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 Ebner's 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.     

Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse

Background: Skeletal myogenesis is extensively influenced by the surrounding environment. However, how the extracellular matrix (ECM) affects morphogenesis of muscles is not well understood. Results: We mapped the three‐dimensional (3D) organization of fibronectin, tenascin, and laminin by immunofluorescence during early epaxial myogenesis in mouse embryos. We define four stages of dermomyotome/myotome development and reveal the 3D organization of myogenic cells within their ECM during those stages. Fibronectin is abundant in all interstitial tissues, while tenascin is restricted to intersegmental borders. Bundles of fibronectin and tenascin also penetrate into the myotome, possibly promoting myocyte alignment. A laminin matrix delineates the dermomyotome and myotome and undergoes dynamic changes, correlating with key developmental events. Conclusion: Our observations cast new light on how myotomal cells interact with their environment and suggest that, as the segmented myotomes transform into the epaxial muscle masses, the laminin matrix disassembles and myocytes use the abundant fibronectin matrix to reach their final organization. Developmental Dynamics 241:350–364, 2012. © 2011 Wiley Periodicals, Inc.     

Integrins in the mouse myotome: developmental changes and differences between the epaxial and hypaxial lineage.

Integrins are cellular adhesion receptors that mediate signaling and play key roles in the development of multicellular organisms. However, their role in the cellular events leading to myotome formation is completely unknown. Here, we describe the expression patterns of the alpha1, alpha4, alpha5, alpha6, and alpha7 integrin subunits in the mouse myotome and correlate them with the expression of several differentiation markers. Our results indicate that these integrin subunits may be differentially involved in the various phases of myogenic determination and differentiation. A detailed characterization of the myogenic cell types expressing the alpha4 and alpha6 subunits showed a regionalization of the myotome and dermomyotome based on cell-adhesion properties. We conclude that alpha6beta1 may be an early marker of epaxial myogenic progenitor cells. In contrast, alpha4beta1 is up-regulated in the intercalated myotome after myocyte differentiation. Furthermore, alpha4beta1 is expressed in the hypaxial dermomyotome and is maintained by early hypaxial myogenic progenitor cells colonizing the myotome.     

Precocious terminal differentiation of premigratory limb muscle precursor cells requires positive signalling.

The timing of myogenic differentiation of hypaxial muscle precursor cells in the somite lags behind that of epaxial precursors. Two hypotheses have been proposed to explain this delay. One attributes the delay to the presence of negative-acting signals from the lateral plate mesoderm adjacent to the hypaxial muscle precursor cells located in the ventrolateral lip of the somitic dermomyotome (Pourquié et al. [1995] Proc. Natl. Acad. Sci. USA 92:3219-3223). The second attributes the delay to an absence of positive-acting inductive signals, similar to those from the axial structures that induce epaxial myotome development (Pownall et al. [1996] Development 122:1475-1488). Because both studies relied principally upon changes in the expression pattern of mRNAs specific to early muscle precursor cell markers, we revisited these experiments using two methods to assess muscle terminal differentiation. First, injection of fluorescent dyes before surgery was used to determine whether ventrolateral lip cells transform from epithelial cells to elongated myocytes. Second, an antibody to a terminal differentiation marker and a new monoclonal antibody that recognises avian and mammalian Pax3 were used for immunohistochemistry to assess the transition from precursor cell to myocyte. The results support both hypotheses and show further that placing axial structures adjacent to the somite ventrolateral lip induces an axial pattern of myocyte terminal differentiation and elongation.     

Developmental fate of the mammalian myotome

The myotome is a segmented paraxial muscle present in all early vertebrate embryos, which in amniotes disappears in mid‐embryogenesis, and is replaced by complex epaxial and hypaxial musculature. Little is known about how this transition occurs. Here, we describe the detailed morphogenesis of the epaxial muscles from the epaxial myotome, in rodent embryos. The results show there is no apoptosis of myotomal fibres during the transition, and that the epaxial muscles arise by translocation, re‐orientation, and elongation of the myotomal myocytes followed by cleavage of the myotomal masses. Myotomal myocytes transit from a mononucleated to a multinucleated state just before onset of this transformation. Each newly‐formed epaxial muscle anlagen includes populations of Pax3‐ and Pax7‐positive muscle progenitors, with different distributions. Using transgenic mouse embryos bearing a GFP marker for Scleraxis, we show that tendon progenitors are tightly associated with the sides and ends of myotomal myocytes as they re‐orient and elongate. Developmental Dynamics 239:2898–2910, 2010. © 2010 Wiley‐Liss, Inc.     

