Migratory and organogenetic capacities of muscle cells in bird embryos

Summary The migratory and organogenetic capacities of muscle cells at different stages of differentiation were tested in heterospecific chick/quail recombinants. Grafts containing muscle cells were taken from the premuscular masses from 4- to 5-day quail embryos, from the limb or trunk muscles of 12-day embryonic and 4-day post-natal quails, and from experimentally produced bispecific premuscular masses in which the myoblasts are of quail origin and the connective tissue cells of chick origin. Grafts were implanted into 2-day chick embryos in place of the somitic mesoderm at the limb level. Hosts were examined 4 to 7 days after operation.After implantation of a piece of premuscular mass, quail cells were found at and around the site of the graft in the truncal region and within the limb as far as the autopod. Quail cells participated predominantly in the trunk and limb musculature, which contained a number of quail myotubes and of bispecific quail/chick myotubes. Apart from skeletal muscles, quail cells contributed sporadically to nerve envelopes and blood vessel walls in the limb.When the graft was of bispecific constitution, quail nuclei in the limb and the trunk were found exclusively in monospecific and bispecific myotubes.After implantation of differentiated embryonic or post-natal muscle tissue, quail cells in the limb contributed only sporadically to nerve envelopes and blood vessel walls, while in the trunk they also participated in the formation of muscles and tendons.It is concluded that the myogenic cells in 4 to 5-day quail premuscular masses are still able to undergo an extensive migration into the limb buds and there participate in the formation of myotubes and anatomically normal muscles. They display developmental potentialities equivalent to those of the somitic myogenic stem cells. These capacities are lost in 12-day embryonic muscles.     

Spatial and temporal patterns of muscle cleavage in the chick thigh and their value as criteria for homology

Regions of lower cell density, called cleavage zones, emerge within the dorsal and ventral muscle masses in the vertebrate limb to separate distinct muscles. In the chick thigh, the stereotyped patterns of separation have been broadly outlined, but differences in interpretation exist because no criteria for separation have been defined, and the tissues of the limb are indistinct early in development. We have examined the cleavage process using modern applications of light microscopy and immunocytochemistry to completely detail the spatial and temporal progression of cleavage in stage 27-32 embryos. We find that each muscle has a complex but characteristic pattern of separation along the proximodistal axis. The complex pattern of separation is not related to the positions of muscles within the thigh, locations of blood vessels, activity patterns of muscles, or innervation patterns. The initial separation patterns are more straightforward than later separations and may be of value in determining the phylogenetic history of limb muscles since the same patterns are common to many tetrapods. Our detailed documentation clarifies the ontogeny of the thigh musculature and reveals more complex separation patterns between muscles than previously described.     

Differences in the Developmental Fate of Cultured and Noncultured Myoblasts When Transplanted into Embryonic Limbs

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.     

Assembly of trunk and limb blood vessels involves extensive migration and vasculogenesis of somite-derived angioblasts

Vascular development requires the assembly of precursor cells into blood vessels, but how embryonic vessels are assembled is not well understood. To determine how vascular cells migrate and assemble into vessels of the trunk and limb, marked somite-derived angioblasts were followed in developing embryos. Injection of avian somites with the cell-tracker DiI showed that somite-derived angioblasts in unperturbed embryos migrated extensively and contributed to trunk and limb vessels. Mouse-avian chimeras with mouse presomitic mesoderm grafts had graft-derived endothelial cells in blood vessels at significant distances from the graft, indicating that mouse angioblasts migrated extensively in avian hosts. Mouse graft-derived endothelial cells were consistently found in trunk vessels, such as the perineural vascular plexus, the cardinal vein, and presumptive intersomitic vessels, as well as in vessels of the limb and kidney rudiment. This reproducible pattern of graft colonization suggests that avian vascular patterning cues for trunk and limb vessels are recognized by mammalian somitic angioblasts. Mouse-quail chimeras stained with both the quail vascular marker QH1 and the mouse vascular marker PECAM-1 had finely chimeric vessels, with graft-derived mouse cells interdigitated with quail vascular cells in most vascular beds colonized by graft cells. Thus, diverse trunk and limb blood vessels have endothelial cells that developed from migratory somitic angioblasts, and assembly of these vessels is likely to have a large vasculogenic component.     

Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud.

