Julien Dubrulle

FGF Signaling Controls Somite Boundary Position and Regulates Segmentation Clock Control of Spatiotemporal Hox Gene Activation

Vertebrate segmentation requires a molecular oscillator, the segmentation clock, acting in presomitic mesoderm (PSM) cells to set the pace at which segmental boundaries are laid down. However, the signals that position each boundary remain unclear. Here, we report that FGF8 which is expressed in the posterior PSM, generates a moving wavefront at which level both segment boundary position and axial identity become determined. Furthermore, by manipulating boundary position in the chick embryo, we show that Hox gene expression is maintained in the appropriately numbered somite rather than at an absolute axial position. These results implicate FGF8 in ensuring tight coordination of the segmentation process and spatiotemporal Hox gene activation.

fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo

Formation and patterning of the vertebrate embryo occur in a head-to-tail sequence. This progressive mode of body formation from the posterior end of the embryo requires a strict temporal coordination of tissue differentiation—a process involving fibroblast growth factor (FGF) signalling. Here we show that transcription of fgf8 messenger RNA is restricted to the growing posterior tip of the embryo. fgf8 mRNA is progressively degraded in the newly formed tissues, resulting in the formation of an mRNA gradient in the posterior part of the embryo. This fgf8 mRNA gradient is translated into a gradient of FGF8 protein, which correlates with graded phosphorylation of the kinase Akt, a downstream effector of FGF signalling. Such a mechanism provides an efficient means to monitor the timing of FGF signalling, coupling the differentiation of embryonic tissues to the posterior elongation of the embryo. In addition, this mechanism provides a novel model for morphogen gradient formation.

Toddler: An Embryonic Signal That Promotes Cell Movement via Apelin Receptors

Introduction Embryogenesis is thought to be directed by a small number of signaling pathways with most if not all embryonic signals having been identified. However, the molecular control of some embryonic processes is still poorly understood. For example, it is unclear how cell migration is regulated during gastrulation, when mesodermal and endodermal germ layers form. The goal of our study was to identify and characterize previously unrecognized signals that regulate embryogenesis. Toddler promotes gastrulation movements via Apelin receptor signaling. Toddler is an essential, short, conserved embryonic signal that promotes cell migration during zebrafish gastrulation. The internalization movement highlighted by the colored cell tracks requires Toddler signaling. Toddler signals via the G-protein–coupled APJ/Apelin receptor and may be one of several uncharacterized embryonic signals. Methods To identify uncharacterized signaling molecules, we mined zebrafish genomic data sets for previously non-annotated translated open reading frames (ORFs). One such ORF encoded a putative signaling protein that we call Toddler (also known as Apela/Elabela/Ende). We analyzed expression, production, and secretion of Toddler using RNA in situ hybridization, mass spectrometry, and Toddler-GFP fusion proteins, respectively. We used transcription activator-like effector (TALE) nucleases to generate frame-shift mutations in the toddler gene. To complement loss-of-function analyses with gain-of-function studies, Toddler was misexpressed through mRNA or peptide injection. We characterized phenotypes using marker gene expression analysis and in vivo imaging, using confocal and lightsheet microscopy. Toddler mutants were rescued thorugh global or localized toddler production. The relationship between Toddler and APJ/Apelin receptors was studied through genetic interaction and receptor internalization experiments. Results We identified several hundred non-annotated candidate proteins, including more than 20 putative signaling proteins. We focused on the functional importance of the short, conserved, and secreted peptide Toddler. Loss or overproduction of Toddler reduced cell movements during zebrafish gastrulation; mesodermal and endodermal cells were slow to internalize and migrate. Both the local and ubiquitous expression of Toddler were able to rescue gastrulation movements in toddler mutants, suggesting that Toddler acts as a motogen, a signal that promotes cell migration. Toddler activates G-protein–coupled APJ/Apelin receptor signaling, as evidenced by Toddler-induced internalization of APJ/Apelin receptors and rescue of toddler mutants through expression of the known receptor agonist Apelin. Discussion These findings indicate that Toddler promotes cell movement during zebrafish gastrulation by activation of APJ/Apelin receptor signaling. Toddler does not seem to act as a chemo-attractant or -repellent, but rather as a global signal that promotes the movement of mesendodermal cells. Both loss and overproduction of Toddler reduce cell movement, revealing that Toddler levels need to be tightly regulated during gastrulation. The discovery of Toddler helps explain previous genetic studies that found a broader requirement for APJ/Apelin receptors than for Apelin. We propose that in these cases, Toddler—not Apelin—activates APJ/Apelin receptor signaling. Our genomics analysis identifying a large number of candidate proteins that function during embryogenesis suggests the existence of other previously uncharacterized embryonic signals. Applying similar genomic approaches to adult tissues might identify additional signals that regulate physiological and behavioral processes. It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mesendodermal cells during zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell movement, suggesting that Toddler is neither an attractant nor a repellent but acts globally as a motogen. Toddler drives internalization of G protein–coupled APJ/Apelin receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes gastrulation movements, and might be the first in a series of uncharacterized developmental signals. A conserved signal is identified that activates G protein–coupled receptors to promote zebrafish gastrulation. Toddler Welcome It has been assumed that most, if not all, major signals that control vertebrate embryogenesis have been identified. Using genomics, Pauli et al. (10.1126/science.1248636, published online 9 January) have now identified several new candidate signals expressed during early zebrafish development. One of these signals, Toddler, is a short, conserved, and secreted peptide that promotes the movement of cells during zebrafish gastrulation. Toddler signals through G protein–coupled receptors to drive internalization of the Apelin receptor, and activation of Apelin signaling can rescue toddler mutants.

