The control of directed myogenic cell migration in the avian limb bud

Summary Interspecific grafting experiments between chick and quail embryos were carried out in order to investigate the mechanism controlling myogenic cell migration in the avian limb bud. In six series, various experimental set-ups were prepared involving different age combinations of donor and host. The migration of the myogenic cells contained nor and host. The migration of the myogenic cells contained in the quail donor could be traced due to the prominent perinucleolar heterochromatin of the quail nucleus. Irrespectively of the presence or absence of the apical ectodermal ridge (AER), myogenic cells were found to migrate distally when implanted at a more distal site or into a younger host. They were even found to migrate in the reverse direction when younger host tissue was located proximal to the graft.From these findings, we conclude that the state of differentiation (juvenility) of the limb bud mesenchyme controls the directed migration of myogenic cells.     

A study on skeletal myogenic cell movement in the developing avian limb bud

Summary Quail limb mesenchyme containing myogenic cells of somitic origin were transplanted into chick limb buds to determine whether cell movement might play a role in avian limb myogenesis. In general, cell displacement was not detected 1-day after implantation: all quail cells were found at the graft site. Migration was evident 2-days after implantation but not all cell types were capable of movement; myogenic cells were very invasive while chondrocytes were relatively immobile. The spreading of myogenic cells was discernible up to 4-days after implantation and specifically in a proximodistal direction towards the apex of the limb.     

An autoradiography study of myogenic cell movement in avian limb buds following heterospecific and homospecific transplantation

Summary Species specificity and the use of quail cells as a marker in the study of myogenic cell movement in the developing avian limb was investigated. In order to establish whether or not observed myogenic cell movement in quail/chick limb transplantation experiments might be an artefact produced by cellular interaction between these cell types a series of homospecific and heterospecific transplantations was performed. Chick wing fragments (staged 20–25 H.H.) were labelled with tritiated thymidine and inserted into unlabelled chick wing bud (homospecific) in ovo. In addition, quail wing fragments were also labelled with tritiated thymidine and transplanted in the same manner into chick (heterospecific), so that the effectiveness of tritium as a marker could be assessed. After 4 days post-incubation, myogenic cell movement was detected in eight out of the ten homospecific transplantions performed. Myogenic cell movement in avian limbs is therefore not produced by interaction between chick and quail cells, as migration was also detected in the chick/chick transplants. Nonetheless, heterospecific transplantation results revealed that autoradiographic methods failed to reveal completely the true extent to which myogenic cell movement occurred, because tritiated thymidine was subject to dilution.     

Study of the origin of connective tissue sheaths of peripheral nerves in the limb of avian embryos

Summary Quail-chick and chick-quail chimeras were constructed by grafting, isotopically, the limb bud of quail embryos into a chick of the same developmental stage and vice versa, prior to the entry of nerve fibres into the limb.After 5–14 days reincubation of the embryos, the components of the connective tissue sheaths of the peripheral nerves were observed by using Feulgen-Rossenbeck staining and light microscopy, in order to distinguish quail cells and chick cells.In all the chimeras studied, the connective tissue sheaths of peripheral nerves (the epineurium, perineurium, perineural septa and endoneural fibroblasts) were formed from the mesenchyme of the limb bud, while Schwann cells were of host origin. Also the outer and inner capsule of muscle spindles originated from the limb bud mesenchyme.These experiments suggest that the connective tissue sheaths of peripheral nerves (at least in the limb region of avian embryos) are not of neural crest origin, but are formed from limb bud mesenchyme.     

Talpid2 mutant chick limb has anteroposterior polarity and altered patterns of programmed cell death

The talpid²(ta²) chick mutant is of interest for the study of limb pattern formation. Talpid² is a simple Mendelian recessive lethal mutation which affects the mesoderm and results in short, spade-like, polydactylous wings and legs. Here, we describe ta² limb development with particular attention to those aspects of ta² which may illuminate the process of normal limb development. From the onset of budding, ta² limb buds are significantly wider than normal buds along the anteroposterior axis. They lack the normal anterior and posterior necrotic zones and have variable development of the central opaque patch. Interdigital programmed cell death is variable and may result in development of distal phalanges without more proximal ones. Talpid² wing vasculature is similar to that of normal wings, but ta² legs are supplied by four large blood vessels. Feathers form regular, parallel rows, similar to normal feathers, but ta² embryos lack the loose mesenchyme which separates the feather buds. Finally, and most significantly, ta² wings and legs display anteroposterior polarity. Anterior and posterior limb skeletal elements can be clearly distinguished from one another within the ta² phenotype. Our observations suggest that the ta² mutant may be useful in analyzing programmed embryonic cell death and anteroposterior limb pattern formation.     

