Blastula stage specification of avian neural crest

Abstract

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of β-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.

Fig. 1.. Neural crest cells are specified at blastula stage in a restricted epiblast region.

(A) Schematic showing hypoblast-free epiblast explants (≈80 μm) from EGK Stage XII chick embryos generated from an equatorial stripe, immersed in collagen gels and cultured under defined non-inducing conditions in isolation. After 25h of culture, lateral explants displayed a robust Pax7+ expression not seen in other explants (n = 5). After 45h, a similar intermediate location displays Pax7+/HNK-1+ and clear double positive migratory cells likely to be NCC (n = 5; scale bar 100 μm). (B) Diagram of a bisection approach to monitor different conditions in original explants. Bisected explants from the same original region generate Pax7+ expressing cells at 25h and migrating Pax7+/HNK-1+ cells at 45h (n = 10; scale bar 100 μm). (C) Images of intermediate explants with colocalization of neural crest markers at 45h (i) Pax7/Sox9, (ii) Snail2/AP2, (iii) Msx1/2/Sox9, (iv) Msx1/2/AP2, (v) Pax7/HNK-1 explant and (va) magnification of the migrating Pax7+/HNK-1+ cells at 45h.

Fig. 2.. Intermediate region of epiblast is specified towards neural crest cell fate independent of neuroectodermal and mesodermal cell fates.

(A) Explants from EGK Stage XII chick embryos, collected after 25h and analyzed for expression of different cell fates; mesodermal (TbxT, (Brachyury) lateral-most), neural (Sox2, medial), and NC (Pax7, intermediate explant) (n = 4). (B) Graphs showing RT-qPCR analysis of twelve explants after 25h of culture, for the expression of Pax7 (NC marker), Sox2/Sox3 (neural marker), and TbxT/Tbx6 (a lateral mesodermal markers). Fold change in expression is represented by normalizing to GAPDH (reference gene) and relative to the positive control region for each gene: cranial neural fold (NF)/neural plate (NP) for Pax7 and Sox2/Sox3 expression and primitive streak (PS) for TbxT/Tbx6. Data from four individual sets of embryos, each with 12 explants and controls, are represented in the RT-qPCR graphs are shown, with error bars representing standard error of mean between four biological replicates (4 embryos).

Fig. 3.. Low density isolated cell analysis of intermediate epiblast region identifies cell autonomous specification of neural crest in culture.

(A) Schematic showing HH stage 8 and EGK stage XII embryos used for low density isolated cell analysis and the workflow. Cells were dissociated from Stage 8 embryo marked by Pax7 expressing neural fold region (boxes sections within the red region) (positive control), plated, fixed and immunostained for Pax7/Sox9. Stage XII embryo explants marked as 1 (intermediate) and 2 (medial) regions were dissociated and cultured at very low density (10–20 cells/cm²) on a thin layer of collagen gel under non-inducing conditions for 30h. (B) Isolated cells in the low density culture were immunostained for Pax7/Sox9 expression from neural fold region, intermediate region (section 1) at time 0 (immediately after plating), intermediate region (section 1) used as no primary control, and from sections 1 and 2. Intermediate region (section 1) had the highest Pax7 positive cells (38%) with 8% Pax+ cells in medial region (n = 4). (C) Explants from EGK St. XII epiblast were dissected, dissociated and cultured for 25h as described in (A) in presence or absence of Wnt-ligand inhibitor (WIF-1) or β-catenin inhibitor (XAV939) and processed for Pax7 immunofluorescence. Six panels from 4 individual embryo explants show distant cells cultured in isolation express Pax7 under control and WIF-1 treated conditions, while no Pax7 expression was seen under XAV939 treatment. (D) Average cell counts for Pax7 positive cells from the three different conditions, untreated control, WIF-1-treated and XAV939-treated (n = 3), and (E) Total DAPI positive cells under these conditions (n = 3). Error bars represent SEM from three separate embryo. Cell counts are provided in supplemental Table 1.

Fig. 4.. Intermediate epiblast consists of precursors of NC and placodal cell fates.

(A) Schematic showing intermediate and medial explant regions from EGK stage XII used for the analysis. (B) Explants from EGK St. XII epiblast were dissociated and cultured for 36hrs. Distant cells in this low-density culture from intermediate explant (explant #1) express Pax7, no ectodermal marker was expressed in these cells. Distant cells in low density culture from medial explants (explant #2) expressed Sox2 and lower levels of Pax7. Expression of placodal gene, Six1, in distant isolated cells was not seen. (C) (i) Images from 3 individual embryos, from few remaining undissociated clusters of cells in the culture corresponding to intermediate epiblast region, analyzed for Six1, Sox2 and Pax7 expression. Six1 is expressed in cells present in clusters. Six1 and Pax7 expression is mutually exclusive. (ii) Undissociated clusters of cells in the culture corresponding to medial epiblast region, display robust Sox2 expression and weak Pax7 expression. This expression pattern was observed in dissociated intermediate explants from 6 individual embryos. Cell counts for Six1+ cells are provided in supplemental Table 1.

Fig. 5.. In vivo lineage tracing validates the contribution of intermediate epiblast region to neural crest lineage.

(A) Schematic summary identifying regions in the epiblast that contributed to NC (n = 8) alone (4) or in combination with other fates (N-neural, NC-neural crest, E-epidermal, M-mesodermal). Chi-Squared analysis identified statistically significant (p<0.05) NC contributions of the cells within the intermediate epiblast region. Embryo labeled with DiI/DiO at time 0 (B) and after 28h of culture at St. 8 (C); sections demonstrating contribution to the CNS by cells in the middle of the embryo labeled with DiI (C′, C”) and contribution to the lateral neural fold (nf) and neural plate border (nbp) by cells in the intermediate epiblast labeled with DiO, colocalized with Pax7 expression or lateral to it (arrowhead and arrow respectively, C′, C”). (D) Second embryo labeled with DiI on intermediate region of epiblast, demonstrating anterior neural fold localization of DiI (St. 10). (D′) Neural tube cross-section at denoted anterior axial level with colocalization of DiI/Pax7 expression domains in migratory and premigratory neural crest cells and some cells in neural epithelium. (D″) An inset of migratory NC region from D′, labeled with DiI in the cell membrane and co-labeled with nuclear DAPI or Pax7.

Fig. 6.. Model of NC cell fate specification from the epiblast.

Beginning with early blastula, we propose a model of early NC cell fate specification from epiblast cells (depicted in orange with underlying hypoblast layer in grey). Based on our study, the model suggests specification of prospective NC (pNC) from the epiblast during blastula stages of embryonic development. The earlier specification of ectodermal and mesodermal fates suggested by various studies are also reflected in the model. pNE, prospective neural ectoderm; pNNE, prospective non-neural ectoderm; pPE, pre-placodal ectoderm; pM, prospective mesoderm; pE, prospective endoderm; PGC, primordial germ cells.