Search

During development, tightly regulated gene expression programs control cell fate and patterning. A key regulatory step in eukaryotic transcription is the assembly of the pre-initiation complex (PIC) at promoters. The PIC assembly has mainly been studiedin vitro, and little is known about its composition during development.In vitrodata suggests that TFIID is the general transcription factor that nucleates PIC formation at promoters. Here we show that TAF10, a subunit of TFIID and of the transcriptional co-activator SAGA, is required for the assembly of these complexes in the mouse embryo. We performedTaf10conditional deletions during mesoderm development and show thatTaf10loss in the presomitic mesoderm (PSM) does not prevent cyclic gene transcription or PSM segmental patterning, while lateral plate differentiation is profoundly altered. During this period, global mRNA levels are unchanged in the PSM, with only a minor subset of genes dysregulated. Together, our data demonstrate that the TAF10-containing canonical TFIID and SAGA complexes, are dispensable for early paraxial mesoderm development, arguing against the generic role in transcription proposed for these fully assembled holo complexes.

Neuromesodermal (NM) stem cells generate neural and paraxial presomitic mesoderm (PSM) cells, which are the respective progenitors of the spinal cord and musculoskeleton of the trunk and tail. The Wnt-regulated basic helix-loop-helix (bHLH) transcription factor mesogenin 1 (Msgn1) has been implicated as a cooperative regulator working in concert with T-box genes to control PSM formation in zebrafish, although the mechanism is unknown. We show here that, in mice, Msgn1 alone controls PSM differentiation by directly activating the transcriptional programs that define PSM identity, epithelial-mesenchymal transition, motility and segmentation. Forced expression of Msgn1 in NM stem cells in vivo reduced the contribution of their progeny to the neural tube, and dramatically expanded the unsegmented mesenchymal PSM while blocking somitogenesis and notochord differentiation. Expression of Msgn1 was sufficient to partially rescue PSM differentiation in Wnt3a−/− embryos, demonstrating that Msgn1 functions downstream of Wnt3a as the master regulator of PSM differentiation. Our data provide new insights into how cell fate decisions are imposed by the expression of a single transcriptional regulator.

Vertebrate segments called somites are generated by periodic segmentation of the anterior extremity of the presomitic mesoderm (PSM). During somite segmentation in zebrafish, mesp-b determines a future somite boundary at position B-2 within the PSM. Heat-shock experiments, however, suggest that an earlier future somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Here, we characterized fibroblast growth factor (FGF) signal activity within the PSM, and demonstrated that an anterior limit of downstream Erk activity corresponds to the future B-5 somite boundary. Moreover, the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. Our results provide the first molecular evidence of the future somite boundary at B-5, and we propose that clock-dependent cyclic inhibition of the FGF/Erk signal is a key mechanism in the generation of perfect repetitive structures in zebrafish development.

During vertebrate development, the primary body axis elongates towards the posterior and is periodically divided into somites, which give rise to the vertebrae, skeletal muscles and dermis. Somites form periodically from anterior to posterior, and the anterior somites form in a more rapid cycle than the posterior somites. However, how this anteroposterior (AP) difference in somitogenesis is generated and how it contributes to the vertebrate body plan remain unclear. Here, we show that the AP difference in zebrafish somitogenesis originates from a variable overlapping segmentation period between one somite and the next. The AP difference is attributable to spatiotemporal inhibition of the clock gene her1 via retinoic acid (RA) regulation of the transcriptional repressor ripply1. RA depletion thus disrupts timely somite formation at the transition, eventually leading to the loss of one somite and the resultant cervical vertebra. Overall, our results indicate that RA regulation of the AP difference is crucial for proper linkage between the head and trunk in the vertebrate body plan.

The segmental pattern of somites is generated by sequential conversion of the temporal periodicity provided by the molecular clock. Whereas the basic structure of this clock is conserved among different species, diversity also exists especially in terms of the molecular network. The temporal periodicity is subsequently converted into the spatial pattern of somites, and Mesp2 plays critical roles in this conversion in the mouse. However, it remains unclear whether Mesp plays similar roles in other vertebrates. In this study, we generated zebrafish mutants lacking all 4 zebrafish mesp genes by using TALEN-mediated genome editing. Contrary to the case for the mouse Mesp2 mutant, in the zebrafish mesp quadruple mutant embryos, the positions of somite boundaries were clearly determined and morphological boundaries were formed, although their formation was not completely normal. On the other hand, each somite was caudalized similarly to the mouse Mesp2 mutant and superficial horizontal myoseptum, and lateral line primordia were not properly formed in the quadruple mutants. These results clarify the conserved and species-specific roles of Mesp in the connection of the molecular clock to somite morphogenesis.

