Bodo Christ

Early stages of chick somite development

We report on the formation and early differentiation of the somites in the avian embryo. The somites are derived from the mesoderm which, in the body (excluding the head), is subdivided into four compartments: the axial, paraxial, intermediate and lateral plate mesoderm. Somites develop from the paraxial mesoderm and constitute the segmental pattern of the body. They are formed in pairs by epithelialization, first at the cranial end of the paraxial mesoderm, proceeding caudally, while new mesenchyme cells enter the paraxial mesoderm as a consequence of gastrulation. After their formation, which depends upon cell-cell and cell-matrix interactions, the somites impose segmental pattern upon peripheral nerves and vascular primordia. The newly formed somite consists of an epithelial ball of columnar cells enveloping mesenchymal cells within a central cavity, the somitocoel. Each somite is surrounded by extracellular matrix material connecting the somite with adjacent structures. The competence to form skeletal muscle is a unique property of the somites and becomes realized during compartmentalization, under control of signals emanating from surrounding tissues. Compartmentalization is accompanied by altered patterns of expression of Pax genes within the somite. These are believed to be involved in the specification of somite cell lineages. Somites are also regionally specified, giving rise to particular skeletal structures at different axial levels. This axial specification appears to be reflected in Hox gene expression. MyoD is first expressed in the dorsomedial quadrant of the still epithelial somite whose cells are not yet definitely committed. During early maturation, the ventral wall of the somite undergoes an epithelio-mesenchymal transition forming the sclerotome. The sclerotome later becomes subdivided into rostral and caudal halves which are separated laterally by von Ebners fissure. The lateral part of the caudal half of the sclerotome mainly forms the ribs, neural arches and pedicles of vertebrae, whereas within the lateral part of the rostral half the spinal nerve develops. The medially migrating sclerotomal cells form the peri-notochordal sheath, and later give rise to the vertebral bodies and intervertebral discs. The somitocoel cells also contribute to the sclerotome. The dorsal half of the somite remains epithelial and is referred to as the dermomyotome because it gives rise to the dermis of the back and the skeletal musculature. The cells located within the lateral half of the dermomyotome are the precursors of the muscles of the hypaxial domain of the body, whereas those in the medial half are precursors of the epaxial (back) muscles. Single epithelial cells at the cranio-medial edge of the dermomyotome elongate in a caudal direction, beneath the dermomyotome, and become anchored at its caudal margin. These post-mitotic and muscle protein-expressing cells form the myotome. At limb levels, the precursors of hypaxial muscles undergo an epithelio-mesenchymal transition and migrate into the somatic mesoderm, where they replicate and later differentiate. These cells express the Pax-3 gene prior to, during and after this migration. All compartments of the somite contribute endothelial cells to the formation of vascular primordia. These cells, unlike all other cells of the somite, occasionally cross the midline of the developing embryo. We also suggest a method for staging somites according to their developmental age.

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.

From somites to vertebral column

We report on the development and differentiation of the somites with respect to vertebral column formation in avian and human embryos. The somites, which are made up of different compartments, establish a segmental pattern which becomes transferred to adjacent structures such as the peripheral nervous system and the vascular system. Each vertebra arises from three sclerotomic areas. The paired lateral ones give rise to the neural arches, the ribs and the pedicles of vertebrae, whereas the vertebral body and the intervening disc develop from the axially-located mesenchyme. The neural arches originate from the caudal half of one somite, whereas the vertebral body is made up of the adjacent parts of two somites. Interactions between notochord and axial mesenchyme are a prerequisite for the normal development of vertebral bodies and intervening discs. The neural arches form a frame for the neural tube and spinal ganglia. The boundary between head and vertebral column is located between the 5th and 6th somites. In the human embryo, proatlas, body of the atlas segment, and body of the axis fuse to form the axis.

The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.