Evolution and developmental patterning of the vertebrate skeletal muscles: perspectives from the lamprey.

The myotome in gnathostome vertebrates, which gives rise to the trunk skeletal muscles, consists of epaxial (dorsal) and hypaxial (ventral) portions, separated by the horizontal myoseptum. The hypaxial portion contains some highly derived musculature that is functionally as well as morphologically well differentiated in all the gnathostome species. In contrast, the trunk muscles of agnathan lampreys lack these distinctions and any semblance of limb muscles. Therefore, the lamprey myotomes probably represent a primitive condition compared with gnathostomes. In this review, we compare the patterns of expression of some muscle-specific genes between the lamprey and gnathostomes. Although the cellular and tissue morphology of lamprey myotomes seems uniform and undifferentiated, some of the muscle-specific genes are expressed in a spatially restricted manner. The lamprey Pax3/7 gene, a cognate of gnathostome Pax3, is expressed only at the lateral edge of the myotomes and in the hypobranchial muscle, which we presume is homologous to the gnathostome hypobranchial muscle. Thus, the emergence of some part of a hypaxial-specific gene regulatory cascade might have evolved before the agnathan/gnathostome divergence, or before the evolutionary separation of epaxial and hypaxial muscles.     

Temporal sequence in the formation of midline dermis and dorsal vertebral elements in avian embryos

Somites compartmentalize into a dorsal epithelial dermomyotome and a ventral mesenchymal sclerotome. While sclerotomes give rise to vertebrae and intervertebral discs, dermomyotomes contribute to skeletal muscle and epaxial dermis. Bone morphogenetic protein (BMP)-signals from the lateral mesoderm induce the lateral portion of the dermomyotome to form chondrogenic precursor cells, forming the cartilage of the scapula blade. The fact that BMPs are expressed in the roof plate of the neural tube where they induce cartilage formation led to the question why cells migrating from the medial part of the dermomyotome do not undergo chondrogenic differentiation and do not contribute to the dorsal part of the vertebrae. In the present study, we traced dermomyotomal derivatives by using the quail-chick marker technique. Our study reveals a temporal sequence in the formation of the vertebral cartilage and the midline dermis. The dorsal mesenchyme overlying the roof plate of the neural tube is formed prior to the de-epithelialization of the dermomyotome. Dermomyotomal cells start to migrate medially into the sub-ectodermal space to form the midline dermis after chondrogenesis of the dorsal mesenchyme has occurred. This time delay between chondrogenesis of the dorsal vertebra and dermal formation allows an undisturbed development of these two tissue components within a narrow region of the embryo.     

The teleost dermomyotome.

Recent work in teleosts has renewed interest in the dermomyotome, which was initially characterized in the late 19th century. We review the evidence for the teleost dermomyotome, comparing it to the more well-characterized amniote dermomyotome. We discuss primary myotome morphogenesis, the relationship between the primary myotome and the dermomyotome, the differentiation of axial muscle, appendicular muscle, and dermis from the dermomyotome, and the signaling molecules that regulate myotome growth from myogenic precursors within the dermomyotome. The recognition of a dermomyotome in teleosts provides a new perspective on teleost muscle growth, as well as a fruitful approach to understanding the vertebrate dermomyotome.     

Novel concept for the epaxial/hypaxial boundary based on neuronal development

Trunk muscles in vertebrates are classified as either dorsal epaxial or ventral hypaxial muscles. Epaxial and hypaxial muscles are defined as muscles innervated by the dorsal and ventral rami of spinal nerves, respectively. Each cluster of spinal motor neurons passing through dorsal rami innervates epaxial muscles, whereas clusters traveling on the ventral rami innervate hypaxial muscles. Herein, we show that some motor neurons exhibiting molecular profiles for epaxial muscles follow a path in the ventral rami. Dorsal deep-shoulder muscles and some body wall muscles are defined as hypaxial due to innervation via the ventral rami, but a part of these ventral rami has the molecular profile of motor neurons that innervate epaxial muscles. Thus, the epaxial and hypaxial boundary cannot be determined simply by the ramification pattern of spinal nerves. We propose that, although muscle innervation occurs via the ventral rami, dorsal deep-shoulder muscles and some body wall muscles represent an intermediate group that lies between epaxial and hypaxial muscles.     