Vertebrate somitogenesis is regulated by a segmentation clock. Clock-linked genes exhibit cyclic expression, with a periodicity matching the rate of somite production. In mice, lunatic fringe (Lfng) expression oscillates, and LFNG protein contributes to periodic repression of Notch signaling. We hypothesized that rapid LFNG turnover could be regulated by protein processing and secretion. Here, we describe a novel Lfng allele (Lfng(RLFNG)), replacing the N-terminal sequences of LFNG, which allow for protein processing and secretion, with the N-terminus of radical fringe (a Golgi-resident protein). This allele is predicted to prevent protein secretion without altering the activity of LFNG, thus increasing the intracellular half-life of the protein. This allele causes dominant skeletal and somite abnormalities that are distinct from those seen in Lfng loss-of-function embryos. Expression of clock-linked genes is perturbed and mature Hes7 transcripts are stabilized in the presomitic mesoderm of mutant mice, suggesting that both transcriptional and post-transcriptional regulation of clock components are perturbed by RLFNG expression. Contrasting phenotypes in the segmentation clock and somite patterning of mutant mice suggest that LFNG protein may have context-dependent effects on Notch activity.

Somites are formed from the presomitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscles. The PSM is dynamic; somites are generated at the anterior end, while the posterior end is continually renewed with new cells entering from the tailbud progenitor region. Which genes control the conversion of tailbud progenitors into PSM and how is this process coordinated with cell movement? Using loss- and gain-of-function experiments and heat-shock transgenics we show in zebrafish that the transcription factor Mesogenin 1 (Msgn1), acting with Spadetail (Spt), has a central role. Msgn1 allows progression of the PSM differentiation program by switching off the progenitor maintenance genes ntl, wnt3a, wnt8 and fgf8 in the future PSM cells as they exit from the tailbud, and subsequently induces expression of PSM markers such as tbx24. msgn1 is itself positively regulated by Ntl/Wnt/Fgf, creating a negative-feedback loop that might be crucial to regulate homeostasis of the progenitor population until somitogenesis ends. Msgn1 drives not only the changes in gene expression in the nascent PSM cells but also the movements by which they stream out of the tailbud into the PSM. Loss of Msgn1 reduces the flux of cells out of the tailbud, producing smaller somites and an enlarged tailbud, and, by delaying exhaustion of the progenitor population, results in supernumerary tail somites. Through its combined effects on gene expression and cell movement, Msgn1 (with Spt) plays a key role both in genesis of the paraxial mesoderm and in maintenance of the progenitor population from which it derives.

The transcription factors Nanog and Oct4 regulate pluripotency in the pre-implantation epiblast and in derivative embryonic stem cells. During post-implantation development, the precise timing and mechanism of the loss of pluripotency is unknown. Here, we show that in the mouse, pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis expression of both Nanog and Oct4 is regionalized. We show that pluripotency tracks the in vivo level of Oct4 and not Nanog by assessing the ability to reactivate or maintain Nanog expression in cell culture. Enforced Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitates moribund pluripotency. Our data suggest that decreasing Oct4 expression is converted to a sudden drop in competence to maintain pluripotency gene regulatory network activity that is subsequently stabilized by epigenetic locks.

In mouse, Hedgehog (Hh) signalling is required for most ventral spinal neurons to form. Here, we analyse the spinal cord phenotype of zebrafish maternal-zygotic smoothened (MZsmo) mutants that completely lack Hh signalling. We find that most V3 domain cells and motoneurons are lost, whereas medial floorplate still develops normally and V2, V1 and V0v cells form in normal numbers. This phenotype resembles that of mice that lack both Hh signalling and Gli repressor activity. Ventral spinal cord progenitor domain transcription factors are not expressed at 24 hpf in zebrafish MZsmo mutants. However, pMN, p2 and p1 domain markers are expressed at early somitogenesis stages in these mutants. This suggests that Gli repressor activity does not extend into zebrafish ventral spinal cord at these stages, even in the absence of Hh signalling. Consistent with this, ectopic expression of Gli3R represses ventral progenitor domain expression at these early stages and knocking down Gli repressor activity rescues later expression. We investigated whether retinoic acid (RA) signalling specifies ventral spinal neurons in the absence of Hh signalling. The results suggest that RA is required for the correct number of many different spinal neurons to form. This is probably mediated, in part, by an effect on cell proliferation. However, V0v, V1 and V2 cells are still present, even in the absence of both Hh and RA signalling. We demonstrate that Gli1 has a Hh-independent role in specifying most of the remaining motoneurons and V3 domain cells in embryos that lack Hh signalling, but removal of Gli1 activity does not affect more dorsal neurons.

Hepatic competence, or the ability to respond to hepatic-inducing signals, is regulated by a number of transcription factors broadly expressed in the endoderm. However, extrinsic signals might also regulate hepatic competence, as suggested by tissue explant studies. Here, we present genetic evidence that Fgf signaling regulates hepatic competence in zebrafish. We first show that the endoderm posterior to the liver-forming region retains hepatic competence: using transgenic lines that overexpress hepatic inducing signals following heat-shock, we found that at late somitogenesis stages Wnt8a, but not Bmp2b, overexpression could induce liver gene expression in pancreatic and intestinal bulb cells. These manipulations resulted in the appearance of ectopic hepatocytes in the intestinal bulb. Second, by overexpressing Wnt8a at various stages, we found that as embryos develop, the extent of the endodermal region retaining hepatic competence is gradually reduced. Most significantly, we found, using gain- and loss-of-function approaches, that Fgf10a signaling regulates this gradual reduction of the hepatic-competent domain. These data provide in vivo evidence that endodermal cells outside the liver-forming region retain hepatic competence and show that an extrinsic signal, Fgf10a, negatively regulates hepatic competence.