Development of the musculature in chick limbs involves tissue and cellular patterning. Patterning at the tissue level leads to the precise arrangement of specific muscles; at the cellular level patterning gives rise to the fibre type diversity in muscles. Although the data suggests that the information controlling muscle patterning is localised within the limb mesenchyme and not in the somitic myogenic precursor cells themselves, the mechanisms underlying muscle organisation have still to be elucidated. The anterior-posterior axis of the limb is specified by a group of cells in the posterior region of the limb mesenchyme, called the zone of polarizing activity (ZPA). When polarizing-region cells are grafted to the anterior margin of the bud, they cause mirror-image digit duplications to be produced. The effect of ZPA grafts can be reproduced by application of retinoic acid (RA) beads and by grafting sonic hedgehog (SHH)-expressing cells to the anterior margin of the limb. Although most previous studies have looked at changes of the skeletal patterning, ZPA and RA also affect muscle patterning. In this report, we investigated the role of SHH in tissue and cellular patterning of forearm wing muscles. Ectopic application of a localised source of SHH to the anterior margin of the wing, leading to complete digit duplication, is able to transform anterior forearm muscles into muscles with a posterior identity. Moreover, the ectopic source of SHH induces a mirror image duplication of the normal posterior muscles fibre types in the new posterior muscles. The reorganisation of the slow fibres can be detected before muscle mass cleavage has started; suggesting that the appropriate fibre type arrangement is in place before the splitting process can be observed.     

The influence of presumptive limb connective tissue on motoneuron axon guidance

During embryogenesis in the chick, the lumbosacral (LS) somatopleure gives rise to the connective tissue and the epidermis of the limb. We wished to determine if the LS somatopleure was a primary source of guidance cues for motoneuron pathway choices along the anteroposterior axis of the limb. At stage (st) 15, prior to its population by muscle cell precursors and the neural crest, the LS somatopleure was shifted anteriorly. This surgery resulted in the development of limbs that were shifted one to four segments into the thoracic region. Muscles within the anterior thigh of the shifted limb were normally patterned and of composite origin: connective tissues were of LS origin, while muscle cells were of LS and thoracic origin. Retrograde HRP labeling at st 35-37 indicated that motoneuron pools to these anterior thigh muscles were located within LS rather than thoracic cord segments. Pools to individual muscles were smaller than normal but occupied segmental and transverse positions in the LS cord that generally matched those of normal embryos. These findings suggest that individual muscles within somatopleure-shifted limbs are innervated specifically and are in accord with their connective tissue (and epidermal) level of origin. Reconstructions of nerve patterns at st 28-31 suggested that LS motoneurons corrected for the shift by altering their pathways at midthigh regions. We conclude that the somatopleure, and most likely its connective tissue component, contains the information for setting up a specific axon guidance system in the developing limb.     

Six1 is not involved in limb tendon development, but is expressed in limb connective tissue under Shh regulation

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3-/- mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1-/- mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.     

Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Local extrinsic signals determine muscle and endothelial cell fate and patterning in the vertebrate limb

Both the muscle and endothelium of the vertebrate limb derive from somites. We have used replication-defective retroviral vectors to analyze the lineage relationships of these somite-derived cells in the chick. We find that myogenic precursors in the somites or proximal limb are not committed to forming slow or fast muscle fibers, particular anatomical muscles, or muscles within specific proximal/distal or dorsal/ventral limb regions. Somitic endothelial precursors are uncommitted to forming endothelium in particular proximal/distal or dorsal/ventral limb regions. Surprisingly, we also find that myogenic and endothelial cells are derived from a common somitic precursor. Thus, local extrinsic signals are critical for determining muscle and endothelial patterning as well as cell fate in the limb.     

Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.     

Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.

Development of a vascular system involves the assembly of two principal cell types - endothelial cells and vascular smooth muscle cells/pericytes (vSMC/PC) - into many different types of blood vessels. Most, if not all, vessels begin as endothelial tubes that subsequently acquire a vSMC/PC coating. We have previously shown that PDGF-B is critically involved in the recruitment of pericytes to brain capillaries and to the kidney glomerular capillary tuft. Here, we used desmin and alpha-smooth muscle actin (ASMA) as markers to analyze vSMC/PC development in PDGF-B-/- and PDGFR-beta-/- embryos. Both mutants showed a site-specific reduction of desmin-positive pericytes and ASMA-positive vSMC. We found that endothelial expression of PDGF-B was restricted to immature capillary endothelial cells and to the endothelium of growing arteries. BrdU labeling showed that PDGFR-beta-positive vSMC/PC progenitors normally proliferate at sites of endothelial PDGF-B expression. In PDGF-B-/- embryos, limb arterial vSMC showed a reduced BrdU-labeling index. This suggests a role of PDGF-B in vSMC/PC cell proliferation during vascular growth. Two modes of vSMC recruitment to newly formed vessels have previously been suggested: (1) de novo formation of vSMC by induction of undifferentiated perivascular mesenchymal cells, and (2) co-migration of vSMC from a preexisting pool of vSMC. Our data support both modes of vSMC/PC development and lead to a model in which PDGFR-beta-positive vSMC/PC progenitors initially form around certain vessels by PDGF-B-independent induction. Subsequent angiogenic sprouting and vessel enlargement involves PDGF-B-dependent vSMC/PC progenitor co-migration and proliferation, and/or PDGF-B-independent new induction of vSMC/PC, depending on tissue context.     