The relationship between limb muscle and endothelial cells migrating from single somite

Somites contribute myogenic and endothelial precursor cells to the limb bud. Transplantations of single somites have shown the pattern of muscle cell distribution from individual somites to individual limb muscles. However, the pattern of the endothelial cell distribution from individual somites to the limb has not been characterized. We have mapped quail muscle and endothelial cell distribution in the distal part of the chick limb after single somite transplantation to determine if there is a spatial relationship between muscle and endothelial cells originating from the same somite. Single brachial somites from quail donor embryos were transplanted into chick embryos, and, following incubation, serial sections were stained with a quail-endothelial cell-specific monoclonal antibody (QH-1), an anti-quail antibody (QCPN) and an anti-desmin antibody to distinguish the quail endothelial and muscle cells from chick cells. Our results show that transplants of somite 16-21 each gave rise to quail endothelial cells in the wing. The anterioposterior position of the blood vessels formed by somitic endothelial cells corresponded to the craniocaudal position of the somite from which they have originated. Endothelial cells were located not only in the peri- and endomysium but also in the subcutaneous, intermuscular, perineural and periost tissues. There was no strict correlation between the distribution of muscle and endothelial cell from a single transplanted somite. Blood vessels formed by grafted quail endothelial cells could invade the muscle that did not contain any quail muscle cells, and conversely a muscle composed of numerous quail muscle cells was lacking any endothelial cells of quail origin. Furthermore, a chimeric limb with very little quail muscle cells was found to contain numerous quail endothelial cells and vice versa. These results suggest that muscle and endothelial cells derived from the same somite migrate on different routes in the developing limb bud.     

An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos

The present study describes a simple, rapid protocol for culture for limb tissue from individual 10.5-day post coitum mouse embryos that supports cartilage differentiation over a six-day period. This technique differs from other commonly used methods utilizing pooled limb tissue in that: 1) forelimbs from individual embryos were used as donor tissue; 2) limb tissue was dissociated by very gentle enzymatic digest (0.1% trypsin, 5 min); and, 3) cell suspensions were plated at a lower density (1 × 10⁷ vs. 2 × 10⁷ cells/ml) in a reduced volume of 3–5 l. Under these modified conditions to increase limb cell yield from each embryo, histochemical and immunohistochemical analyses demonstrated reproducible for-mation of precartilage aggregates and subsequent overt chondrogenesis over a predictable time course. Using this culture protocol, analysis of limb mesenchyme from heterozygous hdf embryos, which bear an insertional mutation of the Cspg2 gene encoding the core protein of the chondroitin sulfate proteoglycan, versican, revealed an overall similar chondrogenic potential to that observed for wild-type littermates. This technique readily enables in vitro culture of limb bud mesenchyme from individual mouse embryos at this developmental stage and may be utilized by investigators to study the effects of the hdf and other transgenic mutations on mammalian limb development in vitro.     

Failure of centrosome migration causes a loss of motile cilia in talpid3 mutants

Background: Loss of function mutations in the centrosomal protein TALPID3 (KIAA0586) cause a failure of primary cilia formation in animal models and are associated with defective Hedgehog signalling. It is unclear, however, if TALPID3 is required only for primary cilia formation or if it is essential for all ciliogenesis, including that of motile cilia in multiciliate cells. Results: FOXJ1, a key regulator of multiciliate cell fate, is expressed in the dorsal neuroectoderm of the chicken forebrain and hindbrain at stage 20HH, in areas that will give rise to choroid plexuses in both wt and talpid³ embryos. Wt ependymal cells of the prosencephalic choroid plexuses subsequently transition from exhibiting single short cilia to multiple long motile cilia at 29HH (E8). Primary cilia and long motile cilia were only rarely observed on talpid³ ependymal cells. Electron microscopy determined that talpid³ ependymal cells do develop multiple centrosomes in accordance with FOXJ1 expression, but these fail to migrate to the apical surface of ependymal cells although axoneme formation was sometimes observed. Conclusions: TALPID3, which normally localises to the proximal centrosome, is essential for centrosomal migration prior to ciliogenesis but is not directly required for de novo centriologenesis, multiciliated fate, or axoneme formation. Developmental Dynamics 242:923–931, 2013. © 2013 Wiley Periodicals, Inc.     

Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation

Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context.     

