20 mitoses, P=0.3; Fig. 5I). A cell division bias didpersist in the 44 hpf fin bud, where it was comparable to that of 35hpf embryos (n=2, 70 mitoses, P=0.6; Fig. 5J). Collectively, thesedata indicate that distinct preferences in cell division plane occurbetween lateral plate mesoderm and the early limb bud. DISCUSSION We have investigated the individual cell behaviours driving thetissue-scale morphogenetic mechanisms by which the vertebratelimb bud arises. Our findings challenge the notion that cellmovement is a feature of only lower vertebrate early limboutgrowth (Rallis et al.,2003). Our data explain the previouslyobserved caudal displacement of lateral plate mesoderm (Chaube,1959) and of the Tbx5 expression domain that marks the forelimbfield prior to initiation (Agarwal et al.,2003; Zhao et al., 2009).Importantly, cell motion and division planes are reoriented duringearly limb outgrowth. Taken together with previous fate mapping 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 transgeniczebrafish chromatin undergoing mitosis (arrowheads) is visualised onsuccessive frames. The angle of a line joining the daughter chromatincentres at telophase is measured with reference to the longitudinalaxis of the lateral plate mesoderm (fourth frame down). The level atwhich the limb bud protruded beyond the lateral plate was selectedas the boundary between these two regions on every individual z-stack (long white line, fourth frame down). Every mitotic anglevisualised on all z-stacks, together encompassing the entire limbbud, was measured over the whole duration of each time-lapseexperiment. (B-J) Polar plot representations of cell division planes inwhich the rostrocaudal axis is defined at 0/180° and the lateral andfuture distal axis of the limb bud is defined at 90°. The proportion ofcell divisions in each 30° segment is represented by the length of thesegment.(B) During mouse Theiler stage 14 (18 somites), lateralplate mesoderm adjacent to somites 8-12 exhibits a preferentialplane of cell division that is parallel to the rostrocaudal embryo axis.At Theiler stage 15 (21-25 somites),although the same preferentialplane of division is found in the lateral plate (C), cells in the earlylimb bud exhibit a different orientation of division that isperpendicular to the rostrocaudal axis, but parallel to the direction ofbud outgrowth (D). In Wnt5a--mutants, orientation of cell divisionin the lateral plate is less apparent (E), although some orientation inthe limb bud is evident (F). This finding correlates with the severeshortening and lack of tissue movement in the lateral platemesoderm of Wnt5a--mutants,whereas budding of the limbs takesplace with evident cell movement, as seen in Fig. 1. In zebrafish at35 hpf, the orientation of cell division is similar to that of the mouseembryo (G), although more pronounced in the limb bud (H). By 44hpf, orientation of cell division in the lateral plate is lost (l), while itpersists in the fin bud (J). of the later limb bud (Saunders, 1948; Vargesson et al., 1997) andgenetic studies (Hasson et al., 2007; Naiche and Papaioannou,2007; Ros et al., 1996), our results suggest that limb initiation andsubsequent outgrowth represent distinct lateral plate-dependent and-independent phases of development. Our comparative analyses reveal that cell movement occurssimilarly in the mouse and chick. Mesoderm movement also occursduring early fin outgrowth, albeit in a different pattern that laterbecomes analogous to that of the mouse. Our data confirm that theapparent condensation of the tbx5 expression domain duringzebrafish pectoral fin initiation (Ahn et al., 2002) is secondary tocell movement, rather than to the loss and gain of expressionamong different cell populations. Differences in tissue movementduring early forelimb outgrowth between the mouse and zebrafishmight be attributable to differences in the position of the limb budrelative to the active site of rostrocaudal axis elongation at the tailend. In zebrafish, the posterior growing end of the embryo is, inrelative terms, further away from the pectoral fin field, and thelateral plate mesoderm adjacent to the pectoral fin bud does notexhibit caudal-ward motion. The pre-existing caudal-ward vectoradjacent to the mouse forelimb field is likely to influence cellmovements into the early bud. An active transition of cell polarity might provide the necessarycoordinate behaviour that underlies initial bud outgrowth. Wefound that the oriented migration and division of mesoderm cellsare linked. Disruption of cell polarity in the Wnt5a mouse mutantssimultaneously resulted in the loss of longitudinal cell organisation,of oriented cell division and of directional movement. Althoughmultiple forces, such as oriented cell division and directional cellmovement, are likely to contribute to limb budding, these Wnt5amutant data suggest that all such behaviours result from positionalpolarity possessed by mesoderm cells. Our data are consistent with a model in which Wnt5a is involvedboth in establishing mesodermal cell orientation and in signallingthe transition ofcell polarity that is necessary to generate a limbbud. Expression of Wnt5a in the elongating tail bud is likely tocontribute to the orientation of cells in the lateral plate mesoderm.By virtue of a new expression domain in the nascent limb field(Gavin et al., 1990), Wnt5a is likely to provide a repolarisation cuefor mesodermal cells to move toward the new Wnt5a source. Thereare likely to be redundant factors contributing to this transition, asthe limb bud was not as strongly affected as the lateral platemesoderm in the absence of Wnt5a.A subset of the numerous Wntand Frizzledgenes that are expressed in the limb bud(Summerhurst et al., 2008) might perform this function, amongother candidates. Although the mechanism linking cell polarity with directionalmovement through cytoskeletal changes in response to Wnt5a hasbeen explored in depth (Witze et al., 2008), the relationship oforiented division to directional movement remains unclear. Wespeculate that cell division generates a force that is capable ofinfluencing the direction of movement of neighbouring cells inearly mesoderm, where they are closely apposed. Although Fgf8was not found to be a chemoattractant in our assay, it is likely tofacilitate the entry of cells into the nascent limb bud by renderingthe limb field mesoderm more fluid in nature (Damon et al., 2008)and by promoting cell survival (Sun et al., 2002). Directionalmovement and increased rates of proliferation also explain why thelimb field, which exhibits higher tissue cohesiveness, is notengulfed by the lateral plate mesoderm. Our data therefore suggestthat multiple mechanisms contribute to early limb bud outgrowth.Improvements in our ability to visualise and measure physical forces in living embryos will facilitate further dissection of thenature and complexity of the morphogenetic mechanisms thatgenerate the limb bud. Acknowledgements We thank Dr Terry P. Yamaguchi for Wnt5a mutant mice; Dr Jacek Topczewskiand the late Dr Jose A. Campos-Ortega for Bactin:hras-egfp and h2af/z:gfptransgenic zebrafish lines, respectively; Drs Gail Martin and Didier Stainier forguidance and review of the manuscript; Drs Sean Egan, Chi-chung Hui,JanetRossant, James Sharpe, Patrick Tam, Rudolph Winklbauer and Yogiro Yamanakafor helpful discussions and review of the manuscript. This work was supportedin part by Research Grant 5-FY07-652 from the March of Dimes Birth DefectsFoundation and by a Pediatric Orthopaedic Society of North America AngelaKuo Young Investigator Award (to S.H.). K.M.V. was supported by an NSF Pre-doctoral fellowship and his work in the Martin and Stainier labs was supportedby the NIH. Work in B.G.C.'s laboratory is supported by The Terry FoxFoundation. Work in M.W.'s laboratory is supported by NSERC and CFI. Work inA.-K.H.s laboratory is supported by the NIH (RO1-HD052115 and RO1-DK084391)and NYSTEM. 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