Getting a head start: Craniofacial heterochrony in marsupials involves dynamic changes to molecular and cellular mechanisms underlying neural crest development

Getting a head start: Craniofacial heterochrony in marsupials involves dynamic changes to molecular and cellular mechanisms underlying neural crest development | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Getting a head start: Craniofacial heterochrony in marsupials involves dynamic changes to molecular and cellular mechanisms underlying neural crest development View ORCID Profile Axel H Newton , Ella R Farley , Andrew T Major , Jennifer C Hutchison , Ben M Lawrence , Karen E Sears , Marilyn B Renfree , Aiden M C Couzens , Geoff Shaw , Sara Ord , View ORCID Profile Richard A Schneider , View ORCID Profile Andrew J Pask doi: https://doi.org/10.1101/2025.09.23.678178 Axel H Newton 1 The School of BioSciences, University of Melbourne , Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Axel H Newton For correspondence: axel.newton{at}unimelb.edu.au Ella R Farley 1 The School of BioSciences, University of Melbourne , Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andrew T Major 1 The School of BioSciences, University of Melbourne , Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer C Hutchison 1 The School of BioSciences, University of Melbourne , Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ben M Lawrence 1 The School of BioSciences, University of Melbourne , Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karen E Sears 2 Department of Ecology and Evolutionary Biology, University of California at Los Angeles , Los Angeles, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marilyn B Renfree 1 The School of BioSciences, University of Melbourne , Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aiden M C Couzens 2 Department of Ecology and Evolutionary Biology, University of California at Los Angeles , Los Angeles, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Geoff Shaw 1 The School of BioSciences, University of Melbourne , Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sara Ord 3 Colossal BioSciences , Dallas, Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Richard A Schneider 4 Department of Orthopaedic Surgery, University of California at San Francisco , San Francisco, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Richard A Schneider Andrew J Pask 1 The School of BioSciences, University of Melbourne , Victoria, Australia 3 Colossal BioSciences , Dallas, Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrew J Pask Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The neural crest is a vertebrate innovation central to craniofacial development and evolution. While the gene regulatory networks guiding neural crest development are well characterized, the mechanisms generating species-specific craniofacial diversity remain poorly understood. Marsupials provide a unique model for studying neural crest plasticity, having evolved accelerated patterns of craniofacial development during embryogenesis. This adaptation arises in response to marsupials being born altricial after a short gestation yet require well-developed mouthparts to attach to a teat and continue development in the pouch. However, how marsupials achieve this heterochronic shift in neural crest development is largely unknown. In this study, we investigate the cellular and molecular mechanisms underlying their distinct heterochrony, revealing that marsupials produce dense pre-migratory aggregates of neural crest cells which undergo collective migration as epithelial-like sheets, potentially facilitating rapid establishment of the facial prominences. These cellular behaviours are unique amongst amniotes but resemble patterns in anamniotes which similarly exhibit accelerated craniofacial development to support early feeding. Marsupials appear to have evolved a similar mechanism of neural crest migration to facilitate their developmental heterochrony. These findings suggest that vertebrate neural crest migration may be shaped by the pace of craniofacial development during embryogenesis rather than phylogeny, providing new perspectives on neural crest plasticity and the developmental mechanisms driving craniofacial diversity across vertebrates. Introduction Marsupial mammals have evolved a reproductive strategy characterized by a short gestation followed by a prolonged postnatal growth, generally in the mother’s pouch. This strategy imposes distinct functional constraints, as neonates require well-developed forelimbs and orofacial structures to crawl to the teat and begin suckling despite an otherwise embryonic-like state 1 . In an attempt to explain this phenomenon, comparative embryologists J. P. Hill and K. P. Watson meticulously documented the early development of Australian marsupials, uncovering a striking anomaly. They observed that the rapid formation of orofacial structures appeared to be driven by mesenchymal cells migrating from the neural plate epithelium 2 . In a letter to Watson, Hill wrote: “ the flat somiteless embryo … is the most interesting not to say puzzling and… unique [as] I verily begin to believe the head mesoderm is derived from the ectoderm !” (quoted in 2 ). Though unpublished and largely overlooked at the time, these observations revealed an unusually early emigration of cranial neural crest cells in marsupial embryos, occurring when the embryo was relatively flat, featureless, and before neural tube closure 2 – a considerable deviation from patterns in other vertebrates 3 . Modern studies have since confirmed that marsupials likely evolved their specialized feeding strategy by accelerating cranial neural crest migration and facial morphogenesis through heterochrony, a change in the timing of developmental events 4 – 10 . Yet, despite decades of research into marsupial biology, the cellular and molecular mechanisms underlying the early emergence of cranial neural crest cells in marsupial embryos remain poorly understood. This derived condition likely requires differential regulation of the genetic circuitry controlling neural crest specification, migration and differentiation. Cranial neural crest cells are migratory, multipotent progenitors that generate the bone, cartilage, muscle, and teeth of the orofacial region 11 – 15 . The cranial neural crest is central to vertebrate development, facilitating the evolution of morphological novelty through the structural and functional integration of the