Hox genes specify vertebral types in the presomitic mesoderm

We show here that expression of Hoxa10 in the presomitic mesoderm is sufficient to confer a Hox group 10 patterning program to the somite, producing vertebrae without ribs, an effect not achieved when Hoxa10 is expressed in the somites. In addition, Hox group 11-dependent vertebral sacralization requires Hoxa11 expression in the presomitic mesoderm, while their caudal differentiation requires that Hoxa11 is expressed in the somites. Therefore, Hox gene patterning activity is different in the somites and presomitic mesoderm, the latter being very prominent for Hox gene-mediated patterning of the axial skeleton. This is further supported by our finding that inactivation of Gbx2, a homeobox-containing gene expressed in the presomitic mesoderm but not in the somites, produced Hox-like phenotypes in the axial skeleton without affecting Hox gene expression.     

Visualizing the lateral somitic frontier in the Prx1Cre transgenic mouse

Changes in the organization of the musculoskeletal system have accounted for many evolutionary adaptations in the vertebrate body plan. The musculoskeletal system develops from two mesodermal populations: somitic mesoderm gives rise to the axial skeleton and all of the skeletal muscle of the body, and lateral plate mesoderm gives rise to the appendicular skeleton. The recognition of embryonic domains resulting from the dynamics of morphogenesis has inspired new terminology based on developmental criteria. Two mesodermal domains are defined, primaxial and abaxial. The primaxial domain includes musculoskeletal structures comprising just somitic cells. The abaxial domain contains somitic myoblasts in connective tissue derived from lateral plate mesoderm, as well as lateral plate-derived skeletal structures. The boundary between these two domains is the lateral somitic frontier. Recent studies have described the developmental relationship between these two domains in the chick. In the present study, we describe the labelling pattern in the body of the Prx1/Cre/Z/AP compound transgenic mouse. The enhancer employed in this transgenic leads to reporter expression in the postcranial, somatic lateral plate mesoderm. The boundary between labelled and unlabelled cell populations is described at embryonic day (E)13.5 and E15.5. We argue that the distribution of labelled cells is consistent with the somatic lateral plate lineage, and therefore provides an estimate of the position of the lateral somitic frontier. The role of the frontier in both development and evolution is discussed.     

Experimental analysis of the origin of the wing musculature in avian embryos

Summary Interspecific grafts of somites, as well as parts of the somatic plate mesoderm, have been made between quail and chick embryos (stages 12–14 H.H.) at the level of the prospective wing bud in order to examine the relationship between somites and wing bud myogenesis. The stability of the natural quail nuclear labelling makes it possible to follow the developmental fate of grafted mesodermal cells in the host embryo. Embryos examined after subsequent incubation periods of 3–7 days show the following distribution of somatic and somitic cells within the wing bud: as soon as the three zones of different cell density within the mesoderm can be distinguished, cells of somitic origin are limited to the prospective myogenic are which is made up of a mixed population of somatic and somitic cells, whereas the prospective chondrogenic area as well as the subectodermal zone only consists of cells originated from the somatic plate mesoderm. After further incubation, single muscle blastema are present which were also seen to be a mixture of somatic and somitic cells. The cells of muscular bundles are of somitic origin, while the muscle connective tissue cells are derived from the somatic plate mesoderm. After grafting into the coelomic cavity or on the chorio-allantoic membrane, fragments of the somatic plate mesoderm previously isolated from quail embryos (stage 14 H.H.) at the level of the prospective wing bud exhibit well developed skeletal elements, but fail to differentiate any musculature. These experimental investigations support previous evidence for a somitic origin of wing bud myogenic cells. Histological and scanning electron microscopic studies of the brachial somites and the adjacent somatic plate mesoderm of chick embryo (stages 13–15 H.H.) reveal that migration of still undifferentiated somitic cells into the brachial somatic plate mesoderm begins to take place in embryos at stage 14.     

Amniote somite derivatives.