The spatiotemporally dynamic distribution of instructive ligands within embryonic tissue, and their feedback antagonists, including inherent stabilities and rates of clearance, are affected by interactions with cell surfaces or extracellular matrix (ECM). Nodal (here, Xnr1 or Nodal1 in Xenopus) and Lefty interact in a cross-regulatory relationship in mesendoderm induction, and are the conserved instructors of left-right (LR) asymmetry in early somitogenesis stage embryos. By expressing Xnr1 and Lefty proproteins that produce mature functional epitope-tagged ligands in vivo, we found that ECM is a principal surface of Nodal and Lefty accumulation. We detected Lefty moving faster than Nodal, with evidence that intact sulfated proteoglycans in the ECM facilitate the remarkable long distance movement of Nodal. We propose that Nodal autoregulation substantially aided by rapid ligand transport underlies the anteriorward shift of Nodal expression in the left LPM (lateral plate mesoderm), and speculate that the higher levels of chondroitin-sulfate proteoglycan (CSPG) in more mature anterior regions provide directional transport cues. Immunodetection and biochemical analysis showed transfer of Lefty from left LPM to right LPM, providing direct evidence that left-side-derived Lefty is a significant influence in ensuring the continued suppression of right-sided expression of Nodal, maintaining unilateral expression of this conserved determinant of asymmetry.

Handedness of the vertebrate body plan critically depends on transient embryonic structures/organs that generate cilia-dependent leftward fluid flow within constrained extracellular environments. Although the function of ciliated organs in laterality determination has been extensively studied, how they are formed during embryogenesis is still poorly understood. Here we show that Kupffer's vesicle (KV), the zebrafish organ of laterality, arises from a surface epithelium previously thought to adopt exclusively extra-embryonic fates. Live multi-photon confocal imaging reveals that surface epithelial cells undergo Nodal/TGFβ signalling-dependent ingression at the dorsal germ ring margin prior to gastrulation, to give rise to dorsal forerunner cells (DFCs), the precursors of KV. DFCs then migrate attached to the overlying surface epithelium and rearrange into rosette-like epithelial structures at the end of gastrulation. During early somitogenesis, these epithelial rosettes coalesce into a single rosette that differentiates into the KV with a ciliated lumen at its apical centre. Our results provide novel insights into the morphogenetic transformations that shape the laterality organ in zebrafish and suggest a conserved progenitor role of the surface epithelium during laterality organ formation in vertebrates.

Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis,functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1-/-. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle,somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.

Anterior to posterior growth of the vertebrate body is fueled by a posteriorly located population of bipotential neuro-mesodermal progenitor cells. These progenitor cells have a limited rate of proliferation, and their maintenance is critical for completion of the anterior-posterior axis. How these cells leave the progenitor state and commit to differentiation is largely unknown, in part because widespread modulation of factors essential for this process causes organism-wide effects. Using a novel assay, we show that Tbx16 (Spadetail) is capable of advancing mesodermal differentiation cell-autonomously. We find that Tbx16 locks cells into the mesodermal state by not only activating downstream mesodermal genes, but also by repressing bipotential progenitor genes, in part through a direct repression of sox2. We demonstrate that tbx16 is activated as cells move from an intermediate Wnt environment to a high Wnt environment, and show that Wnt signaling activates the tbx16 promoter. Importantly, high-level Wnt signaling is able to accelerate mesodermal differentiation cell-autonomously, just as we observe with Tbx16. Finally, because our assay for mesodermal commitment is quantitative, we show that the acceleration of mesodermal differentiation is surprisingly incomplete, implicating a potential separation of cell movement and differentiation during this process. Together our data suggest a model in which high levels of Wnt signaling induce a transition to mesoderm by directly activating tbx16, which in turn acts to irreversibly flip a bistable switch, leading to maintenance of the mesodermal fate and repression of the bipotential progenitor state, even as cells leave the initial high Wnt environment.

In vertebrate somitogenesis, FGF and Wnt signals constitute a morphogenetic gradient that controls the maturation of the presomitic mesoderm (PSM) as well as the transition to segmental units. It remains unclear, however, whether there is a regulatory mechanism that promotes the transition by a direct regulation of FGF and Wnt signaling in the PSM. Here we show that Shisa2, a member of a novel Shisa gene family, plays an essential role in segmental patterning during Xenopus somitogenesis. Shisa2 encodes an endoplasmic reticulum (ER) protein that cell-autonomously inhibits FGF and Wnt signaling by preventing the maturation and the cell-surface expression of their receptors. Shisa2 is expressed in the PSM and its knockdown caused a reduction in somite number by the delayed maturation of PSM and anterior shift of the transition; however, the phase of the segmental clock remained intact. These phenotypes were abolished by the inhibition of both FGF and Wnt signals, but by neither alone. We therefore propose that the individual inhibition of both types of signaling by the regulation of receptor maturation in the ER plays an essential role in the establishment of proper segmental patterning.

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn(Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophiladespite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.