Essential role of endothelial Smad4 in vascular remodeling and integrity

New blood vessels are formed through the assembly or sprouting of endothelial cells (ECs) and become stabilized by the formation of perivascular matrix and the association with supporting mural cells. To investigate the role of endothelial Smad4 in vascular development, we deleted the Smad4 gene specifically in ECs using the Cre-LoxP system. EC-specific Smad4 mutant mice died at embryonic day 10.5 due to cardiovascular defects, including attenuated vessels sprouting and remodeling, collapsed dorsal aortas, enlarged hearts with reduced trabeculae, and failed endocardial cushion formation. Noticeably, Smad4-deficient ECs demonstrated an intrinsic defect in tube formation in vitro. Furthermore, the mutant vascular ECs dissociated away from the surrounding cells and suffered from impaired development of vascular smooth muscle cells. The disturbed vascular integrity and maturation was associated with aberrant expression of angiopoietins and a gap junction component, connexin43. Collectively, we have provided direct functional evidence that Smad4 activity in the developing ECs is essential for blood vessel remodeling, maturation, and integrity.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.     

Fibroblast-growth-factor-induced additional limbs in the study of initiation of limb formation, limb identity, myogenesis, and innervation

In this review, we focus on the additional limb induced by members of the fibroblast growth factor (FGF) family in the flank of chick embryos. The "additional limb" was first reported 73 years ago by Balinsky in 1925. He grafted otic vesicle to the flank of newt embryos and observed the formation of the "additional limb." In 1995, formation of an additional limb was found to be induced by FGF in the chick embryo. This finding subsequently led to the recent understanding of how the limb bud is initially formed, how the limb position is determined, and how the limb identity is determined. Thus, the additional limb has been recognized as a useful experimental system for the study of limb development and its relation to the regionalization of the body. Furthermore, since limb muscles are formed from cells which have migrated from somites and innervation to them takes place from the spinal cord, the additional limb would also be a powerful tool with which to study the relation of limb morphogenesis to developmental processes of the spinal cord and somites. This review consists of five sections: (1) "Introduction," (2) "How to make additional limbs," (3) "Characteristics of the additional limb," (4) "Studies with the additional limb," and (5) "Concluding remarks." In the second section, techniques to make additional limbs are reviewed, showing that additional limbs can be made by fairly easy manipulation of the chick embryo. In the third section, the characteristics analyzed so far of the additional limb are summarized, focusing on its morphology. In the fourth section, recent studies on the use of the additional limb are reviewed: experiments on the additional limb have been performed to elucidate the mechanisms governing determination of limb identity by Hox codes and the Tbx family and initiation of limb formation by FGF10. In addition, the roles of SF/HGF in the formation of limb muscles have also been investigated using the additional limb. In the near future, the additional limb will be also used in the study of innervation from the spinal cord, and probably migration of neural crest cells.     

Topsy-turvy: turning the counter-current heat exchange of leatherback turtles upside down

Counter-current heat exchangers associated with appendages of endotherms feature bundles of closely applied arteriovenous vessels. The accepted paradigm is that heat from warm arterial blood travelling into the appendage crosses into cool venous blood returning to the body. High core temperature is maintained, but the appendage functions at low temperature. Leatherback turtles have elevated core temperatures in cold seawater and arteriovenous plexuses at the roots of all four limbs. We demonstrate that plexuses of the hindlimbs are situated wholly within the hip musculature, and that, at the distal ends of the plexuses, most blood vessels supply or drain the hip muscles, with little distal vascular supply to, or drainage from the limb blades. Venous blood entering a plexus will therefore be drained from active locomotory muscles that are overlaid by thick blubber when the adults are foraging in cold temperate waters. Plexuses maintain high limb muscle temperature and avoid excessive loss of heat to the core, the reverse of the accepted paradigm. Plexuses protect the core from overheating generated by muscular thermogenesis during nesting.     

Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.