craniofacial complex 15 – 18 . The intrinsic capability of these cells have been demonstrated through cross-species grafting experiments, revealing that transplanted neural crest executes autonomous molecular programs to control the timing of morphogenesis and confer species-specific anatomy 19 – 24 . Whilst relying on a deeply conserved gene regulatory network (GRN) governing their induction, specification, migration, and differentiation 25 – 31 , cranial neural crest cells additionally display wide variation in the timing and mode of migration across different taxonomic groups 29 – 31 . Yet how these programs diverge to produce distinct morphological outcomes remains unclear. We hypothesize that, in marsupials, the premature emergence and migration of cranial neural crest cells stem from differential expression or redeployment of neural crest GRN components, accelerating specification, migration, and facial morphogenesis. Consistent with this, evolutionary changes to the SOX9 E3-E5 enhancer drive early SOX9 activation and precocious cranial neural crest development in the marsupial opossum Monodelphis domestica 32 , 33 . However, this alone is unlikely to account for the accelerated patterns of neural crest development observed across marsupials, where the broader molecular and cellular mechanisms underlying these shifts remain unresolved. To address this issue, we compared cranial neural crest development among distantly related marsupials, focusing on species that differ along the altricial–precocial spectrum: G1 are the most altricial, G2 are intermediate and G3 are the most precocial at birth. We examined the fat-tailed dunnart ( Sminthopsis crassicaudata , Order Dasyuromorphia), a small Australian marsupial that gives birth to highly altricial young (G1) after a short 13–14 day gestation 2 , 34 – 36 ; the gray short-tailed opossum ( Monodelphis domestica , Order Didelphimorphia), a small-medium sized American marsupial born at a relatively more advanced state (G2) after 14–15 days 37 ; and the tammar wallaby ( Notamacropus eugenii , Order Diprotodontia) which is the most precocial at birth (G3) after 25 to 28 days of gestation 35 , 38 , 39 . These species exhibit known differences in the rate of neural crest development 2 , offering a natural system to assess how evolutionary variation in gestation length may correlate with cellular and molecular changes in neural crest biology. Here, we characterize the spatiotemporal dynamics of marsupial cranial neural crest specification and migration, comparing these patterns to other vertebrates. Our analyses reveal differential deployment of key neural crest specifier genes and distinct cellular behaviours likely underlying the heterochrony observed in marsupials. These cell biological features are reminiscent of strategies employed by basal vertebrates with similar functional feeding constraints and accelerated craniofacial development. Collectively, our results provide new insight into mechanisms driving craniofacial heterochrony and plasticity, illustrating how evolutionary pressures may shape neural crest development in response to life history constraints. Results Neural crest markers reveal accelerated orofacial development in marsupials The neural crest gene regulatory network is hierarchically organized into sequential modules. 1) Inductive morphogens pattern the ectoderm and create a permissive environment for neural crest induction at the neural plate border (via FGFs, WNTs, and BMPs). 2) Transcription factors respond and define the neural plate border and prime cells for a neural crest fate (via MSX, PAX, ZIC, DLX, and TFAP2 family members), then 3) specify neural crest identity and regulate delamination from the neuroepithelium (e.g., SOX9/10, FOXD, SNAIL, ETS, and TFAP2 family members). 5) Finally, additional factors facilitate the epithelial to mesenchymal transition required for neural crest cells to migrate throughout the embryo (e.g., SOX10, LMO4, and TFAP2A) 14 , 25 , 26 , 40 , 41 . To assess patterns of neural crest and craniofacial development across marsupials, we utilized whole-mount fluorescent imaging of genes across the neural crest gene regulatory network, first in McCrady 42 early-somite stage embryos of Sminthopsis crassicaudata (fat-tailed dunnart), Monodelphis domestica (Gray short-tailed opossum), and Notamacropus ( Macropus ) eugenii (tammar wallaby). To contrast these patterns more broadly across tetrapods, we included the laboratory mouse ( Mus musculus ) and chicken ( Gallus gallus ), which possess similar gestation times of ~19 to 21 days and are comparatively more precocial at birth than any marsupial ( Figure 1 ). Download figure Open in new tab Figure 1. Comparative craniofacial formation across marsupials and amniotes. Neural crest marker gene expression in McCrady stage 24 A) Sminthopsis , B) Monodelphis , C) Macropus , and roughly stage matched E8.5 D) Mus musculus embryos showing cranial neural cell migration and early patterning of the orofacial prominences. While the FNP neural crest is present in each species, relative formation and migration of PA1-3 neural crest is accelerated in the G1 ultra-altricial Sminthopsis , compared with G3 Macropus or mouse. Neural crest migration in each marsupial species can be seen as a collective of cells populating the developing orofacial prominences. E) Quantification of the relative area of neural crest cell-domains across marsupial embryos. The area of the FNP, PA1 (MX/MD), and PA2 was divided against total head area to determine the relative size of neural crest cell-derived domains. This revealed formation of the domains was associated with the relative altriciality of marsupials at birth, confirming the G1 Sminthopsis possessed the largest MD/MX domain, compared to G3 Macropus or mouse. Neural crest marker gene expression in later McCrady stage 27 F) Sminthopsis and G) Monodelphis embryos. H) Stage 27 Sminthopsis head compared to roughly stage matched mouse and chicken embryos showing relative patterning and outgrowth of the orofacial prominences, emphasizing the rapid outgrowth of the maxillary and mandibular primordia in marsupials. FNP = frontonasal process, MX = maxillary process, MD = mandibular process, PA = pharyngeal arch. Visualization of module-specific marker genes associated with early neural crest and craniofacial development confirmed accelerated patterns in marsupials. In each marsupial species, large accumulations of migratory neural crest cells (as