The pattern of MyoD and contractile protein localization in primary epaxial myotome reflects the dynamic progression of nascent myoblast differentiation.

The localization of contractile and regulatory proteins in early stages of epaxial primary myotome development was analyzed by immunofluorescence microscopy. Contractile proteins that appear in an ordered sequence in the rostro-caudal axis of somite development were found to reiterate that sequence in the dorso-medial-to-ventro-lateral axis of primary epaxial myotome development. Pair-wise localization of MyoD-titin, desmin-titin, and desmin-myosin defined three zones extending from the dermomyotome dorso-medial lip (DML) into the primary myotome layer. Zones M1 and M2, which were positive for MyoD + titin and MyoD + titin + desmin, respectively, were restricted to the dorso-medial-most extremity of the myotome layer and did not expand during the course of myotome development. Zone M3 was positive for MyoD, desmin, titin, myosin, and cardiac troponin T and was the only zone that expanded during primary myotome development. Myotome fibers in zone M3 were unit-length, spanning the full rostro-caudal axis of the myotome while fibers in zones M1 and M2 were shorter than unit length. Anti-myoD immunofluorescence, when detected in cells lacking contractile-protein-positive cytoplasm, was restricted to the DML and nascent myotome cells immediately subjacent to the DML. These results demonstrate a dynamic spatio-temporal sequence in the differentiation program of nascent myotome cells as they emerge from the DML; zones M1 and M2 reflect standing waves of sequential contractile protein activation during the maturation of nascent myotomal myoblasts, while the expanding zone M3 reflects the accumulation of mature myotome fibers expressing a full cohort contractile proteins.     

BMP regulation of myogenesis in zebrafish

In amniotes, BMP signaling from lateral plate and dorsal neural tube inhibits differentiation of muscle precursors in the dermomyotome. Here, we show that BMPs are expressed adjacent to the dermomyotome during and after segmentation in zebrafish. In addition, downstream BMP pathway members are expressed within the somite during dermomyotome development. We also show that zebrafish dermomyotome is responsive to BMP throughout its development. Ectopic overexpression of Bmp2b increases expression of the muscle precursor marker pax3, and changes the time course of myoD expression. At later stages, overexpression increases the number of Pax7+ myogenic precursors, and delays muscle differentiation, as indicated by decreased numbers of MEF2+ nuclei, decreased number of multi‐nucleated muscle fibers, and an increased myotome angle. In addition, we show that while BMP overexpression is sufficient to delay myogenic differentiation, inhibition of BMP does not detectably affect this process, suggesting that other factors redundantly inhibit myogenic differentiation. Developmental Dynamics 239:806–817, 2010. © 2010 Wiley‐Liss, Inc.     

Topographic arrangement of the projection from the anterior thalamic nuclei to the cingulate cortex in the cat

After injection of HRP into the cingulate and its adjacent cortical areas, neuronal cells were retrogradely labeled ipsilaterally in the anterior, lateral, ventral, intralaminar and midline thalamic nuclei. Most of them occurred in the anterior nuclei, and a topographic correlation was revealed in the projections from the anterior thalamic nuclei to the cingulate gyrus: the rostral part of the cingulate gyrus receives fibers predominantly from the lateral part of the AM and additionally from the caudomedial part of the AV. The caudal part of the cingulate gyrus receives fibers from the medial part of the anteromedial nucleus (AM) and the rostromedial part of the anteroventral nucleus (AV). The intermediate part of the cingulate gyrus receives fibers from the intermediate part of the AM and the caudal half of the AV.     

