Fate mapping the late tailbud reveals continued cell ingression.

<p>Schematic of DiI labelling experiment for distinct cell populations in HH20/21 tailbud (NT, neural tube; CNH, chordo-neural-hinge; MP mesoderm progenitors) (A); DiI labelling of NT before incubation (B), fixed and analysed in sections (<i>n</i> = 5/5 confined to NT) (B′); after incubation, DiI is restricted to Sox2 positive NT (B′″); DiI labelling of CNH before incubation (C), fixed and analysed in sections (<i>n</i> = 8/11 confined to CNH) (C′); after incubation, DiI is found in NT, MPs, and their derivatives, including Sox2 positive cells in the MP domain (C′″); DiI labelling of MPs before incubation (D), fixed and analysed in sections (<i>n</i> = 6/6 confined to MPs) (D′); after incubation, DiI is restricted to MPs, presomitic mesoderm, somites, and lateral mesoderm and rarely labels Sox2 positive cells in the MP domain (D′″). White arrow, Sox2-expressing/DiI labelled cells in MP domain; white dashed line, region of Sox2 positive cells in the position of the MP domain. (E) Summary of cell movements following DiI labelling (orange) in NT, CNH, and MP domain.</p

Summary data for fate map of the late tailbud.

<p>Summary of contributions of cell groups DiI labelled in HH20 neural tube (NT), chordoneural hinge (CNH), or mesoderm progenitors (MP) to tissues in and derived from the tailbud observed at HH24. <i>n</i>, number of embryos labelled at HH20 for each tissue.</p

FGF signalling regulates cell fate assignment in the tailbud.

<p>Schematic of whole tailbud explant assay (A); <i>Spry2</i> expression in tailbud explants following exposure to vehicle control DMSO (A′), FGFR inhibitor PD173074 (A″), or MEK antagonist PD184352 (A′″); <i>Bra</i> expression in tailbud explants following exposure to DMSO (B), PD173074 (B′), or PD184352 (B″). Arrow indicates normal down regulation of <i>Bra</i> in CNH in DMSO control; nc, notochord; mp, mesoderm progenitors. <i>Sox2</i> expression in tailbud explants following exposure to DMSO (C), PD173074 (C′), or PD184352 (C″); <i>Tbx6L</i> expression in tailbud explants following exposure to DMSO (D), PD173074 (D′), or PD184352 (D″); <i>Wnt3a</i> expression in tailbud explants following exposure to DMSO (E), PD173074 (E′), or PD184352 (E″). In all tailbud explant experiments, treatment groups were processed and scored alongside control DMSO tailbuds; for simplicity of presentation, one representative DMSO explant is shown here for each marker gene. Each tailbud was scored for gene expression in caudal, middle, and rostral thirds. Schematic of bead implantation experiment and image of tailbud with implanted bead prior to incubation (F); vehicle control BSA-only soaked bead does not induce <i>Bra</i> (F′) and sagittal section (F″); FGF4 delivering bead eliciting ectopic <i>Bra</i> expression (F′″) and sagittal section (F″″); BSA beads do not elicit ectopic <i>Sox2</i> (G), but FGF4 beads can induce a small patch of <i>Sox2</i> (G′); BSA beads do not elicit ectopic Sox2/Bra cell foci (H–H′), but these are detected around FGF4 beads (I–I′″); note polarised organisation with co-expressing cells flanked by Sox2 and Bra only expressing cells (I′ and I′″) (arrows in F′, F′″, G′, G′″, ectopic expression domains; asterisk, beads; white dashed line, H′ endogenous CNH or ectopic CNH-like cell group I′,I′″). (J) Summary diagrams depicting combined results of FGF gain- and loss-of-function experiments. Gain of FGF function depicts effects of local exposure to FGF delivered by an implanted bead.</p

Regulation of cell death in the tailbud by retinoid signalling.

