Normal development in Xenopus laevis: A complementary staging table for the skull based on cartilage and bone.

BACKGROUND: Xenopus laevis is a widely used model organism in the fields of genetics and development, and more recently evolution. At present, the most widely used staging table for X. laevis is based primarily on external features and does not describe the corresponding skull development in detail. Here, we describe skull development in X. laevis, complete with labeled figures, for each relevant stage in the most widely used staging table. RESULTS: We find skull development in X. laevis is, for the most part, distinct at each of the previously established stages based on external anatomy. However, variation does exist in the timing of onset of ossification of certain bones in the skull, which results in a range of stages where a skull element first ossifies. The overall sequence of ossification is less variable than the timing of ossification onset. CONCLUSIONS: While events in skull development vary somewhat between specimens, and in comparison, to external events, this staging table is useful in showing both when bones first appear and for documenting the range of temporal variance in X. laevis skull development more accurately than previously done. Furthermore, when only skull data are available, the approximate stage of a specimen can now be determined.

The histology of sutures in chicken skulls: Types, conservation, and ontogeny.

Sutures are fibrous joints that occur between bone elements in vertebrate skulls, where they play a variety of roles including facilitating skull growth and function. In addition, a variety of studies examining sutures from diverse perspectives in many taxa have enabled the determination of anatomical homologs. Surprisingly, one important aspect of sutures-histology-remains unknown in the key model organism of the chicken. To fill this gap in our knowledge, we generated histological sections of six different cranial sutures across a range of developmental stages in embryonic chicken. Despite having a skull that is largely co-ossified or fused as an adult, we found that the types, components, and ontogeny of sutures in chicken skulls are very similar to sutures in other vertebrates. We did, however, find a new transient stage in the ontogeny of sutures between endochondral bone elements, in which one element has ossified and one was still cartilaginous. Moreover, to better understand the morphogenetic events at the onset of suture formation, we compared the developmental histology of six sutures with that of the space between the two ossification centers of the frontal-a location expected to be void of suture structures. We found that the mesenchymal cells in sutures condense and form a middle vascular layer. This was not found to be the case in the space between the two ossifications of the frontal, where instead only osteoid occurs.

Development of the chicken skull: A complement to the external staging table of Hamburger and Hamilton.

Embryonic staging tables provide a standard of developmental stages that can be used by individual investigators and provide approximate time points for the study of developmental phenomena. Surprisingly, despite the presence of a plethora of studies on the chicken skull and its role as a model species in developmental research, a staging table of the development of the chicken skull remains lacking. A detailed photographic staging table of the osseous portion of the chicken skull is thus presented here based on cleared and stained HH stages spanning HH 35 (first appearance of skull ossification) to the final stage before hatching (HH 45). This table documents the development of most of the cranial elements in the skull from the start of ossification until the element takes its final shape. The table shows that the elements of the lower jaw and ventral side of the skull begin ossifying before the skull roof and that most elements take roughly 5 days to reach their final shape, whereas others take up to 9 days (e.g., the frontal). The obtained results lead to several hypotheses about chicken skull development and provide a timeframe for future studies on chicken skull development.

Carm1 regulates Pax7 transcriptional activity through MLL1/2 recruitment during asymmetric satellite stem cell divisions.

In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.

Muscle satellite cell and atypical myogenic progenitor response following exercise.

Skeletal muscle satellite cells play an essential role in muscle regeneration and exercise adaptation. In recent years atypical myogenic progenitors (non-satellite-cell muscle stem cells) have been identified in skeletal muscle and have been hypothesized to play an important role in the process of muscle regeneration. It remains unknown, however, whether any populations other than satellite cells play a significant role in repair and adaptation following exercise-induced damage. We assessed the response of the satellite cell population and the CD45+:Sca-1+ cell population, previously shown to support muscle regeneration following cardiotoxin-induced injury, after acute eccentrically biased exercise in wild-type mice. We observed evidence of focal muscle damage and repair following the exercise protocol using electron microscopy, hematoxylin-eosin staining, and single-fiber analysis. In addition, we observed an approximately sixfold increase in the number of Myf5-expressing cells by 48 h, which remained elevated until at least 96 h following exercise. We did not, however, observe any significant expansion of the CD45+:Sca-1+ cell population or commitment of resident CD45+:Sca-1+ cells to the myogenic lineage. Furthermore, expression of Wnt gene family members, previously associated with myogenic specification of CD45+:Sca-1+ cells, did not differ following exercise. Therefore, we conclude that muscle satellite cells are the primary responders to exercise-induced stress and that the CD45+:Sca-1+ myogenic progenitors do not contribute to muscle repair/adaptation following exercise.

Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex.