Somites are segments of paraxial mesoderm that give rise to a multitude of tissues in the vertebrate embryo. Many decades of intensive research have provided a wealth of data on the complex molecular interactions leading to the formation of various somitic derivatives. In this review, we focus on the crucial role of the somites in building the body wall and limbs of amniote embryos. We give an overview on the current knowledge on the specification and differentiation of somitic cell lineages leading to the development of the vertebral column, skeletal muscle, connective tissue, meninges, and vessel endothelium, and highlight the importance of the somites in establishing the metameric pattern of the vertebrate body.     

Wnt signaling in somite development.

During vertebrate embryogenesis, specialized mesodermal structures, called somites, give rise to a variety of mesodermal tissues including skeletal muscles, vertebrae and dermis. Development of the somites is a rhythmic process that involves a series of steps including segmentation of the paraxial mesoderm, epithelialization, somite formation, somite maturation, somite patterning and differentiation of somitic cells into different lineages. Wnt signaling has been found to play crucial roles in multiple steps of somite development. In this review, we present a brief overview of current knowledge on Wnt signaling events during the development of somites and their derivatives.     

The eventful somite: patterning, fate determination and cell division in the somite

The segmental somites not only determine the vertebrate body plan, but also represent turntables of cell fates. The somite is initially naive in terms of its fate restriction as shown by grafting and rotation experiments whereby ectopically grafted or rotated tissue of newly formed somites yielded the same pattern of normal derivatives. Somitic derivatives are determined by local signalling between adjacent embryonic tissues, in particular the neural tube, notochord, surface ectoderm and the somitic compartments themselves. The correct spatio-temporal specification of the deriving tissues, skeletal muscle, cartilage, endothelia and connective tissue is achieved by a sequence of morphogenetic changes of the paraxial mesoderm, eventually leading to the three transitory somitic compartments: dermomyotome, myotome and sclerotome. These structures are specified along a double gradient from dorsal to ventral and from medial to lateral. The establishment and controlled disruption of the epithelial state of the somitic compartments are crucial for development. In this article, we give a synopsis of some of the most important signalling events involved in somite patterning and cell fate decisions. Particular emphasis has been laid on the issue of epithelio-mesenchymal transition and different types of cell division in the somite.     

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using <Emphasis Type="Italic">EGFP</Emphasis> transfection

Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.     

Sonic hedgehog promotes somitic chondrogenesis by altering the cellular response to BMP signaling

Previous work has indicated that signals from the floor plate and notochord promote chondrogenesis of the somitic mesoderm. These tissues, acting through the secreted signaling molecule Sonic hedgehog (Shh), appear to be critical for the formation of the sclerotome. Later steps in the differentiation of sclerotome into cartilage may be independent of the influence of these axial tissues. Although the signals involved in these later steps have not yet been pinpointed, there is substantial evidence that the analogous stages of limb bud chondrogenesis require bone morphogenetic protein (BMP) signaling. We show here that presomitic mesoderm (psm) cultured in the presence of Shh will differentiate into cartilage, and that the later stages of this differentiation process specifically depend on BMP signaling. We find that Shh not only acts in collaboration with BMPs to induce cartilage, but that it changes the competence of target cells to respond to BMPs. In the absence of Shh, BMP administration induces lateral plate gene expression in cultured psm. After exposure to Shh, BMP signaling no longer induces expression of lateral plate markers but now induces robust chondrogenesis in cultured psm. Shh signals are required only transiently for somitic chondrogenesis in vitro, and act to provide a window of competence during which time BMP signals can induce chondrogenic differentiation. Our findings suggest that chondrogenesis of somitic tissues can be divided into two separate phases: Shh-mediated generation of precursor cells, which are competent to initiate chondrogenesis in response to BMP signaling, and later exposure to BMPs, which act to trigger chondrogenic differentiation.     

