Development

The rapid growth of single-cell transcriptomic technology has produced an increasing number of datasets for both embryonic development and in vitro pluripotent stem cell derived models. This avalanche of data about pluripotency and the process of lineage specification has meant it has become increasingly difficult to define specific cell types or states and compare these to in vitro differentiation. Here we utilize a set of deep learning (DL) tools to integrate and classify multiple datasets. This allows for the definition of both mouse and human embryo cell types, lineages and states, thereby maximising the information one can garner from these precious experimental resources. Our approaches are built on recent initiatives for large scale human organ atlases, but here we focus on the difficult to obtain and process material that spans early mouse, and in particular, human development. Using publicly available data for these stages, we test different deep learning approaches and develop both a model to classify cell types in an unbiased fashion and define the set of genes required to identify lineages, cell types and states. We have used our predictions to probe pluripotent stem cell models for both mouse and human development, showcasing the importance of this resource as a dynamic reference for early embryogenesis.

Cranial ganglia are aggregates of sensory neurons that mediate distinct types of sensation. It is little understood how individual neurons coalesce, distribute and shape the ganglion. The statoacoustic ganglion (SAG) displays several lobes spatially arranged to properly connect with hair cells of the inner ear. To investigate the cellular behaviors involved in the 3D organization of the SAG, we use high resolution confocal imaging of single cell labeled zebrafish neuroblasts (NB), photoconversion, photoablation and genetic perturbations. We find that otic NB delaminate out of the otic epithelium in an EMT-like manner, rearranging apical polarity and primary cilia proteins. We also show that, once delaminated, NB migrate directionally and actively, requiring RhoGTPases. Interestingly, cell tracking of individual delaminated NB reveals that NB migrate and coalesce around a small population of pioneer SAG neurons. These pioneer SAG neurons are not from otic placode origin and populate the coalescence region before otic neurogenesis begins. Upon ablation of these cells, migratory pathways of delaminated NB are disrupted and, consequently, SAG shape is affected. Altogether, this work shows for the first time the role of pioneer SAG neurons in orchestrating SAG development. Summary StatementLittle is known how cranial sensory ganglia organize in 3D. We unveil the repertoire of cellular behaviours underlying statoacoustic morphogenesis and its dependence on relevant pioneer neurons.

The sclerotome in vertebrates comprises an embryonic population of cellular progenitors that give rise to diverse adult tissues including the axial skeleton, ribs, intervertebral discs, connective tissue, and vascular smooth muscle. In the thorax, this cell population arises in the ventromedial region of each of the segmented tissue blocks known as somites. How and when sclerotome adult tissue fates are specified and how the gene signatures that predate those fates are regulated has not been well studied. We have identified a previously unknown role for Ca2+/calmodulin-dependent protein kinase II (CaMKII) in regulating sclerotome patterning in zebrafish. Mechanistically, CaMKII regulates the activity of parallel signaling inputs that pattern sclerotome gene expression. In one downstream arm, CaMKII regulates distribution of the established sclerotome-inductive morphogen sonic hedgehog (Shh), and thus Shh-dependent sclerotome genes. In the second downstream arm, we show a previously unappreciated inductive requirement for Bmp signaling, where CaMKII activates expression of bmp4 and consequently Bmp activity. Bmp activates expression of a second subset of stereotypical sclerotome genes, while simultaneously repressing Shh-dependent markers. Our work demonstrates that CaMKII promotes parallel Bmp and Shh signaling as a mechanism to first promote global sclerotome specification, and that these pathways subsequently regionally activate and refine discrete compartmental genetic programs. Our work establishes how the earliest unique gene signatures that likely drive distinct cell behaviors and adult fates arise within the sclerotome.

Stem cell-based embryo models are promising alternatives for investigating early embryogenesis. We introduce two distinct models to replicate the dynamics between extra-embryonic endoderm and epiblast during mouse embryonic development. Inducible Gata6 (iGata6) embryoid bodies (EBs), exclusively derived from iGata6 embryonic stem (ES) cells, are valuable for modeling the position-dependent development of the primitive endoderm. Inner cell mass (ICM) embryoids, conversely, efficiently formed by aggregating wild-type and iGata6 ES cells, accurately and at a comparable pace simulate in vivo development from E3.5 to E7.5. Notably, ICM embryoids model cell sorting and through a rosette-like stage, the transition of the epiblast from naive to primed pluripotency. Furthermore, the absence of extra-embryonic ectoderm-like cells in this model, directs both the epiblast and visceral endoderm towards an anterior developmental fate. As such, iGata6 EBs and ICM embryoids are powerful tools for advancing our understanding of cell fate decisions during early embryonic development in mice.

