Vertebrate segmentation: is cycling the rule?

Vertebrate segmentation initiates with the subdivision of the paraxial mesoderm into a regular array of somites. Recent evidence suggests that the segmentation clock - a biochemical oscillator acting in the unsegmented paraxial mesoderm cells in most vertebrates - controls cyclic Notch signalling, resulting in periodic formation of somite boundaries.     

The forkhead genes, Foxc1 and Foxc2, regulate paraxial versus intermediate mesoderm cell fate

During vertebrate embryogenesis, the newly formed mesoderm is allocated to the paraxial, intermediate, and lateral domains, each giving rise to different cell and tissue types. Here, we provide evidence that the forkhead genes, Foxc1 and Foxc2, play a role in the specification of mesoderm to paraxial versus intermediate fates. Mouse embryos lacking both Foxc1 and Foxc2 show expansion of intermediate mesoderm markers into the paraxial domain, lateralization of somite patterning, and ectopic and disorganized mesonephric tubules. In gain of function studies in the chick embryo, Foxc1 and Foxc2 negatively regulate intermediate mesoderm formation. By contrast, their misexpression in the prospective intermediate mesoderm appears to drive cells to acquire paraxial fate, as revealed by expression of the somite markers Pax7 and Paraxis. Taken together, the data indicate that Foxc1 and Foxc2 regulate the establishment of paraxial versus intermediate mesoderm cell fates in the vertebrate embryo.     

Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak

Vertebrate somitogenesis is associated with a molecular oscillator, the segmentation clock, which is defined by the periodic expression of genes related to the Notch pathway such as hairy1 and hairy2 or lunatic fringe (referred to as the cyclic genes) in the presomitic mesoderm (PSM). Whereas earlier studies describing the periodic expression of these genes have essentially focussed on later stages of somitogenesis, we have analysed the onset of the dynamic expression of these genes during chick gastrulation until formation of the first somite. We observed that the onset of the dynamic expression of the cyclic genes in chick correlated with ingression of the paraxial mesoderm territory from the epiblast into the primitive streak. Production of the paraxial mesoderm from the primitive streak is a continuous process starting with head mesoderm formation, while the streak is still extending rostrally, followed by somitic mesoderm production when the streak begins its regression. We show that head mesoderm formation is associated with only two pulses of cyclic gene expression. Because such pulses are associated with segment production at the body level, it suggests the existence of, at most, two segments in the head mesoderm. This is in marked contrast to classical models of head segmentation that propose the existence of more than five segments. Furthermore, oscillations of the cyclic genes are seen in the rostral primitive streak, which contains stem cells from which the entire paraxial mesoderm originates. This indicates that the number of oscillations experienced by somitic cells is correlated with their position along the AP axis.     

The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis

Orthotopic grafts of [3H]thymidine-labelled cells have been used to demonstrate differences in the normal fate of tissue located adjacent to and in different regions of the primitive streak of 8th day mouse embryos developing in vitro. The posterior streak produces predominantly extraembryonic mesoderm, while the middle portion gives rise to lateral mesoderm and the anterior region generates mostly paraxial mesoderm, gut and notochord. Embryonic ectoderm adjacent to the anterior part of the streak contributes mainly to paraxial mesoderm and neurectoderm. This pattern of colonization is similar to the fate map constructed in primitive-streak-stage chick embryos. Similar grafts between early-somite-stage (9th day) embryos have established that the older primitive streak continues to generate embryonic mesoderm and endoderm, but ceases to make a substantial contribution to extraembryonic mesoderm. Orthotopic grafts and specific labelling of ectodermal cells with wheat germ agglutinin conjugated to colloidal gold (WGA-Au) have been used to analyse the recruitment of cells into the paraxial mesoderm of 8th and 9th day embryos. The continuous addition of primitive-streak-derived cells to the paraxial mesoderm is confirmed and the distribution of labelled cells along the craniocaudal sequence of somites is consistent with some cell mixing occurring within the presomitic mesoderm.     

Protogenin mediates cell adhesion for ingression and re-epithelialization of paraxial mesodermal cells

In the avian embryo, precursor cells of the paraxial mesoderm that reside in the epiblast ingress through the primitive streak and migrate bilaterally in an anterolateral direction. Herein, we report on the roles of Protogenin (PRTG), an immunoglobulin superfamily protein expressed on the surface of the ingressing and migrating cells that give rise to the paraxial mesoderm, in paraxial mesoderm development. An aggregation assay using L-cells showed that PRTG mediates homophilic cell adhesion. Overexpression of PRTG in the presumptive paraxial mesoderm delayed mesodermal cell migration due to augmented adhesiveness. In contrast, siRNA knockdown of PRTG impaired successive ingression of epiblast cells and disorganized the epithelial structure of the somites. These results suggest that PRTG mediates cell adhesion to regulate continuous ingression of cells giving rise to the paraxial mesodermal lineage, as well as tissue integrity.     

Generation of segment polarity in the paraxial mesoderm of the zebrafish through a T-box-dependent inductive event

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state.     

BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites

Bmpr1a encodes the BMP type IA receptor for bone morphogenetic proteins (BMPs), including 2 and 4. Here, we use mosaic inactivation of Bmpr1a in the epiblast of the mouse embryo (Bmpr-MORE embryos) to assess functions of this gene in mesoderm development. Unlike Bmpr1a-null embryos, which fail to gastrulate, Bmpr-MORE embryos initiate gastrulation, but the recruitment of prospective paraxial mesoderm cells to the primitive streak is delayed. This delay causes a more proximal distribution of cells with paraxial mesoderm character within the primitive streak, resulting in a lateral expansion of somitic mesoderm to form multiple columns. Inhibition of FGF signaling restores the normal timing of recruitment of prospective paraxial mesoderm and partially rescues the development of somites. This suggests that BMP and FGF signaling function antagonistically during paraxial mesoderm development.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Segmentation in vertebrates: a molecular clock linked to periodic somite formation]

In the vertebrate embryo, somites constitute the basis of the segmental body pattern. They give rise to the axial skeleton, the dermis of the back and all striated muscles of the body. In the chick embryo, a pair of somites buds off, in a highly coordinated fashion, every 90 minutes, from the cranial end of the presomitic mesoderm (PSM) while new mesenchymal cells enter the paraxial mesoderm as a consequence of gastrulation. The processes leading to the segmentation of the somite are not yet understood. We have identified and characterised c-hairy1, an avian homologue of the Drosophila segmentation gene, hairy. c-hairy1 is strongly expressed in the presomitic mesoderm where its mRNA exhibits a cyclic posterior-to-anterior wave of expression whose periodicity corresponds to the formation time of one somite (90 min). Fate mapping of the rostral half of the PSM using the quail-chick chimera technique supports a model of cryptic segmentation within the presomitic mesoderm, and indicates that c-hairy1 expression dynamics are not due to massive cell displacement. Analysis of in vitro cultures of isolated presomitic mesoderm demonstrates that rhythmic c-hairy1 mRNA production and degradation is an autonomous property of the paraxial mesoderm. Rather than resulting from the caudal-to-rostral propagation of an activating signal, it arises from pulses of c-hairy1 expression that are coordinated in time and space. Blocking protein synthesis does not alter the propagation of c-hairy1 expression, indicating that negative autoregulation of c-hairy1 expression is unlikely to control its periodic expression. Most of the segmentation models proposed for somite formation rely on the existence of an internal clock coordinating the cells to segment together to form a somite. These results provide the first molecular evidence of a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved.     

Level-specific role of paraxial mesoderm in regulation of Tbx5/Tbx4 expression and limb initiation

Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the paraxial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and down regulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and down regulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation.     

BMP4 and noggin control embryonic blood vessel formation by antagonistic regulation of VEGFR-2 (Quek1) expression

Regulation of VEGFR-2 (Quek1) is an important mechanism during blood vessel formation. In the paraxial mesoderm, Quek1 expression is restricted to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube. By grafting of either intermediate mesoderm or BMP4 beads into the paraxial mesoderm, we show that BMP4 is a positive regulator of VEGFR-2 (Quek1) expression in the quail embryo. Separation of somites from intermediate mesoderm leads to down-regulation of Quek1 expression. The expression of Quek1 in the medial somite half is normally repressed by the notochord and becomes up-regulated and lateromedially expanded after separation of the notochord. Our results show that up-regulation of BMP4 leads to an increase of the number of blood vessels, whereas inhibition of BMP4 by noggin results in a reduction of blood vessels.     

Programming neural Hoxd10: in vivo evidence that early node-associated signals predominate over paraxial mesoderm signals at posterior spinal levels

Studies of the programming of Hox patterns at anterior spinal levels suggest that these events are accomplished through an integration of Hensen's node-derived and paraxial mesoderm signaling. We have used in vivo tissue manipulation in the avian embryo to examine the respective roles of node- derived and other local signals in the programming of a Hox pattern at posterior spinal levels. Hoxd10 is highly expressed in the lumbosacral (LS) spinal cord and adjacent paraxial mesoderm. At stages of LS neural tube formation (stages 12-14), the tailbud contains the remnants of Hensen's node and the primitive streak. Hoxd10 expression was analyzed after transposition of LS neural segments with and without the tailbud, after isolation of normally positioned LS segments from the stage 13 tailbud, and after axial displacement of posterior paraxial mesoderm. Data suggest that inductive signals from the tailbud are primarily responsible for the programming of Hoxd10 at neural plate and the earliest neural tube stages. After these stages, the LS neural tube appears to differ from more anterior neural segments in its lack of dependence on Hox-inductive signals from local tissues, including paraxial mesoderm. Our data also suggest that a graded system of repressive signals for posterior Hox genes is present at cervical and thoracic levels and likely to originate from paraxial mesoderm.     

The somitogenetic potential of cells in the primitive streak and the tail bud of the organogenesis-stage mouse embryo.