Regulation of Wnt signaling is essential for embryonic patterning. Sfrps are secreted Wnt antagonists that directly interact with the Wnt ligand to inhibit signaling. Here, we show that Sfrp1 and Sfrp2 are required for anteroposterior (AP) axis elongation and somitogenesis in the thoracic region during mouse embryogenesis. Double homozygous mutations in Sfrp1 and Sfrp2 lead to severe shortening of the thoracic region. By contrast, a homozygous mutation in one or the other exerts no effect on embryogenesis, indicating that Sfrp1 and Sfrp2 are functionally redundant. The defect of a shortened thoracic region appears to be the consequence of AP axis reduction and incomplete somite segmentation. The reduction in the AP axis is partially due to abnormalities in cell migration of pre-somitic mesoderm from the end of gastrulation. Aberrant somite segmentation is associated with altered oscillations of Notch signaling, as evidenced by abnormal Lfng and Hes7 expression during somitogenesis in the thoracic region. This study suggests that Wnt regulation by Sfrp1 and Sfrp2 is required for embryonic patterning.

Fibroblast growth factor receptor 1 (Fgfr1) plays pleiotropic roles during embryonic development, but the mechanisms by which this receptor signals in vivo have not previously been elucidated. Biochemical studies have implicated Fgf receptor-specific substrates (Frs2, Frs3) as the principal mediators of Fgfr1 signal transduction to the MAPK and PI3K pathways. To determine the developmental requirements for Fgfr1-Frs signaling, we generated mice (Fgfr1(Delta)Frs/DeltaFrs) in which the Frs2/3-binding site on Fgfr1 is deleted. Fgfr1(Delta)Frs/DeltaFrs embryos die during late embryogenesis, and exhibit defects in neural tube closure and in the development of the tail bud and pharyngeal arches. However, the mutant receptor is able to drive Fgfr1 functions during gastrulation and somitogenesis, and drives normal MAPK responses to Fgf. These findings indicate that Fgfr1 uses distinct signal transduction mechanisms in different developmental contexts, and that some essential functions of this receptor are mediated by Frs-independent signaling.

In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus,morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA,following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2,loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.

To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, we have used a new mouse line,T-Cre, to generate mouse embryos with pan-mesodermal loss of Fgf8expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM),particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. Our data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis.

Bone morphogenetic protein (Bmp) signaling is crucial for the formation and patterning of zebrafish ventral and posterior mesoderm. Mutants defective in the Bmp pathway have expanded trunk muscle, abnormal tails and severely impaired development of ventral mesodermal derivatives such as vasculature,blood and pronephros. As Bmps continue to be expressed in the ventral and posterior mesoderm after gastrulation, it is likely that Bmp signaling continues to play an important developmental role during outgrowth of the posterior body. However, because Bmp signaling plays an essential role during the gastrula stages, it has not been possible with mutants or standard disruption techniques to determine the later functions of the Bmp pathway. To study the role of Bmp signaling in the ventral and posterior mesoderm during trunk and tail outgrowth, we generated a transgenic zebrafish line containing a heatshock-inducible dominant-negative Bmp receptor-GFP fusion. Our data show that Bmps are important for tail organizer formation and for patterning the ventral mesoderm during early gastrulation. However, from mid-gastrulation to the early somitogenesis stages, Bmp signaling is important for ventral tail fin development and for preventing secondary tail formation. We conclude that the role of Bmp signaling in the ventral and posterior mesoderm changes as gastrulation proceeds.

Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs)express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic `organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.

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.

Coordination of morphogenesis and cell proliferation is essential during development. In Xenopus, cell divisions are rapid and synchronous early in development but then slow and become spatially restricted during gastrulation and neurulation. One tissue that transiently stops dividing is the paraxial mesoderm, a dynamically mobile tissue that forms the somites and body musculature of the embryo. We have found that cessation of cell proliferation is required for the proper positioning and segmentation of the paraxial mesoderm as well as the complete elongation of the Xenopusembryo. Instrumental in this cell cycle arrest is Wee2, a Cdk inhibitory kinase that is expressed in the paraxial mesoderm from mid-gastrula stages onwards. Morpholino-mediated depletion of Wee2 increases the mitotic index of the paraxial mesoderm and this results in the failure of convergent extension and somitogenesis in this tissue. Similar defects are observed if the cell cycle is inappropriately advanced by other mechanisms. Thus, the low mitotic index of the paraxial mesoderm plays an essential function in the integrated cell movements and patterning of this tissue.

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. We have isolated a zebrafish deficiency, b567, that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites which are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, we demonstrate that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity.

A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.

Somite formation is thought to be regulated by an unknown oscillator mechanism that causes the cells of the presomitic mesoderm to activate and then repress the transcription of specific genes in a cyclical fashion. These oscillations create stripes/waves of gene expression that repeatedly pass through the presomitic mesoderm in a posterior-to-anterior direction. In both the mouse and the zebrafish, it has been shown that the notch pathway is required to create the stripes/waves of gene expression. However, it is not clear if the notch pathway comprises part of the oscillator mechanism or if the notch pathway simply coordinates the activity of the oscillator among neighboring cells. In the zebrafish, oscillations in the expression of a hairy-related transcription factor, her1 and the notch ligand deltaC precede somite formation. Our study focuses on how the oscillations in the expression of these two genes is affected in the mutants aei/deltaD and des/notch1, in ‘morpholino knockdowns’ of deltaC and her1 and in double ‘mutant’ combinations. This analysis indicates that these oscillations in gene expression are created by a genetic circuit comprised of the notch pathway and the notch target gene her1. We also show that a later function of the notch pathway can create a segmental pattern even in the absence of prior oscillations in her1 and deltaC expression.Supplementary data available at http://www.eb.tuebingen.mpg.de/papers/holley_dev_2002.html

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a ‘wave’ of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.