<p>(A–E′, C″, E″) Detection of cell death using Tunel (Apoptag) in chick tailbud at key stages (A) HH16, (B, B′) HH22, (C–C′″) HH24, and (D–E′″) HH26/27. (C″) Some apoptotic cells are detected in mesoderm progenitors (yellow dashed line) and the CNH (red dashed line), distal notochord (black dashed line) at HH24. (C′″) section as in (C″) DAPI stained nuclei confirm tissue organisation. (E″) Increase in apoptosis in the terminal structures by HH27, distal notochord (black dashed line). (E′″) section as in E″ DAPI stained nuclei. (F–H″) Cell death detection using NucView 488 caspase-3 substrate (green) and actin cytoskeleton counter-labelling with Phalloidin (red) in HH20 explanted tailbuds cultured for 24 h in (G–G′) control DMSO only conditions or in (H–H″) the presence of RAR/RXR antagonists. White dashed line, neural tube outline; red dashed line, CNH. CNH region is not well defined in RAR/RXR treated tails. This may reflect ectopic/increased Bra in these conditions. Scale bar, 100 µm.</p

Endogenous retinoid signalling represses mesodermal and promotes neural cell fate in the tailbud.

<p>Schematic of whole tailbud explant experiment (A). <i>Spry2</i> expression in vehicle control DMSO (A′), retinoic acid (RA) (A″), or retinoic acid synthesis inhibitor Disulfiram (A′″) treated tailbuds. <i>Bra</i> expression in DMSO- (B), RA- (B′), or Disulfiram- (B″) treated tailbuds (nc, notochord; mp, mesoderm progenitors; black arrow in B indicates normal downregulation of <i>Bra</i> in CNH/medial mesoderm progenitors at ∼HH24 and white arrow in B″ indicates continued expression in these tissues when RA synthesis is inhibited); <i>Sox2</i> expression in DMSO- (C), RA- (C′), or Disulfiram- (C″) treated tailbuds. <i>Tbx6L</i> expression in DMSO- (D), RA- (D′), or Disulfiram- (D″) treated tailbuds; Quail embryos at HH14–15 were analysed for marker gene expression following development in normal (NQ) and retinoid deficient (VADQ) conditions; mesodermal marker gene <i>Bra</i> in normal (E, E′) and retinoid deficient embryos (F, F′), where it is ectopically expressed in the dorsal neural tube (arrow). <i>Fgf8</i> in normal (G, G′) and retinoid deficient embryos (H, H′), where it is ectopically expressed in the dorsal neural tube (arrow). Note that the normal quail expressing <i>Fgf8</i> is at a slightly later stage and is larger overall than the retinoid deficient quail, however no ectopic patches of <i>Fgf8</i> are seen in normal quail neural tube (this section is also not perfectly transverse and is slightly caudal to that shown for in H′; this explains the apparent broader ventral expression of <i>Fgf8</i> in the normal quail). Neural progenitor marker <i>Sox2</i> in normal (I, I′) and retinoid-deficient embryos, where it is downregulated in dorsal neural tube (arrow) (J, J′). (E′, F′, G′, H′, I′, J′) are transverse sections through the caudal neural tube that contains a lumen. Schematic of in vivo bead implantation experiment (K). <i>Spry2</i> expression in the tail bud incubated with vehicle control DMSO (L) or RAR/RXR antagonists beads (L′). <i>Bra</i> expression with DMSO (M) or RAR/RXR antagonist (M′) soaked beads. <i>Sox2</i> expression with DMSO (N) or RAR/RXR antagonist (N′) soaked beads (*, beads; arrow, ectopic <i>Bra</i>).</p

Summary of signalling pathway interactions and cell fate changes underlying body axis elongation arrest.

<p>At HH22 <i>Bra</i> is expressed in notochord, CNH, and mesodermal progenitors (overlapping with <i>Sox2</i> in neural CNH) and is maintained by FGF (and Wnt) signalling, while <i>Sox2</i> is confined to neural tube and neural-CNH (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001415#pbio-1001415-g001" target="_blank">Figure 1A</a>). From this stage <i>Raldh2 </i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001415#pbio.1001415-Tenin1" target="_blank">[17]</a> and <i>Crabp2</i> begin to be detected in tailbud mesoderm progenitors (signals that induce <i>Raldh2</i> here remain to be determined). FGF signalling no longer inhibits <i>Raldh2</i> nor represses <i>Crabp2</i>, and <i>Cyp26a</i> is downregulated; consequently, RA signalling begins to rise in the tailbud. FGF signalling is required to maintain <i>Bra</i> (except in proximal notochord) as well as <i>Tbx6L</i> and <i>Wnt3a</i> (not shown) in mesoderm progenitors. At HH24 FGF signalling suddenly declines, leading to loss of <i>Bra</i> and expansion of <i>Sox2</i> expression (dashed arrow) into the previous mesoderm progenitor domain, which reflects continued cell ingression from the CNH. At this stage <i>Crabp2</i> is now detected in the neural-CNH and position of the mesoderm progenitors, indicating rising retinoid signalling, which can repress FGF ligands and signalling. By HH26/27 FGF signalling is not detected and the notochord ends abruptly encircled by <i>Sox2</i> expressing cells. Cell death is widespread in the tailbud and is promoted by retinoid signalling.</p