The developmental potency of cells isolated from the primitive streak and the tail bud of 8.5- to 13.5-day-old mouse embryos was examined by analyzing the pattern of tissue colonization after transplanting these cells to the primitive streak of 8.5-day embryos. Cells derived from these progenitor tissues contributed predominantly to tissues of the paraxial and lateral mesoderm. Cells isolated from older embryos could alter their segmental fate and participated in the formation of anterior somites after transplantation to the primitive streak of 8.5-day host embryo. There was, however, a developmental lag in the recruitment of the transplanted cells to the paraxial mesoderm and this lag increased with the extent of mismatch of developmental ages between donor and host embryos. It is postulated that certain forms of cell-cell or cell-matrix interaction are involved in the specification of segmental units and that there may be age-related variations in the interactive capability of the somitic progenitor cells during development. Tail bud mesenchyme isolated from 13.5-day embryos, in which somite formation will shortly cease, was still capable of somite formation after transplantation to 8.5-day embryos. The cessation of somite formation is therefore likely to result from a change in the tissue environment in the tail bud rather than a loss of cellular somitogenetic potency.     

Clonal separation and regionalisation during formation of the medial and lateral myotomes in the mouse embryo.

In vertebrates, muscles of the back (epaxial) and of the body wall and limbs (hypaxial) derive from precursor cells located in the dermomyotome of the somites. In this paper, we investigate the mediolateral regionalisation of epaxial and hypaxial muscle precursor cells during segmentation of the paraxial mesoderm and myotome formation, using mouse LaacZ/LacZ chimeras. We demonstrate that precursors of medial and lateral myotomes are clonally separated in the mouse somite, consistent with earlier studies in birds. This clonal separation occurs after segmentation of the paraxial mesoderm. We then show that myotome precursors are mediolaterally regionalised and that this regionalisation precedes clonal separation between medial and lateral precursors. Strikingly, the properties of myotome precursors are remarkably similar in the medial and lateral domains. Finally, detailed analysis of our clones demonstrates a direct spatial relationship between the myocytes in the myotome and their precursors in the dermomyotome, and earlier in the somite and presomitic mesoderm, refuting several models of myotome formation, based on permanent stem cell systems or extensive cell mingling. This progressive mediolateral regionalisation of the myotome at the cellular level correlates with progressive changes in gene expression in the dermomyotome and myotome.     

The developmental fate of the cephalic mesoderm in quail-chick chimeras.

The developmental fate of the cephalic paraxial and prechordal mesoderm at the late neurula stage (3-somite) in the avian embryo has been investigated by using the isotopic, isochronic substitution technique between quail and chick embryos. The territories involved in the operation were especially tiny and the size of the transplants was of about 150 by 50 to 60 microns. At that stage, the neural crest cells have not yet started migrating and the fate of mesodermal cells exclusively was under scrutiny. The prechordal mesoderm was found to give rise to the following ocular muscles: musculus rectus ventralis and medialis and musculus oblicus ventralis. The paraxial mesoderm was separated in two longitudinal bands: one median, lying upon the cephalic vesicles (median paraxial mesoderm--MPM); one lateral, lying upon the foregut (lateral paraxial mesoderm--LPM). The former yields the three other ocular muscles, contributes to mesencephalic meninges and has essentially skeletogenic potencies. It contributes to the corpus sphenoid bone, the orbitosphenoid bone and the otic capsules; the rest of the facial skeleton is of neural crest origin. At 3-somite stage, MPM is represented by a few cells only. The LPM is more abundant at that stage and has essentially myogenic potencies with also some contribution to connective tissue. However, most of the connective cells associated with the facial and hypobranchial muscles are of neural crest origin. The more important result of this work was to show that the cephalic mesoderm does not form dermis. This function is taken over by neural crest cells, which form both the skeleton and dermis of the face. If one draws a parallel between the so-called "somitomeres" of the head and the trunk somites, it appears that skeletogenic potencies are reduced in the former, which in contrast have kept their myogenic capacities, whilst the formation of skeleton and dermis has been essentially taken over by the neural crest in the course of evolution of the vertebrate head.     

Generation of transgenic mice expressing Cre recombinase under the control of the Dll1 mesoderm enhancer element

To study paraxial mesoderm formation in the mouse, transgenic lines that can be used to either selectively delete or express genes of interest in the paraxial mesoderm are required. We have generated a transgenic mouse line that expresses Cre recombinase in the paraxial mesoderm (PAM) beginning at e7.5. A lacZ Cre recombinase reporter line showed that in addition to PAM and its derivatives, lateral plate and intermediate mesoderm derivatives were also exposed to Cre activity, while the node, notochord, and cardiac mesoderm were not. We further demonstrate that 70–75% of the fibroblasts generated from Dll1‐msd Cre, ROSA26‐rtTA embryos possess Cre recombinase activity. These mice can therefore be used in combination with tet‐responsive transgenic lines to generate mesoderm‐derived embryonic fibroblasts that inducibly express a gene of interest. genesis 47:309–313, 2009. © 2009 Wiley‐Liss, Inc.