The initiation of embryogenesis in the kelp Saccharina latissima is accompanied by significant anisotropy in cell shape. Using monoclonal antibodies, we show that this anisotropy coincides with a spatio-temporal pattern of accumulation of alginates in the cell wall of the zygote and embryo. Alginates rich in guluronates as well as sulphated fucans show a homogeneous distribution in the embryo throughout Phase I of embryogenesis, but mannuronate alginates accumulate mainly on the sides of the zygote and embryo, disappearing as the embryo enlarges at the start of Phase II. This pattern depends on the presence of cortical actin filaments. In contrast, within the embryo lamina, the alginate composition of the walls newly formed by cytokinesis is not affected by the depolymerisation of actin filaments. Thus, in addition to revealing the existence of a mannuronate-rich alginate corset that may restrict the enlargement of the zygote and the embryo, thereby promoting the formation of the apico-basal growth axis, we demonstrate stage- and cytoskeleton-dependent differences in cell wall deposition in Saccharina embryos.

Robust tissue growth control requires long-range communication between the rate of progenitor addition and tissue expansion. However, the regulatory mechanisms that couple these two processes are unknown. In zebrafish, notochord morphogenesis is a principal driver of axis extension through the combined actions of posterior progenitor addition and anterior vacuolation. To elucidate how progenitor dynamics and vacuole-driven cell expansion interact to shape notochord development, we first generated a mathematical model that links progenitor addition rate to the progressive expansion of cells from anterior-to-posterior to simulate vacuolation rate. Comparing this with empirical measurements, we find that progenitor incorporation together with vacuolation, produces a linear gradient in nearest neighbour distance. We next explored the role of YAP/TAZ in regulating the rate of progenitor addition in mutants for YAP/TAZ inhibitor vgll4b. We find that vgll4b expression and YAP activity are enriched in posterior midline progenitors. Loss of vgll4b elevates YAP signaling, enhances progenitor addition, restricts vacuole expansion, and--after a transient buffering phase--compromises A-P axis elongation. These results support a long-range feedback mechanism linking progenitor recruitment to vacuolation, enabling the notochord to balance cellular input with volumetric expansion, thereby maintaining tissue proportions. Graphical abstract. Proposed working model O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/706348v2_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@6c3c6dorg.highwire.dtl.DTLVardef@1f34eb0org.highwire.dtl.DTLVardef@b33a21org.highwire.dtl.DTLVardef@ad582b_HPS_FORMAT_FIGEXP M_FIG C_FIG A. Schematic representation of the authors proposed model illustrating how increased YAP activation in notochord progenitors of vgll4b mutants leads to enhanced progenitor addition to the notochord. This increased incorporation subsequently compromises the ability of notochord cells to undergo proper vacuolation, resulting in reduced axial elongation compared with control embryos.

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system, allows for spatial and temporal control of protein degradation, functioning through the activity of a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID method, utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 minutes of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 interacts with C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the Ftz-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work provides a conceptual improvement to the AID system for dissecting gene function at the single-cell level during C. elegans development.

Bone Morphogenic Protein (BMP) signaling plays an essential and highly conserved role in axial patterning in embryos of many externally developing animal species. However, in mammalian embryos, which develop inside the mother, early development includes an additional stage known as preimplantation. During preimplantation, the epiblast lineage is segregated from the extraembryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling in mouse preimplantation is imprecisely defined. We show that, in contrast to prior reports, BMP signaling (as reported by SMAD1/5/9 phosphorylation) is not detectable until implantation, when it is detected in the primitive endoderm - an extraembryonic lineage. Moreover, preimplantation development appears normal following deletion of maternal and zygotic Smad4, an essential effector of BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extraembryonic cell types drives epiblast morphogenesis post-implantation. Summary StatementGene expression, gene deletion, and pathway visualization evidence show that Smad4-dependent signaling is first active after mouse embryo implantation, when it promotes epiblast morphogenesis non-cell autonomously.

