The FGF8-related signals Pyramus and Thisbe promote pathfinding, substrate adhesion, and survival of migrating longitudinal gut muscle founder cells

Fibroblast growth factors (FGFs) frequently fulfill prominent roles in the regulation of cell migration in various contexts. In Drosophila, the FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths), which signal through their receptor Heartless (Htl), are known to regulate early mesodermal cell migration after gastrulation as well as glial cell migration during eye development. Herein, we show that Pyr and Ths also exert key roles during the long-distance migration of a specific sub-population of mesodermal cells that migrate from the caudal visceral mesoderm within stereotypic bilateral paths along the trunk visceral mesoderm toward the anterior. These cells constitute the founder myoblasts of the longitudinal midgut muscles. In a forward genetic screen for regulators of this morphogenetic process we identified loss of function alleles for pyr. We show that pyr and ths are expressed along the paths of migration in the trunk visceral mesoderm and endoderm and act largely redundantly to help guide the founder myoblasts reliably onto and along their substrate of migration. Ectopically-provided Pyr and Ths signals can efficiently re-rout the migrating cells, both in the presence and absence of endogenous signals. Our data indicate that the guidance functions of these FGFs must act in concert with other important attractive or adhesive activities of the trunk visceral mesoderm. Apart from their guidance functions, the Pyr and Ths signals play an obligatory role for the survival of the migrating cells. Without these signals, essentially all of these cells enter cell death and detach from the migration substrate during early migration. We present experiments that allowed us to dissect the roles of these FGFs as guidance cues versus trophic activities during the migration of the longitudinal visceral muscle founders.     

The RhoGEF Pebble is required for cell shape changes during cell migration triggered by the Drosophila FGF receptor Heartless

The FGF receptor Heartless (HTL) is required for mesodermal cell migration in the Drosophila gastrula. We show that mesoderm cells undergo different phases of specific cell shape changes during mesoderm migration. During the migratory phase, the cells adhere to the basal surface of the ectoderm and exhibit extensive protrusive activity. HTL is required for the protrusive activity of the mesoderm cells. Moreover, the early phenotype of htl mutants suggests that HTL is required for the adhesion of mesoderm cells to the ectoderm. In a genetic screen we identified pebble (pbl) as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. We show that the function of PBL in cell migration is independent of the function of PBL in cytokinesis. Although RHO1 acts as a substrate for PBL in cytokinesis, compromising RHO1 function in the mesoderm does not block cell migration. These data suggest that the function of PBL in cell migration might be mediated through a pathway distinct from RHO1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. We show that PBL is autonomously required in the mesoderm for cell migration. Like HTL, PBL is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of HTL is unable to rescue the early cellular defects in pbl mutants, suggesting that PBL is required for the ability of HTL to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF PBL in HTL-dependent mesodermal cell migration.     

Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.     

Photoactivatable GFP resolves Drosophila mesoderm migration behaviour

Mesoderm migration is a pivotal event in the early embryonic development of animals. One of the best-studied examples occurs during Drosophila gastrulation. Here, mesodermal cells invaginate, undergo an epithelial-to-mesenchymal transition (EMT), and spread out dorsally over the inner surface of the ectoderm. Although several genes required for spreading have been identified, our inability to visualise mesodermal cells in living embryos has left us to speculate about the cell rearrangements involved. Several mechanisms, such as chemotaxis towards a dorsally expressed attractant, differential affinity between mesodermal cells and the ectoderm, and convergent extension, have been proposed. Here we resolve the behaviour of Drosophila mesodermal cells in live embryos using photoactivatable-GFP fused to alpha-Tubulin (PAGFP-Tub). By photoactivating presumptive mesodermal cells before gastrulation, we could observe their migration over non-fluorescent ectodermal cells. We show that the outermost (outer) cells, which are in contact with the ectoderm, migrate dorsolaterally as a group but can be overtaken by more internal (inner) cells. Using laser-photoactivation of individual cells, we then show that inner cells adjacent to the centre of the furrow migrate dorsolaterally away from the midline to reach dorsal positions, while cells at the centre of the furrow disperse randomly across the mesoderm, before intercalating with outer cells. These movements are dependent on the FGF receptor Heartless. The results indicate that chemotactic movement and differential affinity are the primary drivers of mesodermal cell spreading. These characterisations pave the way for a more detailed analysis of gene function during early mesoderm development.     

Specificity of FGF signaling in cell migration in Drosophila.