Development of the epaxial muscles in the human embryo

Although the intrinsic muscles of the back are defined by their embryological origin and innervation pattern, no detailed study on their development is available. Human embryos (5–10 weeks development) were studied, using Amira3D® reconstruction and Cinema4D® remodeling software for visualization. At Carnegie Stage (CS)15, the epaxial portions of the myotomes became identifiable laterally to the developing vertebrae. At CS16, these portions fused starting cranially to form a longitudinal muscle column, which became innervated by the dorsal branches of the spinal nerves. At CS17, the longitudinal muscle mass segregated into medial and lateral columns (completed at CS18). At CS18, the medial column segregated again into intermediate and medial columns (completed at CS20). The lateral and intermediate columns did not separate in the lower lumbar and sacral regions. Between CS20 and CS23, the cervical portions of the three columns segregated again from lateral to medial resulting ventrolaterally in rod‐like continuations of the caudal portions of the columns and dorsomedially in spade‐like portions. The observed topography identifies the iliocostalis and splenius as belonging to the lateral column, the longissimus to the intermediate column, and the (semi‐)spinalis to the medial column. The medial (multifidus) group acquired its transversospinal course during closure of the vertebral arches in the early fetal period. Hence, the anatomical ontology of the epaxial muscles is determined by craniocaudal and lateromedial gradients in development. Three longitudinal muscle columns, commonly referred to as the erector spinae, form the basic architectural design of the intrinsic muscles of the back. Clin. Anat. 29:1031–1045, 2016. © 2016 Wiley Periodicals, Inc.     

Regulation of scapula development

The scapula is a component of the shoulder girdle. Its structure has changed greatly during evolution. For example, in humans it is a large quite flat triangular bone whereas in chicks it is a long blade like structure. In this review we describe the mechanisms that control the formation of the scapula. To assimilate our understanding regarding the development of the scapula blade we start by addressing the issue concerning the origin of the scapula. Experiments using somite extirpation, chick-quail cell marking system and genetic cell labelling techniques in a variety of species have suggested that the scapula had its origin in the somites. For example we have shown in the chick that the scapula blade originates from the somite, while the cranial part, which articulates with the upper limb, is derived from the somatopleure of the forelimb field. In the second and third part of the review we discuss the compartmental origin of this bone and the signalling molecules that control the scapula development. It is very interesting that the scapula blade originates from the dorsal compartment, dermomyotome, which has been previously been associated as a source of muscle and dermis, but not of cartilage. Thus, the development of the scapula blade can be considered a case of dermomyotomal chondrogenesis. Our results show that the dermomyotomal chondrogenesis differ from the sclerotomal chondrogenesis. Firstly, the scapula precursors are located in the hypaxial domain of the dermomyotome, from which the hypaxial muscles are derived. The fate of the scapula precursors, like the hypaxial muscle, is controlled by ectoderm-derived signals and BMPs from the lateral plate mesoderm. Ectoderm ablation and inhibition of BMP activity interfers the scapula-specific Pax1 expression and scapula blade formation. However, only somite cells in the cervicothoracic transition region appear to be committed to form scapula. This indicates that the intrinsic segment specific information determines the scapula forming competence of the somite cells. Taken together, we conclude that the scapula forming cells located within the hypaxial somitic domain require BMP signals derived from the somatopleure and as yet unidentified signals from ectoderm for activation of their coded intrinsic segment specific chondrogenic programme. In the last part we discuss the new data that provides evidence that neural crest contributes for the development of the scapula.     

Topographical projections from the posterior thalamic regions to the striatum in the cat,with reference to possible tecto-thalamo-striatal connections