The transcription factors, MesP1 and MesP2, sharing an almost identical bHLH motif, have an overlapping expression pattern during gastrulation and somitogenesis. Inactivation of the Mesp1 gene results in abnormal heart morphogenesis due to defective migration of heart precursor cells, but somitogenesis is not disrupted because of normal expression of the Mesp2 gene. To understand the cooperative functions of MesP1 and MesP2, either a deletion or sequential gene targeting strategy was employed to inactivate both genes. The double-knockout (dKO) embryos died around 9.5 days postcoitum (dpc) without developing any posterior structures such as heart, somites or gut. The major defect in this double-knockout embryo was the apparent lack of any mesodermal layer between the endoderm and ectoderm. The abnormal accumulation of cells in the primitive streak indicates a defect in the migratory activity of mesodermal cells. Molecular markers employed to characterize the phenotype revealed a lack of the cranio-cardiac and paraxial mesoderm. However, the axial mesoderm, as indicated by brachyury (T) expression, was initially generated but anterior extension was halted after 8.5 dpc. Interestingly, a headfold-like structure developed with right anterior-posterior polarity; however, the embryos lacked any posterior neural properties. The persistent and widely distributed expression of Cerberus-like-1(Cer1), Lim1 and Otx2 in the anterior endoderm might be responsible for the maintenance of anterior neural marker expression. We also performed a chimera analysis to further study the functions of MesP1 and MesP2 in the development of mesodermal derivatives. In the chimeric embryos, dKO cells were scarcely observed in the anterior-cephalic and heart mesoderm, but they did contribute to the formation of the somites, notochord and gut. These results strongly indicate that the defect in the cranial-cardiac mesoderm is cell-autonomous, whereas the defect in the paraxial mesoderm is a non-cell-autonomous secondary consequence.

Pax6 is a paired-type homeobox gene expressed in discrete regions of the central nervous system. In the spinal cord of 7- to 10-somite-stage chicken embryos, Pax6 is not detected within the caudal neural plate, but is progressively upregulated in the neuroepithelium neighbouring each newly formed somite. In the present study, we accumulate data suggesting that this initial activation of Pax6 is controlled via the paraxial mesoderm in correlation with somitogenesis. First, we observed that high levels of Pax6 expression occur independently of the presence of SHH-expressing cells when neural plates are maintained in culture in the presence of paraxial mesoderm. Second, grafting a somite caudally under a neural plate that has not yet expressed the gene induces a premature activation of Pax6. Furthermore, after the graft of a somite, a period of incubation corresponding to the individualization of a new somite in the host embryo produces an appreciable activation of Pax6. Conversely, Pax6 expression is delayed under conditions where somitogenesis is retarded, i.e., when the rostral part of the presomitic mesoderm is replaced by the same tissue isolated more caudally. Finally, Pax6 transcripts disappear from the neural tube when a somite is replaced by presomitic mesoderm, suggesting that the somite is also involved in the maintenance of Pax6 expression in the developing spinal cord. All together these observations lead to the proposal that Pax6 activation is triggered by the paraxial mesoderm in phase with somitogenesis in the cervical spinal cord.

The aim of these experiments was to determine the endogenous distribution of retinoic acid (RA) across a wide range of embryonic stages in the chick embryo. By high pressure liquid chromatography, it was revealed that didehydroRA is the most prevalent retinoic acid in the chick embryo and that the tissues of the stage 24 embryo differed widely in their total RA content (didehydroRA + all-trans-RA). Some tissues such as the heart had very little RA and some such as the neural tube had very high levels, the total variation between these two being 29-fold. We showed that these tissues also synthesised RA and released it into the medium, thus validating the use of the F9 reporter cell system for further analyses of younger staged embryos. With these F9 cells, we showed that, at stage 4, the posterior end of the embryo had barely detectably higher levels of RA than the anterior end, but that a significant level of RA generation was detected as soon as somitogenesis began. Then a sharp on/off boundary of RA was present at the level of the first somite. We could find no evidence for a posterior-to-anterior gradient of RA. Throughout further development, various consistent observations were made: the developing brain did not generate RA, but the spinal part of the neural tube generated it at very high levels so there must be a sharp on/off boundary in the region of the hindbrain/spinal cord junction; the mesenchyme surrounding the hindbrain generated RA whereas the hindbrain itself did not; there was a variation in RA levels from the midline outwards with the highest levels of RA in the spinal neural tube followed by lower levels in the somites followed by lower levels in the lateral plate; the posterior half of the limb bud generated higher levels than the anterior half. With these observations, we were able to draw maps of endogenous RA throughout these early stages of chick embryogenesis and the developmental implications of these results are discussed.