Muscle morphogenesis in the absence of myogenic cells

Summary Experimental evidence indicates, that the myogenic cells themselves are not responsible for the muscle pattern formation. We report on a chance observation that reveals that muscle pattern formation can occur even in the absence of myogenic cells. Epiblastic cells from a quail embryo in the primitive streak stage were implanted into the wing bud of a chick embryo. The grafted quail cells developed into mononucleate, fibroblast-like cells that formed the muscle belly of the extensor medius longus muscle. This showed essentially normal form and topography as revealed by computer-aided 3D-reconstruction. This finding shows, that the formation of muscles does not depend on the presence of myogenic cells.     

The Splotch mutation interferes with muscle development in the limbs

Summary Homozygosity for the Splotch mutation causes neural tube and neural crest defects in mice. It has been demonstrated that Splotch mutant mice carry mutations in the homeodomain of the Pax-3 gene. Pax-3 is expressed in the neural tube, some neural crest derivatives, the mesenchyme of the limb bud and the somites. We have examined the development of the somite-derived skeletal muscles in homozygotes carrying the Splotch (Sp^1H) mutation. Our results suggest that the Splotch mutation affects the development of skeletal muscles in a region-specific way: 1. The expression of the CMZ transgene in homozygotes reveals a disorganisation of the dermomyotome in whole stained embryos. 2. The axial musculature is reduced in size along a rostro-caudal gradient. 3. The muscle anlagen in the limbs develop much more slowly. Muscles of the head and the ventral body wall are normally developed in the mutant on day 13.5 of gestation. Recently, it has been shown that the myogenic precursors of the limbs are derived from the lateral half of the somite. The specific disturbance of muscle development in the limbs of Splotch mutants thus suggests a role for Pax-3 in the organisation of the somite, the production of trophic factors in the limb mesenchyme or an alteration of myogenic and mesenchymal cells.     

Experimental analysis of blood vessel development in the avian wing bud

Shortly after its appearance, the avian limb bud becomes populated by a rich plexus of vascular channels. Formation of this plexus occurs by angiogenesis, specifically the ingrowth of branches from the dorsal aorta or cardinal veins, and by differentiation of endogenous angioblasts within limb mesoderm. However, mesenchyme located immediately beneath the surface ectoderm of the limb is devoid of patent blood vessels. The objective of this research is to ascertain whether peripheral limb mesoderm lacks angioblasts at all stages or becomes avascular secondarily during limb development. Grafts of core or peripheral wing mesoderm, identified by the presence or absence of patent channels following systemic infusion with ink, were grafted from quail embryos at stages 16–26 into the head region of chick embryos at stage 9–10. Hosts were fixed 3–5 days later and sections treated with antibodies that recognize quail endothelial cells and their precursors. Labeled endothelial cells were found intercalated into normal craniofacial blood vessels both nearby and distant from the site of implantation following grafting of limb core mesoderm from any stage. Identical results were obtained following grafting of limb peripheral mesoderm at stages 16–21. However, peripheral mesoderm from donors older than stage 22 did not contain endothelial precursors. Thus at the onset of appendicular development angioblasts are present throughout the mesoderm of the limb bud. During the fourth day of incubation, these cells are lost from peripheral mesoderm, either through emigration or degeneration.     

Experimental analysis of blood vessel development in the avian wing bud.

Shortly after its appearance, the avian limb bud becomes populated by a rich plexus of vascular channels. Formation of this plexus occurs by angiogenesis, specifically the ingrowth of branches from the dorsal aorta or cardinal veins, and by differentiation of endogenous angioblasts within limb mesoderm. However, mesenchyme located immediately beneath the surface ectoderm of the limb is devoid of patent blood vessels. The objective of this research is to ascertain whether peripheral limb mesoderm lacks angioblasts at all stages or becomes avascular secondarily during limb development. Grafts of core or peripheral wing mesoderm, identified by the presence or absence of patent channels following systemic infusion with ink, were grafted from quail embryos at stages 16-26 into the head region of chick embryos at stages 9-10. Hosts were fixed 3-5 days later and sections treated with antibodies that recognize quail endothelial cells and their precursors. Labeled endothelial cells were found intercalated into normal craniofacial blood vessels both nearby and distant from the site of implantation following grafting of limb core mesoderm from any stage. Identical results were obtained following grafting of limb peripheral mesoderm at stages 16-21. However, peripheral mesoderm from donors older than stage 22 did not contain endothelial precursors. Thus at the onset of appendicular development angioblasts are present throughout the mesoderm of the limb bud. During the fourth day of incubation, these cells are lost from peripheral mesoderm, either through emigration or degeneration.     