Detection of lymphatic endothelal cells (LECs) has been problematic because of the lack of specific markers. The homeobox transcription factor Prox1 is expressed in LECs of murine and avian embryos. We have studied expression of Prox1 in human tissues with immunofluorescence. In 19‐wk‐old human fetuses, Prox1 and vascular endothelial growth factor receptor‐3 (VEGFR‐3) are coexpressed in LECs of lymphatic trunks and lymphatic capillaries. Prox1 is located in the nucleus, and its expression is mutually exclusive with that of the blood vascular marker PAL‐E. Prox1 is a constitutive marker of LECs and is found in tissues of healthy adults and lymphedema patients. Blood vascular endothelial cells (BECs) of hemangiomas express CD31 and CD34, but not Prox1. A subset of these cells is positive for VEGFR‐3. Lymphatics in the periphery of hemangiomas express Prox1 and CD31, but not CD34. In lymphangiomas, LECs express Prox1, CD31, and VEGFR‐3, but rarely CD34. In the stroma, spindle‐shaped CD34‐positive cells are present. We show that Prox1 is a reliable marker for LECs in normal and pathologic human tissues, coexpressed with VEGFR‐3 and CD31. VEGFR‐3 and CD34 are less reliable markers for LECs and BECs, respectively, because exceptions from their normal expression patterns are found in pathologic tissues.

The importance of timing differentiation during limb muscle development

BACKGROUND Skeletal muscle of trunk, limbs and tongue develops from a small population of cells that originates from somites. Although promoters and inhibitors of muscle differentiation have been isolated, nothing is known about how the amplification of the muscle precursor pool is regulated; this amplification provides muscle mass during development. Furthermore, little is known about how cells accumulate in the pre-muscle masses in the limbs. We investigated the role of bone morphogenetic protein (BMPs) and Sonic hedgehog (Shh) during proliferation, differentiation and positioning of muscle. RESULTS The proliferation of muscle precursors in limbs was linked to Pax-3 expression. Ectoderm removal downregulated Pax-3 expression, arrested proliferation and prematurely initiated muscle differentiation which exhausted the muscle precursor pool and prevented further muscle growth. BMP-2, BMP-4 and BMP-7 had a dose-dependent effect on pre-myogenic cells: low concentrations maintained a Pax-3-expressing proliferative population, substituting for ectoderm-derived proliferative signals and delaying differentiation, whereas high concentrations prevented muscle development, probably by inducing apoptosis. In the limb, Shh upregulated Bmp-2 and Bmp-7 expression which delayed muscle differentiation, upregulated Pax-3, amplified the muscle precursor population and stimulated excessive muscle growth. CONCLUSIONS These data indicate that embryonic muscle growth requires muscle differentiation to be delayed. Muscle differentiation may occur through a default pathway after cells escape proliferative signals. Positioning of muscle is regulated by high concentrations of BMPs, thus a single type of signalling molecule can determine crucial steps in muscle development: when and where to proliferate, and when and where to differentiate.

Formation and differentiation of the avian sclerotome.

The avian sclerotome forms by epitheliomesenchymal transition of the ventral half-somite. Sclerotome development is characterized by a craniocaudal polarization, resegmentation, and axial identity. Its formation is controlled by signals from the notochord, the neural tube, the lateral plate mesoderm, and the myotome. These signals and crosstalk between somite cells lead to the separation of various subdomains, such as the central and ventral sclerotomes that express Pax1 under the control of Sonic hedgehog and Noggin, and the dorsal and lateral sclerotome that do not express Pax1 and are controlled by Bmp-4. Further subdomains that give rise to specific derivatives are the syndetome, neurotome, meningotome, and arthrotome. The molecular control of subdomain formation and cell type specification is discussed.

In vivo effects of vascular endothelial growth factor on the chicken chorioallantoic membrane.