visualized by either localization of SOX9 protein or SOX10 mRNA expression) formed the presumptive mesenchyme of the frontonasal process (FNP), the first pharyngeal arch (PA1) that is subdivided into the maxillary and mandibular primordia, and the second pharyngeal arch (PA2) ( Figure 1a-c ). However, in the approximately stage-matched mouse embryo (i.e., similar somite numbers and posteriorly open neural tube), the orofacial prominences were less formed, notably PA1 was only just migrating lacking distinct maxillary and mandibular subdivisions, and there was little outgrowth of PA2 ( Figure 1D ). Furthermore, we observed variation in the relative formation of each head and pharyngeal arch neural crest domain in G1-G3 marsupials ( Figure 1E , Table S1), where the relative area of the maxillary and mandibular neural crest cell domains (those responsible for forming the jaws), as well as development of the second and third pharyngeal arches, were the largest in the G1 Sminthopsis , followed by G2 Monodelphis and then G3 Macropus embryos, despite the Macropus embryos being significantly larger overall ( Figure 1D ). Further visualization of neural crest gene expression in stage 27 Sminthopsis and Monodelphis pre-implantation embryos revealed extraordinary outgrowth of the craniofacial complex, especially the mandibular and maxillary aspects of PA1 ( Figure 1F,G ). These regions emerged as distinct prominences taking up a substantial majority of the total volume of the head, with correspondingly large domains of neural crest gene expression. For example, SOX10 labelled a vast population of migratory neural crest in the head, whereas in the trunk SOX10 only labelled narrow bands of migrating neural crest, which highlights how further along neural crest migration is in the head relative to that in the trunk ( Figure 1F,G ). Moreover, comparisons to approximately stage-matched E9.5 mouse and Hamburger Hamilton (HH) stage 43 18 chicken embryos, further demonstrated the rapid developmental trajectory and substantial formation of the orofacial prominences in marsupials ( Figure 1H-J ). Together, these data confirm the heterochrony of neural crest and craniofacial development in marsupials 2 , 4 , 5 relative to eutherians and birds 6 , 10 ; but also uncover interspecific differences in the timing and outgrowth of orofacial prominences relative to the degree of altriciality at birth within marsupials. Neural crest specification genes are co-expressed and accelerated in marsupials To identify molecular mechanisms potentially underlying the craniofacial heterochrony observed in marsupials, we examined a range of neural crest markers. While previous reports have shown that SOX9 in Monodelphis is expressed early in cranial neural crest 32 , 33 , other gene expression patterns are not clear. We therefore sought to define the initial onset of genes associated with neural crest cell specification. We focused on the earliest stages of neural crest cell induction in McCrady stage 19 primitive streak embryos of Sminthopsis and Monodelphis . Wholemount fluorescent imaging of the stem cell/epiblast factor POU5F1 was compared alongside neural crest specification module genes FOXD3, SOX9 , and TFAP2A ; and delamination/migration module genes SNAI2 and SOX10 , to visualize the onset of neural crest cell induction and the progression through defined developmental stages 25 , 26 . While POU5F1 predictably marked the Sminthopsis and Monodelphis epiblast ( Figure 2A,B ), SOX9 and FOXD3 were specifically co-expressed within the neural plate epithelium, but not the surrounding non-neural ectoderm, as seen in whole mount and optical sections ( Figure 2B, B’ ). In the subsequent stage, SOX9 became regionalized to the neural plate borders ( Figure 1C )—supporting previous patterns observed in Monodelphis 32 )—with co-expression of the neural plate border specifier TFAP2A observed in both species ( Figure 2D,E ). Download figure Open in new tab Figure 2. Neural crest specification within the neural plate borders of marsupial embryos. Whole-mount visualization of genes associated with pluripotency, neural plate border (NPB) and neural crest cell specification in marsupial embryos. A-B) HCR labelling of POU5F1, SOX9 and FOXD3 RNA in either Sminthopsis or Monodelphis primitive-streak embryos revealed the boundaries between the neural plate epithelium (NP) and non-neural ectoderm (NNE) in the marsupial epiblast. FOXD3 and SOX9 are restricted to the forming neural plate in Monodelphis (B’). C-D) In the elongating stage 20 Sminthopsis embryo, SOX9 becomes restricted to the neural plate borders (NPB) 32 , alongside expression of TFAP2A . E) TFAP2A expression was similar in the elongating stage 20 Monodelphis embryo, and additional examination of SNAI2 and SOX10 expression showed while SNAI2 was restricted to the underlying mesoderm, SOX10 showed unexpected, paired expression domains in the neural plate borders (asterisk, E’). This suggests pre-migratory neural crest cells are first specified in the flattened, late primitive-streak stage marsupial embryo. Remarkably however, we additionally detected paired domains of SOX10 expression in the neural plate borders of the stage 20 Monodelphis embryo, as visualized by whole mount and optical sections, with SNAI2 restricted to the underlying mesoderm ( Figure 2E, E’ ). Together, these results reveal that while the typical transition from pluripotency to neural plate border formation shows an inductive timeline similar to other vertebrates 44 – 46 , early co-expression of the neural crest specification genes SOX9, FOXD3, TFAP2A and SOX10 within the neural plate borders of the primitive streak-stage embryo may represent initial molecular mechanism underlying the accelerated formation of cranial neural crest cells in marsupials. Cell biological changes accompany premature neural crest formation in marsupials To determine the consequence of these accelerated neural crest gene expression patterns on later developmental stages, we examined stage 21 embryos during the process of neurulation, where the planar embryo develops raised and protruding headfolds 34 , 37 . First, we examined the wholemount mRNA expression profile of definitive neural crest specification and migration genes SNAI2, TFAP2A and SOX10 , as well as localization of SOX9 protein. These analyses demonstrated that marsupial head folds are comprised of densely packed presumptive cranial neural crest cells within the neural plate borders ( Figure 3A,B ), which protruded dorsally from the flattened embryo ( Figure 3C ). The cranial neural crest cell domain, which corresponded to the future frontonasal process (FNP) and first pharyngeal arch (PA1), was present as a collective of densely packed SOX9, TFAP2A, SNAI2 , and SOX10 -positive cells ( Figure 3C ). Analysis of the SNAI2 and SOX10 expression profiles revealed that the headfold domains were additionally arranged into two distinct subdomains ( Figure 3A ), showing a mediolateral gradient of SNAI2:SOX10 positive cells from the midline towards the leading edges of the neural plate borders ( Figure 3A,D,E ). Download figure Open in new tab Figure 3. Formation of dense pre-migratory cranial neural crest aggregates. A) Whole mount visualization of SNAI2, TFAP2A & SOX10 expression , and B) SOX9 localization in Sminthopsis embryos showed the presence of dense headfolds full of presumptive, pre-migratory neural crest cells. The thickened headfolds were densely packed with SOX9 positive cells, which protruded from the dorsal surface of the flattened embryo (B’). C) Quantification of SNAI2 and SOX10 fluorescence intensity per cell in 3D renders revealed the pre-migratory neural crest cells were arranged in SNAI2:SOX10 positive expression gradient along the mediolateral axis of the embryo. Tissue sections confirmed these observations, where the headfolds were thickened relative to the neural plate epithelium. SNAI2, SOX10 and SOX9-positive cells are present as dense pre-migratory aggregates (D-F) , which had yet to break through the basement membrane into the underlying ECM (G) , and possessed E-cadherin junctions (white arrows), but not N-cadherin at the leading edges (black arrows) (H). I) Summarized model of marsupial pre-migratory neural crest cell aggregate formation prior to delamination. To determine the morphogenetic mechanisms underlying the rapid formation of the headfolds, we performed tissue section histology on the cranial neural crest cell domains. These analyses confirmed the mediolateral SNAI2:SOX10 gradient of the head folds ( Figure 3D,G ), but also that these were thickened aggregates comprised of densely packed cells ( Figure 3E ). Staining for extracellular matrix proteins laminin and fibronectin confirmed that the neural crest aggregates were pre-migratory, and had not yet broken through the basement membrane into the underlying extracellular matrix ( Figure 3H ); and staining for both E-cadherin and N-cadherin cell-cell junctions demonstrated the SOX9-positive cranial neural crest cells retained an epithelial morphology and thus had yet to undergo an epithelial-to-mesenchymal transition and delaminate ( Figure 2F-H ). Together, these data demonstrate that marsupial cranial neural crest cells initially form as dense, pre-migratory epithelial-like aggregates within the neural plate borders ( Figure 3H ). Marsupial cranial neural crest cells collectively migrate as epithelial-like sheets To characterize the properties of the cranial neural crest aggregates, we examined cell delamination and migration in subsequent stages. Wholemount and section immunostaining of SNAI2 and SOX10 positive cells in stage 22 and 23 Sminthopsis embryos revealed that the pre-migratory aggregates appeared to delaminate and migrate as densely packed sheets of SOX10 positive cells ( Figure 4A,B ). By stage 24, SOX9-positive neural crest cells had already proliferated to form a robust PA1 subdivided into the maxillary and mandibular primordia, despite the neural plate epithelium remaining open ( Figure 4C ). During delamination, cranial neural crest cells at the edges of the neural plate borders showed repression of SNAI2, with complete supression in the densely packed migratory leader cells ( Figure 4A,B ). Download figure Open in new tab Figure 4. Delamination and migration of cranial neural crest cells. Initiation of Sminthopsis cranial neural crest migration was visualized across stage 22-24 embryos. A-B) At stage 22-23, continuous sheets of SOX10-positive migratory neural crest cells could be seen leaving the lateral edge of the neural plate borders, with delaminating cells repressing SNAI2 (coloured arrows). Tissue sections through pharyngeal arch 1 and 2 (PA1/2) showed cranial neural crest cells became ventrally displaced and expanded as densely packed sheets. A’-B’) staining for E-cadherin, N-cadherin and vimentin revealed the densely packed SOX10-positive migratory cells retained strong E-cadherin junctions (black arrows), but not N-cadherin or vimentin (white arrows), suggesting retention of an epithelial-like morphology. C) By stage 24, SOX9+ cranial neural crest cells had begun filling and demarcating the developing orofacial prominences, showing distinct frontonasal (FNP), and PA1-derived maxillary (MX) and mandibular (MD) primordia. Tissue sections confirmed that while cells within the orofacial prominences maintained SOX9, those within the core of the mandibular primordia began repressing SOX10. Notably, despite abundance cranial neural crest, trunk neural crest was only just being specified and migrating down the posterior axis. D-E) Summarized model of marsupial cranial neural crest cells delamination and migration as epithelial-like aggregates. During initial delamination and migration, the basement membrane is degraded, and the cranial neural crest aggregates migrate as dense sheets wedged between the ectoderm and underlying ECM. To examine whether these migrating cells adopted a mesenchymal morphology, we performed immunostaining for E-cadherin, N-cadherin and vimentin, on stage 22 and 23 cranial tissue sections. Suprisingly, our analyses revealed that the densly packed, newly migratory SNAI2-negative/SOX10-positive cells retained epithelial-like characteristics including E-cadherin high and N-cadherin low cell-cell junctions, with little cytoplasm lacking the mesenchymal intermediate filament vimentin ( Figure 4C,D ). The newly migrating neural crest cells also did not seem to navigate within the fibronectin extracellular matrix, instead appearing to be wedged between the surface ectoderm, endoderm, and mesoderm ( Figure 4F ). Together, our data suggest that cranial neural crest cells in marsupials have evolved unique morphogenetic properties, whereby upon delamination from the neural plate they repress SNAI2, maintain E-cadherin, and migrate in synchrony as densly packed sheets ( Figure 4G ). As marsupial cranial neural crest cells appeared to maintain epithelial-like or