Segmentation of the vertebrate embryo begins when the paraxial mesoderm is subdivided into somites, through a process that remains poorly understood. To study this process, we have characterized X-Delta-2, which encodes the second Xenopus homolog of Drosophila Delta. Strikingly, X-Delta-2 is expressed within the presomitic mesoderm in a set of stripes that corresponds to prospective somitic boundaries, suggesting that Notch signaling within this region establishes a segmental prepattern prior to somitogenesis. To test this idea, we introduced antimorphic forms of X-Delta-2 and Xenopus Suppressor of Hairless (X-Su(H)) into embryos, and assayed the effects of these antimorphs on somite formation. In embryos expressing these antimorphs, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment properly. Both antimorphs also disrupted the segmental expression of X-Delta-2 and Hairy2A, a basic helix-loop-helix (bHLH) gene, within the presomitic mesoderm. These observations suggest that X-Delta-2, via X-Notch-1, plays a role in segmentation, by mediating cell-cell interactions that underlie the formation of a segmental prepattern prior to somitogenesis.

Somitogenesis is the basis of segmentation of the mesoderm in the trunk and tail of vertebrate embryos. Two groups of mutants with defects in this patterning process have been isolated in our screen for zygotic mutations affecting the embryonic development of the zebrafish (Danio rerio). In mutants of the first group, boundaries between individual somites are invisible early on, although the paraxial mesoderm is present. Later, irregular boundaries between somites are present. Mutations in fused somites (fss) and beamter (bea) affect all somites, whereas mutations in deadly seven (des), after eight (aei) and white tail (wit) only affect the more posterior somites. Mutants of all genes but wit are homozygous viable and fertile. Skeletal stainings and the expression pattern of myoD and snail1 suggest that anteroposterior patterning within individual somites is abnormal. In the second group of mutants, formation of the horizontal myoseptum, which separates the dorsal and ventral part of the myotome, is reduced. Six genes have been defined in this group (you-type genes). you-too mutants show the most severe phenotype; in these the adaxial cells, muscle pioneers and the primary motoneurons are affected, in addition to the horizontal myoseptum. The horizontal myoseptum is also missing in mutants that lack a notochord. The similarity of the somite phenotype in mutants lacking the notochord and in the you- type mutants suggests that the genes mutated in these two groups are involved in a signaling pathway from the notochord, important for patterning of the somites.

In a screen for embryonic mutants in the zebrafish a large number of mutants were isolated with abnormal brain morphology. We describe here 26 mutants in 13 complementation groups that show abnormal development of large regions of the brain. Early neurogenesis is affected in white tail (wit). During segmentation stages, homozygous wit embryos display an irregularly formed neural keel, particularly in the hindbrain. Using a variety of molecular markers, a severe increase in the number of various early differentiating neurons can be demonstrated. In contrast, late differentiating neurons, radial glial cells and some non-neural cell types, such as the neural crest-derived melanoblasts, are much reduced. Somitogenesis appears delayed. In addition, very reduced numbers of melanophores are present posterior to the mid-trunk. The wit phenotype is reminiscent of neurogenic mutants in Drosophila, such as Notch or Delta. In mutant parachute (pac) embryos the general organization of the hindbrain is disturbed and many rounded cells accumulate loosely in the hindbrain and midbrain ventricles. Mutants in a group of 6 genes, snakehead(snk), natter (nat), otter (ott), fullbrain (ful), viper (vip) and white snake (wis) develop collapsed brain ventricles, before showing signs of general degeneration. atlantis (atl), big head (bid), wicked brain (win), scabland (sbd) and eisspalte (ele) mutants have different malformation of the brain folds. Some of them have transient pheno-types, and mutant individuals may grow up to adults.

MRF4 (herculin/Myf-6) is one of the four member MyoD family of transcription factors identified by their ability to enforce skeletal muscle differentiation upon a wide variety of nonmuscle cell types. In this study the mouse germline MRF4 gene was disrupted by targeted recombination. Animals homozygous for the MRF4bh1 allele, a deletion of the functionally essential bHLH domain, displayed defective axial myogenesis and rib pattern formation, and they died at birth. Differences in somitogenesis between homozygous MRF4bh1 embryos and their wild-type littermates provided evidence for three distinct myogenic regulatory programs (My1-My3) in the somite, which correlate temporally and spatially with three waves of cellular recruitment to the expanding myotome. The first program (My1), marked initially by Myf-5 expression and followed by myogenin, began on schedule in the MRF4bh1/bh1 embryos at day 8 post coitum (E8). A second program (My2) was highly deficient in homozygous mutant MRF4 embryos, and normal expansion of the myotome failed. Moreover, expression of downstream muscle-specific genes, including FGF-6, which is a candidate regulator of inductive interactions, did not occur normally. The onset of MyoD expression around E10.5 in wild-type embryos marks a third myotomal program (My3), the execution of which was somewhat delayed in MRF4 mutant embryos but ultimately led to extensive myogenesis in the trunk. By E15 it appeared to have largely compensated for the defective My2 program in MRF4 mutants. Homozygous MRF4bh1 animals also showed improper rib pattern formation perhaps due to the absence of signals from cells expressing the My2 program. Finally, a later and relatively mild phenotype was detected in intercostal muscles of newborn animals.