Transcription factors are key players in gene networks controlling cell fate specification during development. In multicellular organisms, they can display complex patterns of expression and binding to their targets, which necessitates tissue-specific characterisation of transcription factor-target interactions. Here, we focus on C. elegans seam cell development, which is used as a model of robust epidermal stem cell patterning. Despite our knowledge of multiple transcription factors playing a role in epidermal development, the composition of the gene network underlying cell fate patterning remains largely unknown. We introduce Targeted DamID (TaDa) that allows tissue-specific transcription factor target identification in intact C. elegans animals without cell isolation. We employ this method to recover putative targets in the epidermis for two transcription factors, the HES1 homologue LIN-22 and the NR5A1/2 nuclear hormone receptor NHR-25. Using single-molecule FISH (smFISH), we validate TaDa predictions and reveal a role for these transcription factors in promoting cell differentiation, as well as an unusual link between a HES factor and the Wnt signalling pathway. Our results expand our understanding of the epidermal gene network and highlight the power of TaDa to dissect the architecture of tissue-specific gene regulatory networks.

The specification and differentiation of atrial and ventricular myocardial cell types during development is incompletely understood. We have previously shown that Foxa2 expression during gastrulation identifies a population of ventricular fated progenitors, allowing for labeling of these cells prior to the morphogenetic events that lead to chamber formation and acquisition of bona fide atrial or ventricular identity. In this study, we performed single cell RNA sequencing of Foxa2Cre;mTmG embryos at the cardiac crescent (E8.25), primitive heart tube (E8.75) and heart tube (E9.25) stage in order to understand the transcriptional mechanisms underlying formation of atrial and ventricular cell types at the earliest stages of cardiac development. We find that progression towards differentiated myocardial cell types occurs primarily based on heart field progenitor identity, and that different progenitor populations contribute to ventricular or atrial identity through separate differentiation mechanisms. We identified a number of candidate markers that define such differentiation processes, as well as differential regulation of metabolic processes that distinguish atrial and ventricular fated cells at the earliest stages of development. We further show that exogenous injection with retinoic acid during formation of the cardiac primordia causes defects in ventricular chamber size and is associated with dysregulation in FGF signaling in anterior second heart field cells and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit in myocardial committed progenitors that result in formation of hypomorphic ventricles with decreased expression of important metabolic processes and sarcomere assembly. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac progenitor cell specification and differentiation, and the precise effects of manipulating cardiac progenitor field patterning via exogenous retinoic acid signaling.

A hallmark of gastrulation is the establishment of germ layers by internalization of cells initially on the exterior. In C. elegans the end of gastrulation is marked by the closure of the ventral cleft, a structure formed as cells internalize during gastrulation, and the subsequent rearrangement of adjacent neuroblasts that remain on the surface. We found that a nonsense allele of srgp-1/srGAP leads to 10-15% cleft closure failure. Deletion of the SRGP-1 C-terminal domain led to a comparable rate of cleft closure failure, whereas deletion of the N-terminal F-BAR region resulted in milder defects. Loss of the SRGP-1 C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/-catenin in surface cells during cleft closure. A mutant form of HMP-1 with an open M domain can suppress cleft closure defects in srgp-1 mutant backgrounds, suggesting that this mutation acts as a gain-of-function allele. Since SRGP-1 binding to HMP-1 is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1 is constitutively open. A good candidate is AFD-1/Afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1 is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/Afadin increases cleft closure defects in srgp-1 and hmp-1R551/554A backgrounds. We propose that SRGP-1 promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1 opens, allowing maturing junctions to transition from recruitment of SRGP-1 to AFD-1. Our work identifies new roles for -catenin interactors during a process crucial to metazoan development.