pseudoepithelial characteristics during delamination and migration ( Figure 4 ), we assessed their spatial distrubution during early patterning of the orofacial prominences. Additional whole-mount immunostaining for SOX10 and E-cadherin was performed on a series of stage 21, 22 and 23 embryos collected from a single litter, highlighting the rapid progression through these stages 34 . Volumetric visualization of E-cadherin and SOX10 using z-stacks, z-slices and 3D reconstruction lent additional support to the newly delaminating SOX10-positive cells exhibiting epithelial-like characteristics. Here, leader cells appeared to migrate collectively from the neural plate borders and expand as a connected sheet with E-cadherin positive cell junctions moving to fill the orofacial prominences ( Figure 5A-C ). As expansion progressed, the E-cadherin-positive cell junctions were notably retained at the leading edges of the sheets ( Figure 5A’-C’ ), supporting earlier observations in E-cadherin tissue sections ( Figure 4D,E ). The collective sheet-like arrangement could be seen in 3D volumetric renders of the SOX10-positive cells ( Figure 4B-C ), which appeared to fan out and help sculpt the developing orofacial prominences. Download figure Open in new tab Figure 5. Collective migration of marsupial neural crest cells as an epithelial-like sheet. Pre-migratory (A) , early migratory (B) , and late migratory (C) embryos stained for SOX10 and E-cadherin, emphasizing the collective and sheet-like migration of cranial neural crest cells. Top panels show z-projection of wholemount confocal stack. A’-C’ shows magnified z-section slice through the coronal plane, and A’’-C’’ shows transverse z-slice through head, demonstrating E-cadherin positive cells at the leading edges. D-F panels show 3D volumetric reconstructions of A-C, with 90-degree rotation. SOX10-positive cells were used to create a region of interest mask over an opaque head volume, to emphasize neural crest cell morphology underneath the surface ectoderm and within the context of the developing head. SOX10-positive, post-migratory neural crest cells were densely packed with continuous E-cadherin-positive cell junctions (arrowheads), suggesting migration as a collective epithelial-like sheet. Leader neural crest cells retain stronger E-cadherin junctions than trailing cells. A=anterior, P=posterior, NPB=neural plate border, ROI=region of interest. Given the significant contribution of cranial neural crest cells to the developing orofacial prominences of marsupials, we further examined proliferation rates within the domains of SOX10-positive neural crest cells. Mitotically active phospho-histone 3 (PH3)-positive cells appeared to be distributed uniformly across the embryonic head region and not specifically elevated within the domains of SOX10-positive neural crest cells (Figure S1). Thus as increased proflieration does not appear to explain rapid facial patterning in marsupials, heterotopic accumulation of these cells prior to migration may instead act as a reservoir to provide increased quantities of migratory NCCs to the forming facial prominences. Taken together, our results suggest that Sminthopsis cranial neural crest cells migrate in a manner consistent with collective migration as dense epithelial-like sheets. Interestingly, while these migratory patterns have not been described in eutherian mammals 46 – 49 , they resemble cranial neural crest migration in the African clawed frog (Xenopus), where during the initial wave of migration, neural crest cells similarly retain a pseudoepithelial morphology with strong cell-cell contacts and sheet-like migration 50 – 52 . Discussion Heterochrony has long been proposed as a mechanism to account for the highly specialized evolutionary innovations found in the craniofacial complex of marsupial mammals 2 , 4 , 5 , 53 – 55 . Owing to their unique reproductive and life-history strategy, marsupials have evolved short gestation times and highly altricial young at birth. Such attributes place distinct functional and anatomical constraints on neonates who must begin feeding immediately for nourishment in the mother’s pouch 53 – 55 . To enable such feeding behaviour, marsupials rapidly form orofacial structures that are derived from and patterned by cranial neural crest cells, including the cartilages, bones, teeth, musculature and connective tissues, as well as additional structures 2 , 4 , 5 , 10 , 56 . The developmental mechanisms which underlie this distinct heterochrony have previously only been explored in a limited way 32 , 33 , 57 . In this study, we provide comparative and detailed analyses of neural crest cells to identify the cellular and molecular events that may underlie the accelerated craniofacial development in marsupials. We show that marsupial evolution involves plasticity in neural crest developmental programs, specifically distinct gene expression profiles and cellular trajectories compared with other amniotes 2 , 4 , 5 , 32 . In Sminthopsis , the timeline from neural crest induction to patterning and separation of the facial prominances occurs over approximately 24 hours 34 . This is not only faster than in Monodelphis 32 but may represent the most rapid craniofacial patterning in any mammal to date. The timing and mode of cranial neural crest migration varies among amniotes. In eutherian mammals, cranial neural crest cells migrate during closure of the neural folds 46 – 48 , 58 , 59 , whereas in birds and reptiles migration occurs after closure of the neural tube 60 – 62 . In contrast, marsupial cranial neural crest cells begin migrating very early, while the embryo and neural plate epithelium remain flat ( Figure 6 ) 2 , 4 , 5 . We also identified the heterotopic formation of dense pre-migratory aggregates within the neural plate borders, which form distinct headfolds protruding from the planar embryo ( Figure 2 ) 2 , 4 , 5 , 34 . Immediately following, presumptive neural crest cells underwent rapid delamination and migration, moving collectively as dense epithelial-like sheets ( Figure 3 , 4 ), quickly populating and shaping the developing orofacial prominences ( Figure 5 , 6 ). These behaviours diverge from the canonical mesenchymal migration seen in amniotes and instead parallel the epithelial-like strategies of anamniotes, such as Xenopus , which similarly exhibits aggregation and collective migration of dense cranial neural crest cell sheets (Figure S2). This contrast with the typical mesenchymal