The Drosophila Delta (Dl) gene is essential for cell-cell communication regulating the determination of various cell fates during development. Dl encodes a transmembrane protein, which contains tandem arrays of epidermal-growth-factor-like repeats in the extracellular domain and directly interacts with Notch, another transmembrane protein with similar structural features, in a ligand-receptor-like manner. Similarly, cell-cell interactions involving Delta-like and Notch-like proteins are required for cell fate determinations in C. elegans. Notch homologues were also isolated from several vertebrate species, suggesting that cell-to-cell signaling mediated by Delta- and Notch-like proteins could also underlie cell fate determination during vertebrate development. However, in vertebrates, no Delta homologues have yet been described. We have isolated a novel mouse gene, Dll1 (delta-like gene 1), which maps to the mouse t-complex and whose deduced amino acid sequence strongly suggests that Dll1 represents a mammalian gene closely related to Drosophila Delta. Dll1 is transiently expressed during gastrulation and early organogenesis, and in a tissue-restricted manner in adult animals. Between day 7 and 12.5 of development, expression was detected in the paraxial mesoderm, closely correlated with somitogenesis, and in subsets of cells in the nervous system. In adult animals, transcripts were detected in lung and heart. Dll1 expression in the paraxial mesoderm and nervous system is strikingly similar to the expression of mouse Notch1 during gastrulation and early organogenesis. The overlapping expression patterns of the Dll1 and Notch1 genes suggest that cells in these tissues can communicate by interaction of the Dll1 and Notch1 proteins. Our results support the idea that Delta- and Notch-like proteins are involved in cell-to-cell communication in mammalian embryos and suggest a role for these proteins in cellular interactions underlying somitogenesis and development of the nervous system.

The segmentation clock is an oscillating genetic network thought to govern the rhythmic and sequential subdivision of the elongating body axis of the vertebrate embryo into somites: the precursors of the segmented vertebral column. Understanding how the rhythmic signal arises, how it achieves precision and how it patterns the embryo remain challenging issues. Recent work has provided evidence of how the period of the segmentation clock is regulated and how this affects the anatomy of the embryo. The ongoing development of realtime clock reporters and mathematical models promise novel insight into the dynamic behavior of the clock. © 2012. Published by The Company of Biologists Ltd. Fil: Oates, Andrew C.. Max Planck Institute Of Molecular Cell Biology And Genetics; Alemania Fil: Morelli, Luis Guillermo. Max Planck Institute Of Molecular Cell Biology And Genetics; Alemania. Universidad de Buenos Aires; Argentina Fil: Ares, Saúl. Max Planck Institute For The Physics Of Complex Systems; Alemania

It is well known that the muscles of the vertebrate body are derived from the somite. Precursor cells within the somite proper form the back or axial muscles while other precursor cells migrate away from the somite to populate the muscle of the limbs and ventral body wall. Although both types of muscle are generally thought of as arising from a common progenitor population, the myotome, recent evidence points to developmental differences in these two groups of muscles which may reflect different developmental lineages. To test the lineage hypothesis, we used microsurgery and the chick-quail nucleolar marker system to follow the developmental fate of the lateral and medial halves of somites at the wing level. The results showed that the structures of the mature somite (myotome and sclerotome) are derived virtually exclusively from cells residing in the medial half of the newly formed somite. On the other hand, virtually all of the cells residing in the lateral half of the newly formed somite are destined to leave the somite proper and populate the limb muscle and, probably, other somite-derived mesenchymal structures in the limb and ventral body wall. Switch-graft experiments show that the two halves of newly formed somites are largely interchangeable demonstrating that their ultimate developmental fate is position-dependent and that it becomes fixed as a result of extrinsic influences which act during later stages of somitogenesis. We conclude that at least two distinct myogenic lineages exist in the somite; one giving rise to the muscles of the back and the other giving rise to the limb musculature.

Somitogenesis in the mouse embryo commences with the generation of presumptive somitic mesoderm at the primitive streak and in the tail-bud mesenchyme. The presumptive somitic mesoderm is then organized into somite primordia in the presomitic mesoderm. These primordia undergo morphogenesis leading to the segmentation of somites at the cranial end of the presomitic mesoderm. Somite sizes at the time of segmentation vary according to the position of the somite in the body axis: the size of lumbar and sacral somites is nearly twice that of upper trunk somites and of tail somites. The size of the presomitic mesoderm, which is governed by the balance between the addition of cells at the caudal end and the removal of somites at the cranial end, changes during embryonic development. Somitogenesis is disturbed during the compensatory growth of mouse embryos which have suffered a drastic size reduction at the primitive-streak and early-organogenesis stages. The formation of somites is retarded and the upper trunk somites are formed at a smaller size. The embryo also follows an entirely different growth profile, but a normal body size is restored by the early foetal stage. The somite number is regulated to normal and this is brought about by an altered rate of somite formation and the adjustment of somite size in proportion to the whole body size. It is proposed that axis formation and somitogenesis are related morphogenetic processes and that embryonic growth controls the kinetics of somitogenesis, namely by regulating the number of cells allocated to each somite and the rate of somite formation.