Summary Projections from the posterior thalamic regions to the striatum were studied in the cat by the anterograde tracing method after injecting wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the caudalmost regions of the lateroposterior thalamic nucleus (caudal LP), suprageniculate nucleus (Sg) and magnocellular division of the medial geniculate nucleus (MGm). The results were further confirmed by the retrograde tracing method after injecting WGA-HRP into the regions of the caudate nucleus (Cd) and putamen (Put) where afferent fibers from the caudal LP, Sg and MGm were distributed. Fibers from the MGm, Sg or caudal LP were distributed mainly in the medial, middle or lateral part of the caudal half of the putamen (caudal Put), respectively. Although there was a considerable overlap, thalamostriatal fibers from the caudal LP terminated more caudally than those from the MGm. On the other hand, thalamocaudate fibers from the MGm, Sg and lateral part of the caudal LP overlapped with each other in the ventrolateral part of the caudal half of the caudate nucleus (caudal Cd). Fibers from the medial part of the caudal LP were distributed in the ventral part of the caudal Cd. In the superior colliculus (SC) of the cats with WGA-HRP injections in the caudal LP, retrogradely labeled neuronal cell bodies were mainly seen ipsilaterally in the superficial SC layer, and simultaneously, anterogradely labeled axon terminals were observed in the striatum. On the other hand, when WGA-HRP was injected into the Sg or MGm, labeled SC neurons were mainly located in the intermediate and deep SC layers. Thus, ascending impulses from the superficial SC layer may possibly be conveyed ipsilaterally via the caudal LP to the ventral and ventrolateral parts of the caudal Cd and the lateral part of the caudal Put, whereas those from the intermediate and deep SC layers may be relayed via the Sg and/or MGm to the ventrolateral part of the caudal Cd and the middle and medial parts of the caudal Put.Abbreviations AC anterior commissure - Am amygdaloid nucleus - Cd caudate nucleus - Ce centromedial nucleus - CL centrolateral nucleus - Cl claustrum - CM-Pf centre médian-parafascicular complex - CP cerebral peduncle - d deep SC layer - EC external capsule - Ep entopeduncular nucleus - GP globus pallidus - i intermediate SC layer - IC internal capsule - Ip interpeduncular nucleus - LG lateral geniculate nucleus - LP lateroposterior nucleus - MD mediodorsal nucleus - MG medial geniculate nucleus - MGm magnocellular division of MG - MGp principal division of MG - NBIC nucleus of brachium of inferior colliculus - O oculomotor nucleus - OT optic tract - Pom medial division of posterior group of thalamus - Pt pretectum - Pul pulvinar nucleus - Put putamen - Pv paraventricular nucleus of thalamus - R reticular nucleus of thalamus - Rh rhomboid nucleus - RN red nucleus - s superficial SC layer - SC superior colliculus - Sg suprageniculate nucleus - SN substantia nigra - SNpc pars compacta of SN - SNpr pars reticulata of SN - V lateral ventricle - VA ventroanterior nucleus - VL ventrolateral nucleus - VM ventromedial nucleus - WGA-HRP wheat germ agglutinin-HRP conjugate     

Morphological analysis of the role of the neural tube and notochord in the development of somites

The differentiation of avian somites and skeletal muscles, which themselves are derived from somites, was studied in ovo after the isolation of the unsegmented segmental plate from the notochord and/or neural tube by surgical operations at the level of the segmental plate. In each experiment, the newly formed somites had a normal histological structure, with an outer epithelial somite and core cells in the somitocoeles. Thereafter, the three derivatives of the somites (dermatome, myotome and sclerotome) reacted differently to the different operations. When the somites developed without the notochord, only somitocoele cells showed massive cell death, and muscles developed regardless of the presence or absence of the neural tube. On the contrary, no cell death was observed in any part of the somites that were formed with the neural tube or the notochord present, and muscle cells developed. However, in those embryos that retained only the notochord, striated muscles developed only in the lateral body wall. In each of the experimental operations, the surface ectoderm always covered the somites, and, regardless of the state of sclerotome and/or myotome differentiation, the dermatome always survived. These histological observations indicate that the first step in somite formation is independent of axial structures. The results further suggest that the notochord may produce diffusible factors that are necessary for the survival and further development of sclerotomal cells, and that both the neural tube and notochord can support muscle differentiation. However, it is likely that each structure has a relationship to the development of epaxial muscles and hypaxial muscles respectively. Furthermore, an intimate relationship may also exist between the surface ectoderm and the development of the dermatome.     

Development of somites,muscle, and skeleton is independent of signals from the wolffian duct

Background : In the vertebrate embryo, skeletal muscle and the axial skeleton arise from the somites. Patterning of the somites into the respective somite compartments, namely dermomyotome, myotome, and sclerotome, depends on molecular signals from neighboring structures, including surface ectoderm, neural tube, notochord, and lateral plate mesoderm. A potential role of the intermediate mesoderm, notably the Wolffian or nephric duct, in somite development is poorly understood. Results : We studied somite compartmentalization as well as muscular and skeletal development after surgical ablation of the early Wolffian duct anlage, which lead to loss of the Wolffian duct and absence of the mesonephros, whereas Pax2 expression in the nephrogenic mesenchyme was temporarily maintained. We show that somite compartments, as well as the somite derivatives, skeletal muscle and the cartilaginous skeleton, develop normally in the absence of the Wolffian duct. Conclusions : Our results indicate that development of the musculoskeletal system is independent of the Wolffian duct as a signaling center. Developmental Dynamics 242:941–948, 2013. © 2013 Wiley Periodicals, Inc.