properties of avian, reptile, and eutherian mammal cranial neural crest cells ( Figure 6 ) 46 – 48 , 58 – 62 highlights the evolution of unique cellular trajectories in marsupials. Download figure Open in new tab Figure 6. Marsupial specific alterations to the vertebrate pan-neural crest GRN. Typical progression through the neural crest gene regulatory network sees activation of inducers, specifiers and migratory factors which drive key stages of neural crest cell development. Simplified diagram of the cellular transitions underlying neural crest development between chicken, mouse and marsupial neural crest development, emphasizing the heterochronic differences in the timing of migration. Marsupials show accelerated neural crest specification and migration, which may be mediated by alterations to the timing of key genes within gene modules, as per our observations and those in 32 . Detailed analysis of marsupial pre-migratory aggregates uncovered putative molecular mechanisms underlying their unique specification and migratory properties. In the canonical neural crest GRN, SOX9 both specifies neural crest identity 41 , 63 , 64 and activates SNAI1/2 and SOX10 to regulate the epithelial-mesenchymal transition, delamination, and migration of neural crest cells into the surrounding extracellular matrix 41 , 65 . While early and persistent SOX9 expression has been linked to accelerated neural crest development in Monodelphis 32 , 33 , we also observed heterochronic activation of SOX10 at the neural plate borders in primitive streak-stage embryos ( Figure 2 , 6 ), supporting early activation of the marsupial neural crest GRN. During cranial neural crest delamination and migration, SNAI2 facilitates the acquisition of mesenchymal properties by repressing E-cadherin and induction of N-cadherin 46 – 48 , 58 – 60 , 64 , 66 , 67 . In marsupials however, SNAI2 is repressed early, and E-cadherin is retained in newly delaminating and migrating neural crest cells ( Figure 3 , 4 ), which may contribute to their distinct collective migratory behaviour. Typically, SNAI2 is maintained in migratory neural crest cells 60 , 68 , as observed in zebrafish, turtles, birds, and mice. Though in Xenopus embryos SNAI2 is repressed and E-cadherin maintained in delaminating cranial neural crest cells, similarly exhibiting collective migration of dense epithelial-like sheets 52 , 69 . Accordingly, marsupials may have similarly evolved early repression of SNAI2 in pre-migratory neural crest cells to retain epithelial-like properties via E-cadherin junctions and facilitate collective sheet migration. It is important to note however, that Snai1 and Snai2 are both dispensable for neural crest cell delamination and migration in mouse 70 , highlighting potential species-specific differences in neural crest GRN requirements. The heterochronic formation, heterotopic accumulation, and collective sheet migration of dense epithelial-like cranial neural crest cells observed in this study is distinct from patterns reported in other well-studied amniotes. To contextualize this finding across less studied taxa, we surveyed reports of cranial neural crest development across diverse vertebrates to define the typical formation and migration patterns represented by each clade (summarized in Table S1 and references therein). Epithelial sheet-like migration has not been previously described in eutherian mammals including rabbits, primates, rodents, and pigs, nor in birds or reptiles. In contrast, collective migration of neural crest cell aggregates is reported in several anamniotes, including amphibians (e.g., Urodeles such as newts and axolotls, and Anurans such as Xenopus 29 , 31 , 69 , 71 , 72 ) and multiple lineages of ray-finned fish 64 , 73 – 77 . Intriguingly, historical descriptions of monotreme (e.g. platypus and echidna) embryology suggest possible cranial neural crest cell aggregates and epithelial sheet-like migration patterns referred to as ‘trigeminal ganglionic primordia’ 78 – 80 . Monotremes, like marsupials, produce highly altricial young and continue development ex utero through extended lactation 78 , 79 , thereby facing comparable early feeding constraints. This raises the intriguing possibility that neural crest cell aggregates and collective sheet migration represent an ancestral mammalian strategy, later reduced or lost in eutherians ( Figure 7 ). Download figure Open in new tab Figure 7. Tracing the mode of neural crest migration across vertebrates. Vertebrate phylogeny showing documented patterns of neural crest cell migration and corresponding life history and mode of reproduction. Neural crest cells and their migration patterns are denoted by green shading. Collective migration of epithelial-like sheets of neural crest cells has evolved in ray-finned fishes (Actinopterygii), amphibians, monotremes and marsupials, which each produce altricial young with immediate independent feeding requirements. In contrast, oviparous sharks (Chondrichthyes), reptiles, birds (Aves) and studied eutherian mammals give birth to more precocial young lacking early feeding constraints, and exhibit migration of distinct streams of mesenchymal neural crest cells. These phylogenetic relationships suggest that epithelial-like neural crest cell aggregates represent an ancestral state that persisted in basal mammalian lineages but was modified in reptiles, birds and eutherians with the evolution of precocial young. When cranial neural crest migratory strategies are mapped across vertebrates, instead of grouping phylogenetically, these different strategies align independently as evolutionary adaptations to altricial feeding ( Figure 7 ). In many anamniote species, heterochronic acceleration and collective migration of epithelial-like cranial neural crest cells enable early formation of skeletal feeding and respiratory structures in altricial larvae 73 , 81 . The functional requirements for a well-formed orofacial apparatus at early, altricial life stages in marsupials may have promoted the evolution of similar neural crest cell migration strategies utilized in anamniotes, representing a type of developmental “atavism” or convergence of ancestral strategies. Support for this hypothesis comes from comparative analyses of facial patterning across G1-G3 marsupial groups, with altricial marsupials (i.e. Sminthopsis ) possessing advanced patterning and outgrowth of the orofacial prominences, as first described by Hill and Watson 2 . These observations suggest