We wanted to investigate the relationship between receptor tyrosine kinase (RTK) activated signaling pathways and the induction of cell migration. Using Drosophila tracheal and mesodermal cell migration as model systems, we find that the intracellular domain of the fibroblast growth factor receptors (FGFRs) Breathless (Btl) and Heartless (Htl) can be functionally replaced by the intracellular domains of Torso (Tor) and epidermal growth factor receptor (EGFR). These hybrid receptors can also rescue cell migration in the absence of Downstream of FGFR (Dof), a cytoplasmic protein essential for FGF signaling. These results demonstrate that tracheal and mesodermal cells respond during a specific time window to a receptor tyrosine kinase (RTK) signal with directed migration, independent of the presence or absence of Dof. We discuss our findings in the light of the recent findings that RTKs generate a generic signal that is interpreted in responding cells according to their developmental history.     

FGF8 spliceforms mediate early mesoderm and posterior neural tissue formation in Xenopus

The relative contributions of different FGF ligands and spliceforms to mesodermal and neural patterning in Xenopus have not been determined, and alternative splicing, though common, is a relatively unexplored area in development. We present evidence that FGF8 performs a dual role in X. laevis and X. tropicalis early development. There are two FGF8 spliceforms, FGF8a and FGF8b, which have very different activities. FGF8b is a potent mesoderm inducer, while FGF8a has little effect on the development of mesoderm. When mammalian FGF8 spliceforms are analyzed in X. laevis, the contrast in activity is conserved. Using a loss-of-function approach, we demonstrate that FGF8 is necessary for proper gastrulation and formation of mesoderm and that FGF8b is the predominant FGF8 spliceform involved in early mesoderm development in Xenopus. Furthermore, FGF8 signaling is necessary for proper posterior neural formation; loss of either FGF8a or a reduction in both FGF8a and FGF8b causes a reduction in the hindbrain and spinal cord domains.     

FGF signalling and the mechanism of mesoderm spreading in Drosophila embryos

FGF signalling is needed for the proper establishment of the mesodermal cell layer in Drosophila embryos. The activation of the FGF receptor Heartless triggers the di-phosphorylation of MAPK in the mesoderm, which accumulates in a graded fashion with the highest levels seen at the dorsal edge of the mesoderm. We have examined the specific requirement for FGF signalling in the spreading process. We show that only the initial step of spreading, specifically the establishment of contact between the ectoderm and the mesoderm, depends upon FGF signalling, and that unlike the role of FGF signalling in the differentiation of heart precursors this function cannot be replaced by other receptor tyrosine kinases. The initiation of mesoderm spreading requires the FGF receptor to possess a functional kinase domain, but does not depend upon the activation of MAPK. Thus, the dispersal of the mesoderm at early stages is regulated by pathways downstream of the FGF receptor that are independent of the MAPK cascade. Furthermore, we demonstrate that the activation of MAPK by Heartless needs additional cues from the ectoderm. We propose that FGF signalling is required during the initial stages of mesoderm spreading to promote the efficient interaction of the mesoderm with the ectoderm rather than having a long range chemotactic function, and we discuss this in relation to the cellular mechanism of mesoderm spreading.     

FGF signal interpretation is directed by Sprouty and Spred proteins during mesoderm formation

Vertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. We identified two genes for each family in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments we show that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca(2+) and PKCdelta signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca(2+) or PKCdelta signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation.     

The role of FGF signaling in guiding coordinate movement of cell groups

Cell migration influences cell-cell interactions to drive cell differentiation and organogenesis. To support proper development, cell migration must be regulated both temporally and spatially. Mesoderm cell migration in the Drosophila embryo serves as an excellent model system to study how cell migration is controlled and influences organogenesis. First, mesoderm spreading transforms the embryo into a multilayered form during gastrulation and, subsequently, cells originating from the caudal visceral mesoderm (CVM) migrate along the entire length of the gut. Here we review our studies, which have focused on the role of fibroblast growth factor (FGF) signaling, and compare and contrast these two different cell migration processes: mesoderm spreading and CVM migration. In both cases, FGF acts as a chemoattractant to guide cells’ directional movement but is likely not the only signal that serves this role. Furthermore, FGF likely modulates cell adhesion properties since FGF mutant phenotypes share similarities with those of cell adhesion molecules. Our working hypothesis is that levels of FGF signaling differentially influence cells’ response to result in either directional movement or changes in adhesive properties.     

The role of FGF signaling in guiding coordinate movement of cell groups: Guidance cue and cell adhesion regulator?

Cell migration influences cell-cell interactions to drive cell differentiation and organogenesis. To support proper development, cell migration must be regulated both temporally and spatially. Mesoderm cell migration in the Drosophila embryo serves as an excellent model system to study how cell migration is controlled and influences organogenesis. First, mesoderm spreading transforms the embryo into a multilayered form during gastrulation and, subsequently, cells originating from the caudal visceral mesoderm (CVM) migrate along the entire length of the gut. Here we review our studies, which have focused on the role of fibroblast growth factor (FGF) signaling, and compare and contrast these two different cell migration processes: mesoderm spreading and CVM migration. In both cases, FGF acts as a chemoattractant to guide cells’ directional movement but is likely not the only signal that serves this role. Furthermore, FGF likely modulates cell adhesion properties since FGF mutant phenotypes share similarities with those of cell adhesion molecules. Our working hypothesis is that levels of FGF signaling differentially influence cells’ response to result in either directional movement or changes in adhesive properties.     