Satellite cells purified from adult skeletal muscle can participate extensively in muscle regeneration and can also re-populate the satellite cell pool, suggesting that they have direct therapeutic potential for treating degenerative muscle diseases. The paired-box transcription factor Pax7 is required for satellite cells to generate committed myogenic progenitors. In this study we undertook a multi-level approach to define the role of Pax7 in satellite cell function. Using comparative microarray analysis, we identified several novel and strongly regulated targets; in particular, we identified Myf5 as a gene whose expression was regulated by Pax7. Using siRNA, fluorescence-activated cell sorting (FACS) and chromatin immunoprecipitation (ChIP) studies we confirmed that Myf5 is directly regulated by Pax7 in myoblasts derived from satellite cells. Tandem affinity purification (TAP) and mass spectrometry were used to purify Pax7 together with its co-factors. This revealed that Pax7 associates with the Wdr5-Ash2L-MLL2 histone methyltransferase (HMT) complex that directs methylation of histone H3 lysine 4 (H3K4, refs 4-10). Binding of the Pax7-HMT complex to Myf5 resulted in H3K4 tri-methylation of surrounding chromatin. Thus, Pax7 induces chromatin modifications that stimulate transcriptional activation of target genes to regulate entry into the myogenic developmental programme.

Muscle stem cells and regenerative myogenesis.

A population of myogenic progenitors termed satellite cells undertakes postnatal development and repair of skeletal muscle. Studies have indicated that atypical myogenic precursors can also participate in muscle regeneration. The source of this regenerative capacity has been attributed to "adult stem cells" that represent poorly understood multipotent cell lineages, believed to reside in all adult tissue populations. Here we review the origin and location of muscle satellite cells and stem cells, as well as the mechanisms by which they may be specified. We discuss how the experimental models utilized raise important questions regarding the validity of extrapolating these findings.

Molecular mechanisms of muscle atrophy.

Skeletal muscle atrophy has extreme adverse consequences. Molecular mechanisms that mediate the process of atrophy are not well defined. Recent studies have focused on diverse molecular cascades that control the activation of ubiquitin ligases, indicating that the involvement of the ubiquitin proteasome may be common to a range of atrophic stimuli.

Sonic Hedgehog functions by localizing the region of proliferation in early developing feather buds.

Feathers are formed following a series of reciprocal signals between the epithelium and the mesenchyme. Initially, the formation of a dense dermis leads to the induction of a placode in the overlying ectoderm. The ectoderm subsequently signals back to the dermis to promote cell division. Sonic Hedgehog (Shh) is a secreted protein expressed in the ectoderm that has previously been implicated in mitogenic and morphogenetic processes throughout feather bud development. We therefore interfered with Shh signaling during early feather bud development and observed a dramatic change in feather form and prominence. Surprisingly, outgrowth did occur and was manifest as irregular, fused, and ectopic feather domains at both molecular and morphological levels. Experiments with Di-I and BrdU indicated that this effect was at least in part caused by the dispersal of previously aggregated proliferating dermal cells. We propose that Shh maintains bud development by localizing the dermal feather progenitors.

EphA4, RhoB and the molecular development of feather buds are maintained by the integrity of the actin cytoskeleton.

The development of feather buds is a highly ordered process involving epithelial-mesenchymal signalling. Cellular morphology is determined by the actin cytoskeleton, which is controlled by networks of regulators such as the GTPases. EphA4 belongs to a receptor tyrosine kinase family that has been consistently shown to regulate the cytoskeleton via Rho family GTPases in neural development and is expressed in early stages of feather bud development though its role has not been defined. We therefore used an in vitro skin culture system to interfere with EphA4 levels in feather buds using anti-sense oligonucleotides, demonstrating a severe effect on both their number and morphological form. Analysis of the Rho family of GTPases revealed that this effect was mediated by the GTPase RhoB, the expression of which was altered in response to altered levels of EphA4. In addition, the inhibition of RhoB mimicked the effects of reduced EphA4 levels on feather development. Significantly, manipulation of cytoskeletal dynamics revealed that those cells undergoing morphogenetic change regulate the patterning signals responsible for initiating feather development. We propose that this molecular maintenance mechanism between EphA4-RhoB and the actin cytoskeleton converges or coordinates with other morphogenic signalling systems to control feather bud development.

Decamethonium bromide-mediated inhibition of embryonic muscle development.

This study determined the effect of decamethonium bromide (DMBr), a non-competitive blocker of the neuromuscular junction, on skeletal muscle development during chick embryogenesis. Decamethonium bromide caused generalized edema and high mortality with treated embryos rarely surviving beyond day 16 of incubation. Muscle degeneration was grossly evident on the muscles of abdomen, pectoral girdle, and leg. Semi-thin sections showed a high infiltration of macrophages in treated embryos and a massive degenerative process. Electron microscopy showed that both fast and slow fibers formed in the control and treated embryos, but those of the treated embryos failed to form myofibrils. Other organ systems, such as the heart and the gut, appeared histologically normal throughout the course of treatment. To investigate possible nerve independent action of DMBr on muscle development we determined the effect of this compound on the growth and differentiation of the C2C12 skeletal muscle cell line. DMBr treatment of C2C12 cell cultures did not affect the growth or survival of the cells, even at a tenfold higher concentration than that used in ovo, but myosin heavy chain expression was dramatically inhibited. We conclude that DMBr has a nerve independent blocking inhibition effect on myosin heavy chain synthesis in the developing avian embryo besides the recognized role as a non-competitive post-synaptic blocker of the neuromuscular junction.

Heparan sulfate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma.

RPTPsigma is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTPsigma or CRYPalpha, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTPsigma are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTPsigma is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTPsigma in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTPsigma. HSPGs are therefore a novel class of heterotypic ligand for cPTPsigma, suggesting that cPTPsigma signaling in axons and growth cones is directly responsive to matrix-associated cues.