Explanted and implanted notochord of amphibian anuran embryos

Summary The notochord of amphibian anuran embryos contains catecholamines during the early developmental stages. In order to determine if these catecholamines are synthesized in situ, the development of their specific histofluorescence was investigated in the notochord alone or the notochord combined with the lateral somitic mesoderm, both explanted at the neurula stage and cultivated in vitro or implanted into the ventral part of early neurulae endoderm. The histofluorescence evolution, on the other hand, was investigated in the notochord alone or combined with myotomes, both explanted after the beginning of catecholamine biosynthesis and cultivated in vitro for one hour, in order to determine the effect of explantation and culture on the accumulation of notochordal catecholamines.The comparative study of catecholamine histofluorescence in these different samples shows that:the notochord is able to perform, on its own, the entire biosynthesis of the catecholamines stored in it during the early developmental stages.the catecholamines generated from isolated notochords tend to diffuse into the culture medium, probably due to a deficiency in the vesicular storage system usually found in the catecholamine-synthesizing cells. This loss of catecholamines in vitro can be obviated by the presence round the notochord of any embryonal tissue (somitic mesoderm, endoderm).Abbreviations CA Catecholamines - FIF Formaldehyde Induced Fluorescence     

The bHLH regulator pMesogenin1 is required for maturation and segmentation of paraxial mesoderm

Paraxial mesoderm in vertebrates gives rise to all trunk and limb skeletal muscles, the trunk skeleton, and portions of the trunk dermis and vasculature. We show here that germline deletion of mouse pMesogenin1, a bHLH class gene specifically expressed in developmentally immature unsegmented paraxial mesoderm, causes complete failure of somite formation and segmentation of the body trunk and tail. At the molecular level, the phenotype features dramatic loss of expression within the presomitic mesoderm of Notch/Delta pathway components and oscillating somitic clock genes that are thought to control segmentation and somitogenesis. Subsequent patterning and specification steps for paraxial mesoderm also fail, leading to a complete absence of all trunk paraxial mesoderm derivatives, which include skeletal muscle, vertebrae, and ribs. We infer that pMesogenin1 is an essential upstream regulator of trunk paraxial mesoderm development and segmentation.     

The ventralizing effect of the notochord on somite differentiation in chick embryos

The dorso-ventral pattern formation of the somites becomes manifest by the formation of the epithelially organized dorsal dermomyotome and the mesenchymal ventrally situated sclerotome. While the dermomyotome gives rise to dermis and muscle, the sclerotome differentiates into cartilage and bone of the axial skeleton. The onset of muscle differentiation can be visualized by immunohistochemistry for proteins associated with muscle contractility, e.g. desmin. The sclerotome cells and the epithelial ventral half of the somite express Pax-1, a member of a gene family with a sequence similarity to Drosophila paired-box-containing genes. In the present study, changes of Pax-1 expression were studied after grafting an additional notochord into the paraxial mesoderm region. The influence of the notochord and the floor-plate on dermomyotome formation and myotome differentiation has also been investigated. The notochord is found to exert a ventralizing effect on the establishment of the dorso-ventral pattern in the somites. Notochord grafts lead to a suppression of the formation and differentiation of the dorsal somitic derivatives. Simultaneously, a widening of the Pax-1-expressing domain in the sclerotome can be observed. In contrast, grafted roof-plate and aorta do not interfere with dorso-ventral patterning of the somitic derivatives.     

Embryonic cells depleted of β‐catenin remain competent to differentiate into dorsal mesodermal derivatives

Disruption of axis specification leads to defects in dorsal tissue patterning and cell movements. Here, we examine how β‐catenin coordinately affects gastrulation movements and dorsal mesoderm differentiation. The reduction of β‐catenin protein levels by morpholino oligonucleotides complementary to β‐catenin mRNA causes a disruption in gastrulation movements. Time‐lapse imaging of β‐catenin morphants during gastrulation reveals that involution occurs simultaneously around the blastopore in the absence of convergent extension cell movements. Transplantation experiments show that morphant cells grafted from the marginal zone into wild‐type hosts differentiate into notochord and muscle. However, wild‐type mesoderm cells grafted to the marginal zone of β‐catenin morphants do not form dorsal tissues. These data argue that β‐catenin is not required for the initial establishment of dorsal mesoderm cell competency, but it is required for the maintenance of that competency. We propose that tissue interactions that occur during convergent extension movements are necessary for maintaining dorsal tissue competency. Developmental Dynamics 236:3007–3019, 2007. © 2007 Wiley‐Liss, Inc.     

Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog

The patterning of vertebrate somitic muscle is regulated by signals from neighboring tissues. We examined the generation of slow and fast muscle in zebrafish embryos and show that Sonic hedgehog (Shh) secreted from the notochord can induce slow muscle from medial cells of the somite. Slow muscle derives from medial adaxial myoblasts that differentiate early, whereas fast muscle arises later from a separate myoblast pool. Mutant fish lacking shh expression fail to form slow muscle but do form fast muscle. Ectopic expression of shh, either in wild-type or mutant embryos, leads to ectopic slow muscle at the expense of fast. We suggest that Shh acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm.     

The metameric pattern of the head mesoderm — does it exist?

It is a long-standing question whether the paraxial head mesoderm of vertebrate embryos is segmentally organized into somites like the trunk or not. On the one hand, no somites are seen in the anterior head mesoderm in vertebrate embryos, on the other hand, such a segmental pattern has been described under the name of somitomeres. In order to investigate the patterning of mesodermal cells in the head of avian embryos we performed scanning electron microscopy, computer assisted reconstructions of the head mesoderm and density analyses of head mesoderm cells. We observed regional differences within the head mesoderm of avian embryos, but we could not see a consistent somitomeric pattern in the head mesoderm. In sum, we consider that the avian head mesoderm is not arranged in a metameric pattern.     

Global patterning of the vertebrate mesoderm.

We describe recent advances in the understanding of patterning in the vertebrate post-cranial mesoderm. Specifically, we discuss the integration of local information into global level information that results in the overall coordination along the anterioposterior axis. Experiments related to the integration of the axial and appendicular musculoskeletal systems are considered, and examples of genetic interactions between these systems are outlined. We emphasize the utility of the terms primaxial and abaxial as an aid to understanding development of the vertebrate musculoskeletal system, and hypothesize that the lateral somitic frontier is a catalyst for evolutionary change.     

On the histogenetic potency of the tailhud mesoderm

Summary The caudalmost part of the tailbud mesoderm (terminal paraxial tailbud mesoderm) does not develop into somites. It is not clear whether this terminal paraxial tailbud mesoderm can be considered to be a part of the segmental plate. To elucidate the nature of the tailbud mesoderm, grafts containing caudal somites, caudal prospective somitic mesoderm and the terminal paraxial tailbud mesoderm were grafted from quail embryos into the wing bud mesoderm of chick embryos. The distinct nuclear difference between quail and chick allows the identification of the grafts on a cellular level. The grafts containing caudalmost somites and the prospective somitic mesoderm differentiate into muscle and cartilage. The terminal paraxial tailbud mesoderm, on the other hand, did not give rise to either of these tissues. From this it can be concluded that the terminal paraxial tailbud mesoderm cannot be considered to be a part of the segmental plate.     

Positional control of mesoderm movement and fate during avian gastrulation and neurulation.

Segments of primitive streak from donor quail embryos at stages of gastrulation and neurulation were transplanted heterotopically and isochronically to primitive streaks of host chick embryos. The subsequent movement and fate of grafted cells was determined using the quail nucleolar marker to define grafted cells. The pattern of movement of grafted cells depended on their new position within the primitive streak, not on their original position. When cells of cranial regions were placed more caudally, they moved to mesodermal subdivisions that were located lateral to those they would have populated if left in their original position. When caudal segments were placed more cranially, they moved to more medial mesodermal subdivisions. Whether the fate of grafted cells corresponded to their original location or their new location depended on both their level of origin and their new position. Cells from heterotopically transplanted Hensen's nodes, which migrated to the somitic and more lateral mesoderm, self-differentiated notochords. Similarly, in some cases, heterotopically transplanted prospective somitic cells, which migrated to lateral plate mesoderm, formed ectopic somites. In other cases, however, grafted cells contributed to the host's somites, intermediate mesoderm, and lateral plate mesoderm. Moreover, prospective somitic cells, which migrated to the extraembryonic lateral plate mesoderm, changed their fate and formed extraembryonic lateral plate mesoderm; and prospective lateral plate mesoderm cells, which migrated to the somitic mesoderm, formed somites as well as intermediate mesoderm and lateral plate mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)