The meninges are a multilayered connective tissue that supports and protects the brain and skull, yet their developmental origins and signaling functions remain poorly understood. Here, we establish zebrafish as a tractable model for defining meningeal development and function across larval and adult stages. Using a restricted foxc1b:Gal4 reporter, we resolve the spatiotemporal emergence of meningeal fibroblasts and demonstrate a conserved, dosage-sensitive requirement for Foxc1 activity during meningeal formation. A targeted pharmacological screen identifies Wnt signaling as essential for timely establishment of the primary meninx. To define adult meningeal diversity, we integrate single-cell multiome profiling with spatial validation and identify multiple transcriptionally distinct populations organized into layered compartments, including pial, arachnoid, dural, and periosteal dura fibroblasts. Finally, inducible larval ablation of meningeal cells reveals limited regenerative capacity following widespread loss and leads to persistent defects in calvarial osteogenesis and brain architecture, including reduced osteoblast differentiation at bone fronts and disrupted tissue organization at sites lacking meningeal recovery. Together, these findings define key features of zebrafish meninges and provide a framework for dissecting meningeal development, regeneration, and meninges-dependent signaling in vivo. Summary StatementThis study uses zebrafish to show how the tissues surrounding the brain develop early and guide proper formation of both the brain and skull.

Pericytes are mural cells that provide support to the endothelium of small blood vessels. Pericyte soma are regularly spaced along vessels, and their processes overlap only slightly. Given that vessel patterning is imprecise, we explore the interplay between vessel growth and pericyte recruitment that leads to even pericyte spacing. After recruitment to the zebrafish brain central arteries (CtAs), pericytes undergo rapid expansion, followed by morphological differentiation. Blocking angiogenesis by reducing Gpr124 (Wnt) or Vegf signaling reduces the length of the vessel network and the number of pericytes, preserving spacing, suggesting proportional recruitment of pericytes to cover the network and the territorial nature of pericytes. However, these initial brain pericytes have low proliferation rates. We demonstrate that additional pericytes are recruited firstly through migration of col5a1- and later col1a2-expressing fibroblasts into the brain. These second-wave pericytes retain some fibroblast properties and show elevated col1a2 levels in a model of pericyte loss (notch3 mutants). Our data provide new insights into the developmental timing, expansion, and novel origins of late-arriving brain pericytes during embryogenesis. SUMMARY STATEMENTThis article demonstrates that brain pericytes originate from multiple sources, including fibroblast-derived populations, and how pericyte numbers are adjusted in proportion to vessel development.

Molecular embryology studies have established that the patterning of the gastrula-stage mouse embryo is dependent on a regulatory network where the WNT, BMP and NODAL signalling pathways cooperate. Still, important aspects of their respective contributions to this process remain unclear. Here, studying their impact on the spatial organization and the developmental trajectories of micro-patterned Epiblast Like Cells (EpiLC) colonies, we show that when BMP is present, it dominates NODAL and WNT and imposes a posterior character to the colonies differentiation. However, the use of two Nodal mutant cell lines allowed us to show that prior to BMP action, NODAL is required to establish the mesendodermal lineage. The fact that mutant phenotypes were more severe in vitro than in vivo suggests that embryonic phenotypes are partially rescued by ligands of extra-embryonic or maternal origin. Our work demonstrates the complementarity of micro-patterned EpiLC colonies to embryological approaches.

Early embryogenesis of the nematode Caenorhabditis elegans progresses in an autonomous fashion within a protective chitin eggshell. Cell division timing and the subsequent, mechanically guided positioning of cells is virtually invariant between individuals, especially before gastrulation. Here, we have challenged this stereotypical developmental program in early stages by mechanically perturbing the embryo, without breaking its eggshell. Compressing embryos to about 2/3 of their unperturbed diameter only resulted in markedly slower cell divisions. In contrast, compressing embryos to half of their native diameter frequently resulted in a loss of cytokinesis, yielding a non-natural syncytium that still allowed for multiple divisions of nuclei. Although the orientation of mitotic axes was strongly altered in the syncytium, key features of division timing and spatial arrangement of nuclei remained surprisingly similar to unperturbed embryos in the first few division cycles. This suggests that few, very robust mechanisms provide a basic and resilient program for safeguarding the early embryogenesis of C. elegans. STATEMENT OF SIGNIFICANCEEarly embryogenesis of the nematode Caenorhabditis elegans progresses in an autonomous fashion within a protective chitin eggshell. Cell division timing and cell positioning seemingly runs on autopilot, yielding a stereotypical development. Compressive forces, a potential hazard in the nematodes native habitat, may jeopardize this. We show that compressing embryos to 2/3 of their native diameter results in markedly slower cell divisions but leaves the early embryonic program otherwise intact. Further compression of embryos impairs the formation of new cells while nuclei still divide in a common cytoplasm (syncytium) with basic features of division timing and spatial arrangement being surprisingly similar to unperturbed embryos. This suggests that few robust mechanisms provide a basic program for the early embryonic autopilot.