Cell-cell adhesion in the anterior portion of the segmental plate plays an important role in the initiation of somitogenesis. Dissociated somites show a marked tendency to reassociate into somite-size clumps within a few hours after dissociation. Segmental plate cells show very little tendency to reassociate, even after 24 h in culture. The cells from the anterior portion of the segmental plate do show a tendency to reassociate. This supports the hypothesis that cell-cell adhesion between the cells in the anterior portion of the segmental plate plays a major role in the initiation of somitogenesis.

The intrasomitic changes in cell arrangement which accompany somite rotation during somitogenesis in Xenopus laevis were analysed with the scanning electron microscope (SEM). Longitudinal, horizontal fractures of whole embryos were examined at various dorsoventral levels of stage-22 to -24 embryos. Observations of the gross morphological features of somitogenesis, and the cellular changes which accompany somite segmentation and somite rotation were made. Several of these observations lead to modifications of previous models for the cellular basis of somitogenesis in Xenopus. Individual cellular rearrangements, rather than simultaneous block rotation of a whole somite, appear to be responsible for the 90° rotation of myotomal cells within a single somite. Cellular arrangments in fused somites were also examined. Some ultraviolet-irradiated embryos displayed a complete lack of a notochord. The somites in those embryos were fused across the midline beneath the neural tube. The dorsal and ventral arms of the somites are not fused. Normal rotation occurs only in the dorsal and ventral arms while, in the majority of cases, cells in the fused region fail to rotate normally. In some cases, individual cells in the fused region undergo partial rearrangement. Those observations support the notion that individual cellular rearrangements account for the rotation of the whole somite.

Somitogenesis in the avian embryo: an experimental study The determinism of somitogenesis has been studied. A complete segmental plate can form somites even when isolated from cord, neural tube and somites. Somites are always formed if the segmental plate remains in continuity with the somites which have just formed. When the segmental plate is separated from the axis and deprived of its apical end, it undergoes no differentiation or differentiates incompletely. In the latter case the quality of differentiation decreases in a cephalo-caudal direction. When the segmental plate, without its apical end, is left in contact with neural tissue or chorda, it forms somites normally. The growth of the segmental plate is reduced whenever it has been isolated from neural tube and chorda altogether. Behind its apical end the segmental plate can regulate excesses as well as deficiencies. It appears, then, that the intersomitic fissures are determined at the apical ends of the segmental plates, but not behind them. This determination is progressively established backwards. It can be transmitted by the last differentiated as well as the differentiating somites. However, the neural tube, and possibly the cord, play the leading role in this process, for the intersomitic clefts are fixed at their proper place in connexion with them.

During gastrulation, embryonic cells become specified into distinct germ layers. In mouse, this continues throughout somitogenesis from a population of bipotent stem cells called neuromesodermal progenitors (NMps). However, the degree of self-renewal associated with NMps in the fast-developing zebrafish embryo is unclear. With a genetic clone tracing method, we labelled early embryonic progenitors and find a strong clonal similarity between spinal cord and mesoderm tissues. We followed individual cell lineages by light-sheet imaging, revealing a common neuromesodermal lineage contribution to a subset of spinal cord tissue across the anterior-posterior body axis. An initial population subdivides at mid gastrula stages and is directly allocated to neural and mesodermal compartments during gastrulation. A second population in the tailbud undergoes delayed allocation to contribute to the neural and mesodermal compartment only at late somitogenesis. Cell tracking and retrospective cell fate assignment at late somitogenesis stages reveal these cells to be a collection of mono-fated progenitors. Our results suggest that NMps are a conserved population of bipotential progenitors, whose lineage varies in a species-specific manner due to vastly different rates of differentiation and growth.

During somitogenesis, epithelial somites form from the pre-somitic mesoderm (PSM) in a periodic manner. This periodicity is regulated by a molecular oscillator, known as the ‘segmentation clock’, that is characterised by an oscillatory pattern of gene expression that sweeps the PSM in a caudal-rostral direction. Key components of the segmentation clock are intracellular components of the Notch, Wnt and FGF pathways, and it is widely accepted that intracellular negative-feedback loops regulate oscillatory gene expression. However, an open question in the field is how intracellular oscillations are coordinated, in the form of spatiotemporal waves of expression, across the PSM. In this study, we provide a potential mechanism for this process. We show at the mRNA level that the Notch1 receptor and Delta-like 1 (Dll1) ligand vary dynamically across the PSM of both chick and mouse. Remarkably, we also demonstrate similar dynamics at the protein level; hence, the pathway components that mediate intercellular coupling themselves exhibit oscillatory dynamics. Moreover, we quantify the dynamic expression patterns of Dll1 and Notch1, and show they are highly correlated with the expression patterns of two known clock components [Lfng mRNA and the activated form of the Notch receptor (cleaved Notch intracellular domain, NICD)]. Lastly, we show that Notch1 is a target of Notch signalling, whereas Dll1 is Wnt regulated. Regulation of Dll1 and Notch1 expression thus links the activity of Wnt and Notch, the two main signalling pathways driving the clock.