During early amniote embryonic development, cells establishing the primitive streak must orchestrate mesendoderm specification and convergent extension within the exact same cell population at roughly the same developmental time. Investigating these dual events in early chick embryos, researchers found that while fibroblast growth factor and cVG1 signalling can each induce markers for both processes, fibroblast growth factor signalling is strictly required for cVG1 to induce streak identity but dispensable for activating planar cell polarity. By monitoring TBXT, a marker of primitive streak identity, and PRICKLE1, a marker of the planar cell polarity pathway driving cell intercalation, the study clarifies how distinct signals coordinate complex cellular behaviours. These findings demonstrate that downstream programs required to construct the primitive streak are genetically separable, offering detailed mechanistic insight into early embryonic patterning and body axis elongation.

In amniote embryos, primitive streak formation establishes the primary anteroposterior body axis and initiates major morphogenetic movements. This developmental process requires embryonic epiblast cells to execute several distinct cellular programs simultaneously, including expressing signature identity markers like Brachyury, also designated TBXT, SNAI2, and TBX6, alongside an epithelial-to-mesenchymal transition leading directly to cell ingression. At the same time, prospective streak cells undergo epithelial cell intercalation, or convergent extension, which narrows the tissue mediolaterally while elongating the streak along the midline. In the disc-shaped epiblast of the chick embryo, streak formation can initiate from any position along the marginal zone circumference. This remarkable developmental flexibility highlights an essential question regarding how a single responding cell population integrates multiple molecular cues so that cell intercalation and lineage specification consistently occur together within the same embryonic territory.

Earlier developmental studies identified two primary signalling candidates governing these events: cVG1, a transforming growth factor-beta superfamily member annotated as GDF3 in chickens though synteny suggests orthology to GDF1, and fibroblast growth factor signals, predominantly FGF8. The factor cVG1 is expressed within the posterior marginal zone and positions primitive streak emergence. Grafting cVG1-expressing cell pellets into non-posterior embryonic regions induces ectopic streak formation, whereas morpholino knockdown of cVG1 disrupts streak establishment. Separately, FGF signalling was implicated in promoting streak elongation by activating WNT-planar cell polarity components like Flamingo, PRICKLE1, and VANGL2, which drive mediolateral intercalation via myosin-II. Although FGF signalling originates largely from the underlying cellular hypoblast, surgically removing the hypoblast still permits chick embryos to form and elongate functional primitive streaks, raising questions about whether hypoblast-derived FGF is truly mandatory for planar polarity induction.

To clarify how these signalling pathways interact during early development, the researchers systematically mapped the expression dynamics of FGF8, TBXT, and PRICKLE1 using whole-mount in situ hybridisation complemented by sectioning across embryonic stages defined by Eyal-Giladi and Kochav. Histological sections allowed the team to pinpoint the tissue layers expressing each transcript. The detailed spatial analysis showed that FGF8 is initially restricted to lower layers at stage EGK XII, concentrating in Koller’s sickle and the area pellucida hypoblast. By stage EGK XIII, FGF8 expression expands into both the area pellucida epiblast and hypoblast. Consistent with earlier RNA sequencing data indicating that FGF8 is the only significantly expressed fibroblast growth factor family member at these pre-streak stages, these observations confirmed that the hypoblast and Koller’s sickle represent the primary local sources of endogenous FGF signalling in the early blastoderm.

Examining downstream target genes revealed precise temporal patterns in prospective primitive streak cells prior to overt morphological changes. Unexpectedly, transcripts for TBXT were detected as early as stage EGK XI to XII within the most posterior epiblast, many hours before primitive streak formation. By stage Hamburger-Hamilton 2, as the streak begins physical emergence, TBXT is expressed in the forming mesendoderm and overlying epiblast. Concurrently, transcripts for PRICKLE1 appear at stage EGK XII to XIII within Koller’s sickle and the posterior area pellucida epiblast, intensifying markedly between stages EGK XIV and HH 2 in both mesendoderm and overlying epiblast. These overlapping spatiotemporal patterns indicated that posterior epiblast cells receive cues from both epiblast-derived cVG1 and lower-layer FGF8 during early pre-streak stages, setting the stage for primitive streak initiation.

The researchers next tested the capacity of cVG1 to activate streak identity and planar cell polarity by grafting transfected cell pellets into anterior embryonic regions. Human embryonic kidney 293T cells transfected with the DMVg1 expression plasmid were cultured in hanging drops to produce pellets of approximately 500 to 1000 cells. When grafted into the anterior marginal zone of stage EGK X to XI embryos, ectopic cVG1 induced expression of both TBXT and PRICKLE1 in adjacent epiblast cells. Time-course in situ hybridisation showed that neither marker was induced after six hours of incubation. By nine hours, however, robust ectopic expression appeared, with five of six embryos showing PRICKLE1 induction and five of five embryos showing TBXT induction. Continued incubation for twelve and fifteen hours yielded consistent induction, showing that cVG1 activates identity specification and planar cell polarity along a synchronized developmental timeline.