a mechanism by which altricial feeding structures may evolve across diverse taxa and highlight cranial neural crest heterotopy and collective migration as potential drivers of adaptive craniofacial evolution. Our findings suggests that shifts in the timing and deployment of key factors within the neural crest GRN underlie the heterotopic accumulation of neural crest cells prior to migration. This reservoir may give marsupials a developmental “head start”, supplying increased numbers of progenitors to the orofacial prominences. The amount of neural crest cells allocated to the orofacial prominences appears to regulate species-specific differences in craniofacial morphology 82 , and neural crest cells are known to control the timing of differentiation as well as the structural and functional integration of the craniofacial musculoskeletal system 18 . Within this framework, our results provide a plausible explanation for how marsupials achieve accelerated formation of the craniofacial complex to support feeding 2 , 4 , 5 . These unique marsupial patterns not only underscore their value as evolutionary developmental models but also reveal fundamental principles underlying adaptive craniofacial diversity across vertebrates. Methods Dunnart Husbandry and Embryo Collection Dunnarts were obtained from an experimental colony run within the School of Biosciences. Female and male dunnarts were allocated into mating pairs in a 3:1 or 2:1 ratio of females to males. Detection of pregnancy for timed embryo collection was performed using recently described methods 34 , where paired females were monitored for weight changes as the primary indicator of pregnancy, and females which showed consecutive days of weight increase after ovulation were used for embryo collection. Dunnarts were humanely killed by cervical dislocation; the paired uteri were then removed from the peritoneum and opened in DEPC-PBS. Pre-implantation embryos (<stage 28) were rolled out of the uterus, while implanted embryos were carefully cut out with their membranes intact. Embryos were transferred into 4% paraformaldehyde (PFA) and left to fix at 4°C overnight or over the weekend. Tammar wallaby embryo Collection Tammar wallabies ( Notamacropus ( Macropus ) eugenii ) from Kangaroo Island South Australia were held in a breeding colony of the University of Melbourne. Pouch young were removed to reactivate their diapausing blastocysts (designated day 0 of pregnancy) 83 and collected as previously described 84 . All animal experiments conformed to the Australian National Health and Medical Research Council (2013) guidelines and were approved by the University of Melbourne Animal Experimentation Ethics Committees. Whole-mount Fluorescent Imaging Gene expression analysis was performed using immunofluorescence and hybridization chain reaction (HCR) 85 . In both cases, whole embryos fixed in PFA were washed in RNAse free 1X PBS with 0.1% Tween-20 (PBST) and then dehydrated in a 25%, 50%, 75%, and 100% methanol series (in PBS) on ice. Embryos were then stored in 100% methanol at −20°C until use. Target genes were chosen to represent key nodes of the neural crest cell gene regulatory network, including pluripotency ( POU5F1 ), initiation ( FOXD3, TFAP2A ), specification ( SOX9, SNAI2 ( SLUG )), migration ( SOX10 ), as well as markers for general morphology (DAPI, F-Actin/phalloidin, E-Cadherin, Fibronectin and Laminin, Table S2). HCR probes (Molecular Instrument, Los Angeles, CA) were initially designed against target gene sequences from the Sminthopsis crassicaudata transcriptome 86 . However, to ensure cross-reactivity between marsupials, probe sequences were BLASTed and retained only if they were specific to the gene of interest and had >85% similarity to Monodelphis (the most distant marsupial relative). HCR was performed using the protocol provided by Molecular Instruments 85 with minor modifications based on the stage of the embryos. Briefly, embryos were rehydrated with the reverse methanol series (75%, 50%, 25%, 100% DEPC-PBST) on ice. Embryos were then incubated with proteinase-K at room temperature for 5 minutes (stages 24-25) or 10 minutes (for stages 27-29), then post-fixed in DEPC-PFA for 20 minutes at room temperature. Embryos at stages prior to stage 24 were not incubated with proteinase-K and were not post-fixed. All embryos were incubated with 10-30pmol of probes for POU5F1, SNAI2, FOXD3, TFAP2A, SOX9 and/or SOX10 in hybridisation buffer at 37°C overnight. On the second day, embryos were washed in wash buffer and DEPC-SSCT and were then incubated with 30pmol of H1 and H2 hairpins in amplification buffer at room temperature overnight. On the third day, embryos were washed in DEPC-SSCT and incubated with DAPI (1:10,000 in PBST), before being washed again in PBST. Methanol dehydrated embryos were used for immunofluorescence but were not rehydrated in a series. Instead, embryos were transferred directly from 100% methanol into blocking buffer (1X PBS with 0.1% Tween-20 (PBST) with 3% BSA) and left to block for at least 1 hour, rotating at 4°C. Primary antibodies (Table S2) were diluted in PBST with 1% BSA and incubated with embryos overnight at 4°C while rotating. On the second day, embryos were washed with PBST to remove any unbound primary antibodies, then incubated with secondary antibodies (diluted at 1:500 in PBST with 1% BSA) at room temperature overnight in the dark while rotating. On the third day, embryos were washed in PBST and then incubated with DAPI (1:10,000 in PBST), before a final wash in PBST prior to mounting. Mounting of Embryos for microscopy Mounting was performed based on the size, stage and shape of the embryo. Mounting of early-stage embryos (stage 19-22) involved positioning the embryo flat on the slides with the dorsal surface facing upwards. Embryos often adopted the curvature of the vesicle and occasionally required further flattening through cutting the extra embryonic membranes. Embryos were then overlaid with glycerol mounting media (90% glycerol in 0.1 M PBS with 50 mg/ml propyl gallate) and a coverslip gently applied. Mounting of stage 23+ embryos required generation of custom silicon wells using aquarium-grade sealant. Embryos were positioned within the well and filled with the same fluorescent mounting media. A cover slip was then placed on top to seal the embryo within the chamber. Tissue Section Immunofluorescence Whole embryos were transferred to 30% sucrose in PBS and allowed to sink, typically