The effect of vascular endothelial growth factor (VEGF165) on the chorioallantoic membrane (CAM) of 13-day-old chick embryos was studied. The factor was applied in doses of 0.5–4 μg for a period of up to 4 days. Macroscopical, histological and immunohistological studies were carried out. The localization of the factor was examined with an anti-VEGF antibody. The mitogenicity of VEGF165 and basic fibroblast growth factor (bFGF) were studied by means of the BrdU-anti-BrdU method. Furthermore, the effect of heparin alone and in combination with VEGF165 was investigated. VEGF165 specifically induces angiogenesis in doses of 0.5 μg and more. A brush-like formation of blood vessels can be seen in the region of the precapillary vessels. Angiogenesis also takes place in the region of the capillaries and the venules. Histologically we found indications of sprouting as well as of intussusceptive capillary growth. The presence of the factor in the application area could be demonstrated with the anti-VEGF antibody for a period of 3 days. The factor is located in the chorionic epithelium and the intraepithelial capillaries. The BrdU-studies show that VEGF165 induces strong endothelial cell proliferation, whereas bFGF elicits fibrocyte proliferation and minor endothelial cell proliferation. Heparin induces squamous metaplasia of the chorionic and allantoic epithelium in combination with an aggregation of fibrocytes. We could not detect any enhancement of VEGF165 by heparin.

The development of the avian vertebral column.

Segmentation of the paraxial mesoderm leads to somite formation. The underlying molecular mechanisms involve the oscillation of ”clock-genes” like c-hairy-1 and lunatic fringe indicative of an implication of the Notch signaling pathway. The cranio-caudal polarity of each segment is already established in the cranial part of the segmental plate and accompanied by the expression of genes like Delta1, Mesp1, Mesp2, Uncx-1, and EphA4 which are restricted to one half of the prospective somite. Dorsoventral compartmentalization of somites leads to the development of the dermomyotome and the sclerotome, the latter forming as a consequence of an epithelio-to-mesenchymal transition of the ventral part of the somite. The sclerotome cells express Pax-1 and Pax-9, which are induced by notochordal signals mediated by sonic hedgehog (Shh) and noggin. The craniocaudal somite compartmentalization that becomes visible in the sclerotomes is the prerequisite for the segmental pattern of the peripheral nervous system and the formation of the vertebrae and ribs, whose boundaries are shifted half a segment compared to the sclerotome boundaries. Sclerotome development is characterized by the formation of three subcompartments giving rise to different parts of the axial skeleton and ribs. The lateral sclerotome gives rise to the laminae and pedicles of the neural arches and to the ribs. Its development depends on signals from the notochord and the myotome. The ventral sclerotome giving rise to the vertebral bodies and intervertebral discs is made up of Pax-1 expressing cells that have invaded the perinotochordal space. The dorsal sclerotome is formed by cells that migrate from the dorso-medial angle of the sclerotome into the space between the roof plate of the neural tube and the dermis. These cells express the genes Msx1 and Msx2, which are induced by BMP-4 secreted from the roof plate, and they later form the dorsal part of the neural arch and the spinous process. The formation of the ventral and dorsal sclerotome requires directed migration of sclerotome cells. The regionalization of the paraxial mesoderm occurs by a combination of functionally Hox genes, the Hox code, and determines the segment identity. The development of the vertebral column is a consequence of a segment-specific balance between proliferation, apoptosis and differentiation of cells.

Molecular and cellular biology of avian somite development

Much of our understanding of early vertebrate embryogenesis derives from experimental work done with the chick embryo. Studies of the avian somite have played a key role in elucidating the developmental history of this important structure, the source of most muscle and bone in the organism. Here we review the development of the avian somite including morphological and molecular data on the origin of paraxial mesoderm, maturation of the segmental plate, specification and formation of somite compartments, and somite cell differentiation into cartilage and skeletal muscle.

The onset of myotome formation in the chick

SummaryThe onset of myotome formation in somites of chick embryos was studied by use of a polyclonal antidesmin antibody and by histochemical demonstration of acetylcholine esterase activity. The myotome cells originate from the dermatome only; sclerotome cells do not contribute to the myotome. The formation of the myotome starts in the craniomedial corner of the dermatome. From there the myotome formation continues simultaneously along the medial and the cranial edge of the dermatome. It was found that only the already longitudinally oriented cells of the cranial dermatome edge give rise to the myotome; the cells of the dorsomedial dermatome edge do not contribute to the myotome. Myotome cells do not originate directly from the surface of the overlying dermatome by delamination.