In parallel gain-of-function assays, the authors applied mouse FGF8b protein to the anterior marginal zone of stage EGK X to XI embryos using heparin-acrylic beads soaked at fifty micrograms per millilitre. After overnight culture, FGF8b induced an ectopic primitive streak expressing TBXT in four of six embryos, confirming its streak-inducing capacity. However, the detailed induction kinetics driven by FGF8 differed from those observed with cVG1 pellets. FGF8b beads induced PRICKLE1 expression within adjacent tissue by six hours in three of five embryos, whereas zero of five showed induction at three hours. In contrast, weak ectopic TBXT expression appeared as early as three hours post-grafting in four of five embryos, intensifying further by six hours in all six tested embryos. Thus, exogenous FGF8 activates transcription of both target genes substantially more rapidly than cVG1.

To test whether endogenous FGF signalling is required for either process, the researchers applied microbeads soaked in SU5402, a pharmacological inhibitor of fibroblast growth factor receptors, at 250 micromolar. Delivering SU5402 beads into the posterior area pellucida of stage EGK X to XI embryos completely abolished endogenous TBXT expression in zero of four embryos, compared to two of two control embryos receiving vehicle dimethyl sulfoxide beads. Crucially, posterior SU5402 treatment failed to suppress endogenous PRICKLE1 expression, with four of four embryos maintaining normal transcript expression alongside three of three controls. In anterior co-grafts with cVG1 pellets, SU5402 blocked cVG1-mediated TBXT induction in zero of six embryos, whereas four of four controls showed normal induction. Conversely, PRICKLE1 induction by cVG1 persisted in two of three SU5402-treated embryos and all six control embryos.

Because fibroblast growth factor receptors signal intracellularly through the mitogen-activated protein kinase pathway via MEK1 and MEK2, the authors verified their findings using U0126, a selective MEK1/2 inhibitor, alongside the inactive analogue U0124. Posterior treatment with U0126 beads severely suppressed TBXT expression in prospective streak tissue, leaving detectable transcripts in only two of twelve embryos, whereas eight of nine control embryos exposed to U0124 maintained normal TBXT expression. Conversely, endogenous PRICKLE1 transcripts persisted in eleven of twelve U0126-treated embryos and five of eight control embryos. In anterior co-grafts with cVG1 pellets, U0126 completely blocked TBXT induction in zero of four embryos while three of four controls showed expression; however, PRICKLE1 induction by cVG1 occurred robustly in four of five U0126-treated embryos and five of five controls, confirming selective MAPK pathway involvement.

To examine how cVG1 coordinates primitive streak development against inhibitory cues, the team evaluated bone morphogenetic protein signalling by grafting BMP4-expressing cell pellets into the posterior area pellucida. Earlier work established that BMP4 antagonizes cVG1 to inhibit primitive streak emergence in the early embryo. Implantation of BMP4 pellets at stage EGK X to XI disrupted PRICKLE1 expression, producing a distinct gap in the transcript domain after nine hours in seven of seven embryos, compared to zero of five controls. Extended overnight culture caused a physical splitting of the forming streak, with five of six embryos developing duplicated or bilateral streaks flanking the pellet. This clear result shows that BMP4 represses PRICKLE1 and prevents streak coherence, consistent with the view that cVG1 counteracts localized BMP signals to coordinate morphogenetic movements during normal primitive streak positioning.

Together, these experiments demonstrate that mesendoderm identity and convergent extension during primitive streak development are regulated through distinct pathways downstream of organizing signals. Fibroblast growth factor signalling through the MAPK pathway is strictly necessary for cVG1 to induce primitive streak identity marked by TBXT, explaining why these two factors collaborate in normal development. In contrast, FGF/MAPK activity is dispensable for cVG1 to induce PRICKLE1, resolving how primitive streaks elongate normally following surgical hypoblast removal. The authors emphasize that primitive streak formation in vivo involves additional concurrent events, including epithelial-to-mesenchymal transition and directed cell ingression, amid complex gradients of WNT, NODAL, and BMP molecules. While these findings clarify that cVG1 serves as a common upstream regulator for streak identity and convergent extension initiation, future experimental work must explore how these distinct signalling networks converge to orchestrate morphogenesis in early embryos.

Subject of Research: Signalling pathways governing mesendoderm specification and planar cell polarity during chick primitive streak formation

Article Title: Exploring the roles of FGF/MAPK and cVG1/GDF signalling on mesendoderm induction and convergent extension during chick primitive streak formation

Article References: Lee, H. C., Oliveira, N. M. M., & Stern, C. D. (2022). Exploring the roles of FGF/MAPK and cVG1/GDF signalling on mesendoderm induction and convergent extension during chick primitive streak formation. Development Genes and Evolution, 232(5-6), 115-123. https://doi.org/10.1007/s00427-022-00696-1

Image Credits: AI Generated

DOI: 10.1007/s00427-022-00696-1

Keywords: chick embryo, primitive streak, FGF8, cVG1, GDF3, TBXT, PRICKLE1, planar cell polarity, convergent extension, mesendoderm specification, SU5402, U0126, MEK1/2, BMP4, area pellucida, hypoblast, epiblast, in situ hybridisation