overnight. Embryos were then positioned within cryomolds in Tissue-Tek O.C.T. mounting media (ProSciTech) and stored at −80 until use. 15-20uM cryosections were cut and placed on alternating superfrost slides for successive immunostaining. Slides were washed in 1% Triton-X in 1X PBS (PBTX) and then blocked for at least 1 hour with 2% BSA in PBS at room temperature in a humidified chamber to prevent drying. Slides were then washed in PBS again and then incubated with primary antibodies diluted in PBS at 4°C overnight. On the second day, sections were washed in PBS and incubated with secondary antibodies (1:500 in PBS containing 1% BSA) at room temperature in the dark for 1 hour. Slides were then washed in PBS and incubated with DAPI (1:10,000 in PBS), before being washed again and mounted with glycerol mounting media and a coverslip. Microscopy and post-processing Embryos and slides were imaged on a Nikon A1R confocal microscope with NIS-Elements software. Whole mount scans were captured with a 10x PL APO Lambda MRD00105 air objective (NA = 0.45), while tissue section images were imaged with a 40x PL FLUO MRH01401 oil objective (NA = 1.3). Image processing and quantification All image post-processing was performed using ImageJ (Fiji) for visualization, z-projection or z-slicing; or Imaris (Oxford Instruments, UK) for 3D volumetric reconstruction and quantification. Confocal imaging data was imported into Imaris v10.2 (Oxford Instruments, UK) for volumetric visualization and surface rendering. Intensity thresholding of SNAI2 and SOX10 expression channels were used to create surfaces for 3D volumetric rendering. To calculate relative channel intensities between cells, the Imaris “cells” module was used to segment cells via DAPI signal (low threshold to capture cells broadly) and generate cell counts/data across the embryonic head encompassing the headfolds. Cells data was exported from Imaris in CSV format, imported into R studio for further analysis and graphing. The average channel intensities per cell for each channel were plotted against cell position in the frontal axis of the embryo. Cell morphology and proliferation were determined within neural crest cells in Imaris via creation of a region of interest and channel mask. Initially, neural crest cells were identified using SOX10-positive signal in the Imaris “spots” module to generate a surface of the migratory neural crest cell domain. To visualize the migratory mass without interference from the overlying ectoderm or surrounding tissues, a mask was created for each channel from the surface, before the background volume made opaque. This allowed specific visualization of cell junctions between neighbouring neural crest cells, with E-cadherin signal from the overlying ectoderm. To determine proliferation rates between neural crest cells and non-neural crest cells, the neural crest surface was used to generate a neural crest cell-specific, and non-neural crest cell mask. The ‘cells’ function was then used on the DAPI channel of each mask to calculate the number of cells within the region of interest, followed by the PH3 channel to calculate the number of proliferating cells. We then calculated the proportion of proliferative cells (proliferative/total*100) within a region to determine the gross proliferation rate. To measure the relative area of FNP & PA1-3 neural crest cell domains between marsupials, area of each domain was calculated from the z-projection images of stage 24 embryos using ImageJ (Fiji). This allowed direct comparisons, irrespective of gene marker or image quality. The total head area was defined as the anterior-most edge of the FNP domain to immediately below PA2, and each neural crest cell-domain was calculated using neural crest cell-gene expression to define the domain boundaries. Total shape was captured using the Polygon-selection tool and area calculated using the Measure function. neural crest cell-domain area was divided by total head area to obtain a % proportion of total head area (Table S3). Author contributions AHN and AJP conceived the study. AHN and ERF performed the experiments. AHN reconstructed and analysed imaging data. AHN and ATM performed image quantification and plotting. AHN, ERF, JCH and BML monitored dunnarts for pregnancy and collected embryos. AMC and KES generated and supplied opossum embryos. GS and MBR generated and supplied tammar wallaby embryos. AHN, ERF, RAS, and AJP wrote the manuscript. All authors reviewed the data and gave final approval of the manuscript. Funding information This research was conducted under research funding through Australian Research Council DP210102645 and DP160103683 to Andrew J. Pask, UoM ECR grant TP605149 to Axel H. Newton, and generous philanthropic funding from the Wilson Family trust and the Colossal Foundation. Competing interests Additional research and salary funding was provided by Colossal BioSciences (Texas, USA). Data availability All data is contained within the manuscript. Figure Legends Figure S1. Accelerated formation of the head folds does not appear to be due increased rates of proliferation. Extension of Figure 4 . Pre-migratory (stage 21), early migratory (stage 22) and late migratory (stage 23) embryos stained for SOX10 and PH3 highlighting uniform cell proliferation during the initial stages of head formation. Top panels show z-projection of wholemount confocal stack. Middle panel shows z-section slice through the coronal plane. Bottom panels show 3D volumetric reconstructions of SOX10-positive region of interest within opaque head volume, to emphasize neural crest cell morphology underneath the surface ectoderm and within the context of the developing head. Quantified proliferation of early migrating (stage 22) SOX10-positive neural crest cell domain does not show increases in proportions of actively diving cells compared to SOX10 negative cells across the embryo. A=anterior, P=posterior, NPB=neural plate border, ROI = region of interest. Figure S2. Re-acquisition of ancestral collective neural crest cell migration behaviours in marsupials Comparative neural crest cell development at key stages between Sminthopsis , mouse, chicken and Xenopus embryos. Sminthopsis and Xenopus show striking convergence in neural crest cell behaviours during neurulation, including collective mass migration of neural crest cells from the flat neural plate epithelium to rapidly produce first arch crest mesenchyme. 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