Coupling segmentation to axis formation.

A characteristic feature of the vertebrate body is its segmentation along the anteroposterior axis, as illustrated by the repetition of vertebrae that form the vertebral column. The vertebrae and their associated muscles derive from metameric structures of mesodermal origin, the somites. The segmentation of the body is established by somitogenesis, during which somites form sequentially in a rhythmic fashion from the presomitic mesoderm. This review highlights recent findings that show how dynamic gradients of morphogens and retinoic acid, coupled to a molecular oscillator, drive the formation of somites and link somitogenesis to the elongation of the anteroposterior axis.

Uncoupling segmentation and somitogenesis in the chick presomitic mesoderm.

Little is known about the tissue interactions and the molecular signals implicated in the sequence of events leading to the subdivision of the somite into its rostral and caudal compartments. It has been demonstrated that rostrocaudal identity of the sclerotome is acquired at the presomitic (PSM) level. However, it is not known whether this compartment specification is fully determined in the PSM or whether it is dependent upon maintenance cues from the surrounding environment, as is the case for somite epithelialization. In this report, we address this issue by examining the expression profiles of C-Delta-1 and C-Notch-1, the avian homologues of mouse Delta-like1 (Delta1) and Notch1 which have been implicated in the specification of the somite rostrocaudal polarity in mouse. In chick, these genes are expressed in distinct but partially overlapping domains in the PSM and subsequently in the caudal regions of the somites. We have used an in vitro assay that consists of culturina PSM explants to examine the regulation of these genes in this tissue. We find that PSM explants cultured without overlying ectoderm continue to lay down stripes of C-Delta-1 expression, although epithelialization is blocked. These results suggest that somite rostrocaudal patterning is an autonomous property of the PSM. In addition, they demonstrate that segmentation is not necessarily coupled with the formation of somites.

Somite formation and patterning.

As a consequence of their segmented arrangement and the diversity of their tissue derivatives, somites are key elements in the establishment of the metameric body plan in vertebrates. This article aims to largely review what is known about somite development, from the initial stages of somite formation through the process of somite regionalization along the three major body axes. The role of both cell intrinsic mechanisms and environmental cues are evaluated. The periodic and bilaterally synchronous nature of somite formation is proposed to rely on the existence of a developmental clock. Molecular mechanisms underlying these events are reported. The importance of an antero-posterior somitic polarity with respect to somite formation on one hand and body segmentation on the other hand is discussed. Finally, the mechanisms leading to the regionalization of somites along the dorso-ventral and medio-lateral axes are reviewed. This somitic compartmentalization is believed to underlie the segregation of dermis, skeleton, and dorsal and appendicular musculature.

Response to Nodal morphogen gradient is determined by the kinetics of target gene induction

Morphogen gradients expose cells to different signal concentrations and induce target genes with different ranges of expression. To determine how the Nodal morphogen gradient induces distinct gene expression patterns during zebrafish embryogenesis, we measured the activation dynamics of the signal transducer Smad2 and the expression kinetics of long- and short-range target genes. We found that threshold models based on ligand concentration are insufficient to predict the response of target genes. Instead, morphogen interpretation is shaped by the kinetics of target gene induction: the higher the rate of transcription and the earlier the onset of induction, the greater the spatial range of expression. Thus, the timing and magnitude of target gene expression can be used to modulate the range of expression and diversify the response to morphogen gradients. DOI: http://dx.doi.org/10.7554/eLife.05042.001

no tail integrates two modes of mesoderm induction

During early zebrafish development the nodal signalling pathway patterns the embryo into three germ layers, in part by inducing the expression of no tail (ntl), which is essential for correct mesoderm formation. When nodal signalling is inhibited ntl fails to be expressed in the dorsal margin, but ventral ntl expression is unaffected. These observations indicate that ntl transcription is under both nodal-dependent and nodal-independent regulation. Consistent with these observations and with a role for ntl in mesoderm formation, some somites form within the tail region of embryos lacking nodal signalling. In an effort to understand how ntl is regulated and thus how mesoderm forms, we have mapped the elements responsible for nodal-dependent and nodal-independent expression of ntl in the margin of the embryo. Our work demonstrates that expression of ntl in the margin is the consequence of two separate enhancers, which act to mediate different mechanisms of mesoderm formation. One of these enhancers responds to nodal signalling, and the other to Wnt and BMP signalling. We demonstrate that the nodal-independent regulation of ntl is essential for tail formation. Misexpression of Wnt and BMP ligands can induce the formation of an ectopic tail, which contains somites, in embryos devoid of nodal signalling, and this tail formation is dependent on ntl function. Similarly, nodal-independent tail somite formation requires ntl. At later stages in development ntl is required for notochord formation, and our analysis has also led to the identification of the enhancer required for ntl expression in the developing notochord.

Welcome to Syndetome: A New Somitic Compartment

Virtually nothing was known about the embryonic origin of tendons, until a recent paper by Brent and colleagues in which they track the origin of tendon progenitors of the body axis and reveal the molecular events and tissue interactions leading to their commitment.