FGF8/17/18 functions together with FGF9/16/20 during formation of the notochord in Ciona embryos

Fibroblast growth factor (FGF) signalling has been implicated in the generation of mesoderm and neural fates in chordate embryos including ascidians and vertebrates. In Ciona, FGF9/16/20 has been implicated in both of these processes. However, in FGF9/16/20 knockdown embryos, notochord fate recovers during later development. It is thus not clear if FGF signalling is an essential requirement for notochord specification in Ciona embryos. We show that FGF-MEK-ERK signals act during two distinct phases to establish notochord fate. During the first phase, FGF signalling is required during an asymmetric cell division to promote notochord at the expense of neural identity. Consistently, ERK1/2 is specifically activated in the notochord precursors following this cell division. Sustained activation of ERK1/2 is then required to maintain notochord fate. We demonstrate that FGF9/16/20 acts solely during the initial induction step and that, subsequently, FGF8/17/18 together with FGF9/16/20 is involved in the following maintenance step. These results together with others' show that the formation of a large part of the mesoderm cell types in ascidian larvae is dependent on signalling events involving FGF ligands.     

Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8

During gastrulation in amniotes, epiblast cells ingress through the primitive streak and migrate away to form endodermal, mesodermal, and extraembryonic structures. Here we analyze the detailed movement trajectories of cells emerging at different anterior-posterior positions from the primitive streak, using in vivo imaging of the movement of GFP-tagged streak cells. Cells emerging at different anterior-posterior positions from the streak show characteristic cell migration patterns, in response to guidance signals from neighboring tissues. Streak cells are attracted by sources of FGF4 and repelled by sources of FGF8. The observed movement patterns of anterior streak cells can be explained by an FGF8-mediated chemorepulsion of cells away from the streak followed by chemoattraction toward an FGF4 signal produced by the forming notochord.     

FGF/MAPK signaling is required in the gastrula epiblast for avian neural crest induction

Neural crest induction involves the combinatorial inputs of the FGF, BMP and Wnt signaling pathways. Recently, a two-step model has emerged where BMP attenuation and Wnt activation induces the neural crest during gastrulation, whereas activation of both pathways maintains the population during neurulation. FGF is proposed to act indirectly during the inductive phase by activating Wnt ligand expression in the mesoderm. Here, we use the chick model to investigate the role of FGF signaling in the amniote neural crest for the first time and uncover a novel requirement for FGF/MAPK signaling. Contrary to current models, we demonstrate that FGF is required within the prospective neural crest epiblast during gastrulation and is unlikely to operate through mesodermal tissues. Additionally, we show that FGF/MAPK activity in the prospective neural plate prevents the ectopic expression of lateral ectoderm markers, independently of its role in neural specification. We then investigate the temporal participation of BMP/Smad signaling and suggest a later involvement in neural plate border development, likely due to widespread FGF/MAPK activity in the gastrula epiblast. Our results identify an early requirement for FGF/MAPK signaling in amniote neural crest induction and suggest an intriguing role for FGF-mediated Smad inhibition in ectodermal development.     

FGF signal regulates gastrulation cell movements and morphology through its target NRH

We used cDNA microarray analysis to screen for FGF target genes in Xenopus embryos treated with the FGFR1 inhibitor SU5402, and identified neurotrophin receptor homolog (NRH) as an FGF target. Causing gain of NRH function by NRH mRNA or loss of NRH function using a Morpholino antisense-oligonucleotide (Mo) led to gastrulation defects without affecting mesoderm differentiation. Depletion of NRH by the Mo perturbed the polarization of cells in the dorsal marginal zone (DMZ), thereby inhibiting the intercalation of the cells during convergent extension as well as the filopodia formation on DMZ cells. Deletion analysis showed that the carboxyl-terminal region of NRH, which includes the "death domain," was necessary and sufficient to rescue gastrulation defects and to induce the protrusive cell morphology. Furthermore, we found that the FGF signal was both capable of inducing filopodia in animal cap cells, where they do not normally form, and necessary for filopodia formation in DMZ cells. Finally, we demonstrated that FGF required NRH function to induce normal DMZ cell morphology. This study is the first to identify an in vivo role for FGF in the regulation of cell morphology, and we have linked this function to the control of gastrulation cell movements via NRH.