Anterior-Posterior axis formation in the mouse embryo requires the active migration of the DVE cell population at E5.5. While intracellular Ca2+ signalling has been shown to control cell migration in multiple cell contexts, it is unknown whether it is required for DVE migration. The pattern of Ca2+ activity in the mouse embryo at early peri-implantation stages is also unknown. Using the GCaMP6f Ca2+ reporter line we performed a detailed assessment of Ca2+ dynamics between E0.5 - E5.5 using live imaging. We find that prior to implantation, Ca2+ transients are rare, but at E5.5 widespread, periodic, Ca2+ transients in extra-embryonic tissues can be observed, including in the VE and ExE. In contrast, cells of the E5.5 epiblast remain relatively quiescent but show sporadic large-scale multi-cellular waves. Inhibition of SERCA at E5.5 abolishes Ca2+ transients and leads to DVE arrest, indicative that these transients are required for axial patterning. Together these results reveal the pattern of Ca2+ handling in the early mouse embryo and a novel requirement in anterior-posterior axis formation.

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi (BC). Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity common to epithelial cells. Whether and how cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Time-course RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feedback to hepatoblast-to-hepatocyte differentiation. Summary statementDifferentiation of liver progenitors to functional hepatocytes requires anisotropic elongation of their nascent apical surfaces into tubular bile canaliculi.

Tudor domain-containing proteins are conserved across the animal kingdom for their function in germline development and fertility. Previously, we demonstrated that Tudor domain-containing protein 5-like (Tdrd5l) plays an important role in the germline where it promotes male identity. However, Tdrd5l is also expressed in both the ovary and testis during later stages of germline development, suggesting that it plays a role in germline differentiation in both sexes. We found that Tdrd5l localizes to a potentially novel germline body and plays a role in post-transcriptional gene regulation. RNA sequencing of Tdrd5l-mutant ovaries compared to wild-type showed that differentially expressed genes were enriched for maternally deposited RNAs. Additionally, embryos laid by Tdrd5l-mutant females exhibited reduced viability and displayed dorsal appendage defects suggesting a failure of proper dorsal-ventral (D/V) patterning. As D/V patterning is dependent on gurken (grk), we examined Grk expression during oogenesis. We observed premature accumulation of Grk protein in nurse cells indicating that translation is no longer properly repressed during mRNA transport to the oocyte. We also observed increased nurse cell accumulation of the cytoplasmic polyadenylation element binding protein Oo18 RNA-Binding Protein (Orb or CPEB), a translational activator of Grk. Decreasing orb function was able to partially rescue the Tdrd5l-mutant phenotype, and so defects in Orb are likely a primary cause of the defects in Tdrd5l mutants. Our data indicate that Tdrd5l is important for translational repression of maternal mRNAs such as orb, and possibly others, following their synthesis in the nurse cells and during their transport to the oocyte.

Studies of cell fate focus on specification, but little is known about maintenance of the differentiated state. We find that TGF{beta} signaling plays an essential role in maintenance of the tendon cell fate. To examine the role TGF{beta} signaling in tenocytes TGF{beta} type II receptor was targeted in the Scleraxis cell lineage. Tendon development was not disrupted in mutant embryos, but shortly after birth tenocytes lost differentiation markers and reverted to a more stem/progenitor state. Targeting of Tgfbr2 using other Cre drivers did not cause tenocyte dedifferentiation suggesting a critical significance for the spatio-temporal activity of ScxCre. Viral reintroduction of Tgfbr2 to mutants was sufficient to prevent and even rescue mutant tenocytes suggesting a continuous and cell-autonomous role for TGF{beta} signaling in cell fate maintenance. These results uncover the critical importance of molecular pathways that maintain the differentiated cell fate and a key role for TGF{beta} signaling in these processes.

Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the promyelocytic leukaemia zinc finger (Plzf) transcription factor is required for this transition to happen in an orderly fashion. Plzf expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Plzf, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in plzf mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.