Basal lamina molecules are concentrated in myogenic regions of the mouse limb bud

 Molecular components of basal lamina, such as laminin, stimulate the differentiation of skeletal muscle cells in culture, while interstitial matrix components such as fibronectin are inhibitory. However, the role of extracellular matrix (ECM) molecules in muscle cell differentiation in the embryo is less well understood. As a first step toward understanding the role of the ECM in embryonic myogenesis, the localization of basal lamina molecules in the mouse limb bud before and during muscle cell differentiation was determined by immunofluorescence. Laminin, collagen type IV and nidogen (entactin) were concentrated in myogenic regions of the limb bud both before and during differentiation of skeletal muscle cells. Punctate immunofluorescence for basal lamina molecules was concentrated in dorsal and ventral premuscle and muscle masses, when compared with other regions of limb mesenchyme. In contrast, immunofluorescence for fibronectin, an interstitial extracellular matrix molecule, was decreased in premuscle and muscle masses. These results suggest that basal lamina components play an important stimulatory role in early stages of skeletal muscle differentiation in the developing mouse limb bud. Accepted: 7 May 1998     

Eya‐six are necessary for survival of nephrogenic cord progenitors and inducing nephric duct development before ureteric bud formation

Background: Specification of the metanephric mesenchyme is a central step of kidney development as this mesenchyme promotes nephric duct induction to form a ureteric bud near its caudal end. Before ureteric bud formation, the caudal nephric duct swells to form a pseudostratified epithelial domain that later emerges as the tip of the bud. However, the signals that promote the formation of the transient epithelial domain remain unclear. Here, we investigated the early roles of the mesenchymal factor Six family and its cofactor Eya on the initial induction of nephric duct development. Results: The nephrogenic progenitor population is initially present but significantly reduced in mice lacking both Six1 and Six4 and undertakes an abnormal cell death pathway to be completely eliminated by ～E10.5–E11.0, similar to that observed in Eya1^‐/‐ embryos. Consequently, the nephric duct fails to be induced to undergo normal proliferation to pseudostratify and form the ureteric bud in Six1^‐/‐;Six4^‐/‐ or Eya1^‐/‐ embryos. Conclusions: Our data support a model where Eya‐Six may form a complex to regulate nephron progenitor cell development before metanephric specification and are critical mesenchymal factors for inducing nephric duct development. Developmental Dynamics 244:866–873, 2015. © 2015 Wiley Periodicals, Inc.     

Characteristics of initiation and early events for muscle development in the Xenopus limb bud.

In Xenopus laevis, limb buds start to develop at a later point of the larval stage, prior to metamorphosis. This onset of limb development in Xenopus is totally different from that in amniotes such as birds and mammals, in which limb buds emerge at an early stage of embryogenesis, in parallel with other organogenesis. We investigated limb myogenesis in Xenopus, focusing on myogenic gene expression, myogenic ability of limb bud cells in the early stage, and the origin of myogenic precursor cells in the limb bud. The Xenopus early limb bud contains myoD/cardiac actin-positive and pax3/pax7-negative cells. Interestingly, results of transplantation experiments have revealed that this early limb bud contains myogenic precursor cells. In order to know the contribution of myogenic cells in somites to myogenic precursor cells in the early limb bud, we used a Cre-LoxP system for tracing over a long period. The results of fate tracing for myogenic cells in somites of the Xenopus embryo suggested that early-specified myogenic cells in somites do not contribute to limb muscle in Xenopus. Taken together, the results suggest that limb muscle development in Xenopus has characteristics of initiation and early events distinct from those of other vertebrate clades.     

Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for β-catenin

Vertebrate muscle arises sequentially from embryonic, fetal, and adult myoblasts. Although functionally distinct, it is unclear whether these myoblast classes develop from common or different progenitors. Pax3 and Pax7 are expressed by somitic myogenic progenitors and are critical myogenic determinants. To test the developmental origin of embryonic and fetal myogenic cells in the limb, we genetically labeled and ablated Pax3⁺ and Pax7⁺ cells. Pax3⁺Pax7⁻ cells contribute to muscle and endothelium, establish and are required for embryonic myogenesis, and give rise to Pax7⁺ cells. Subsequently, Pax7⁺ cells give rise to and are required for fetal myogenesis. Thus, Pax3⁺ and Pax7⁺ cells contribute differentially to embryonic and fetal limb myogenesis. To investigate whether embryonic and fetal limb myogenic cells have different genetic requirements we conditionally inactivated or activated β-catenin, an important regulator of myogenesis, in Pax3- or Pax7-derived cells. β-Catenin is necessary within the somite for dermomyotome and myotome formation and delamination of limb myogenic progenitors. In the limb, β-catenin is not required for embryonic myoblast specification or myofiber differentiation but is critical for determining fetal progenitor number and myofiber number and type. Together, these studies demonstrate that limb embryonic and fetal myogenic cells develop from distinct, but related progenitors and have different cell-autonomous requirements for β-catenin.