FGF and RA pathway interactions during body axis elongation.

<p>Unlike control DMSO beads (A), retinoic acid delivering beads (B) repress <i>Fgf8</i> in the early tailbud. Tailbud explanted from HH16 embryos (C) cultured in control DMSO (D) or RA (9-Cis, 10 µM) (D′) and HH19-22 tailbud explants pairs (E) cultured with DMSO (F, G) or RA (1 µM AT-RA, F′; 100 nM AT-RA, G′) showing RA represses <i>Fgf8</i> and <i>Fgf4</i> in the maturing tailbud. RA inhibited both <i>Fgf8</i> and <i>Fgf4</i> in these tailbud halves at the lowest concentration of 100 nM AT-RA (<i>n</i> = 3/4 explant pairs for each gene). Trunk explant pairs (neural tube flanked by somites and lateral plate mesoderm) from HH16 (H) cultured with BSA vehicle control (I) or FGF8 (I′) lose <i>Raldh2</i> in response to FGF8. Older, HH19-22 trunk explant pairs (J) also downregulate <i>Raldh2</i> in response to FGF, BSA control (K), and FGF8 treated (K′). HH19–22 tailbud explant pairs (J) do not alter <i>Crabp2</i> levels in response to FGF, control BSA (L), and FGF8 (L′). HH19–22 tailbud explant pairs do not alter <i>Raldh2</i> levels in response to FGF, control BSA (M), and FGF8 (M′). Explants in (K), (K′), (M), and (M′) are taken from the same embryo, cultured and processed in parallel. In addition, neither BSA nor FGF4 delivering beads repressed expression of <i>Raldh2</i> (N, N′) or <i>Crabp2</i> (O, O′) expression in the tailbud in vivo. All explants are orientated as in (D). C, Caudal; R, Rostral; asterisks indicate beads.</p

Expression of retinoid pathway genes in the maturing tailbud.

<p><i>Raldh2</i> expression in the maturing tailbud (A–D′). Arrows indicate new tailbud <i>Raldh2</i> domains in the caudo-lateral mesoderm progenitors (C–C″). <i>Cyp26a</i> expression is downregulated in the early tailbud and is absent by HH24 (E–H′). Gradual expansion of <i>Crabp2</i> transcription into chick tailbud from HH19 to HH26 in whole-mount (I–L), analysed in lateral (I′–L′) and medial (I″–L″) sagittal sections (SS) and in transverse sections (TS) of rostral (I′″–L′″) and caudal (I″″–L″″) tailbud. Black dashed lines indicate distal tip of notochord in (SS) and notochord in TS; red dashed lines indicate CNH at stages where this can be defined (HH19, 22, 24). Asterisk in (K′″) indicates neural CNH. Summary of changes in <i>Crabp2</i> expression between HH22 and HH24, indicating increasing RA activity in mesoderm progenitors and neural portion of the CNH and at edges of notochord CNH as body axis elongation ceases at HH24 (M). Bead grafting schematic (N). Expression of <i>Crabp2</i> in chicken embryos grafted with DMSO bead (N′) or retinoic acid (RA) bead (N″), indicating <i>Crabp2</i> increase in response to RA. <i>Crabp2</i> in quail embryos raised on normal (NQ) (O) or Vitamin A–deficient diet (VADQ) (O′), demonstrating RA-dependence of <i>Crabp2</i> transcription in vivo. Tailbud explant pairs (P) cultured in the presence of (P′) control DMSO or (P″) RAR/RXR antagonists shows RA dependence of <i>Crabp2</i> expression in late tailbud tissue. Abbreviations as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001415#pbio-1001415-g001" target="_blank">Figure 1</a>.</p