Roles for GFRalpha1 receptors in zebrafish enteric nervous system development

Components of the zebrafish GDNF receptor complex are expressed very early in the development of enteric nervous system precursors, and are already present as these cells begin to enter the gut and migrate caudally along its length. Both gfra1a and gfra1b as well as ret are expressed at this time, while gfra2 expression, the receptor component that binds the GDNF-related ligand neurturin, is not detected until the precursors have migrated along the gut. Gfra genes are also expressed in regions of the zebrafish brain and peripheral ganglia, expression domains conserved with other species. Enteric neurons are eliminated after injection with antisense morpholino oligonucleotides against ret or against both Gfra1 orthologs, but are not affected by antisense oligonucleotides against gfra2. Blocking GDNF signaling prevents migration of enteric neuron precursors, which remain positioned at the anterior end of the gut. Phenotypes induced by injection of antisense morpholinos against both Gfra orthologs can be rescued by introduction of mRNA for gfra1a or for gfra2, suggesting that GFRalpha1 and GFRalpha2 are functionally equivalent.     

Mandibular arch muscle identity is regulated by a conserved molecular process during vertebrate development

Vertebrate head muscles exhibit a highly conserved pattern of innervation and skeletal connectivity and yet it is unclear whether the molecular basis of their development is likewise conserved. Using the highly conserved expression of Engrailed 2 (En2) as a marker of identity in the dorsal mandibular muscles of zebrafish, we have investigated the molecular signals and tissues required for patterning these muscles. We show that muscle En2 expression is not dependent on signals from the adjacent neural tube, pharyngeal endoderm or axial mesoderm and that early identity of head muscles does not require bone morphogenetic pathway, Notch or Hedgehog (Hh) signalling. However, constrictor dorsalis En2 expression is completely lost after a loss of fibroblast growth factor (Fgf) signalling and we show that is true throughout head muscle development. These results suggest that head muscle identity is dependent on Fgf signalling. Data from experiments performed in chick suggest a similar regulation of En2 genes by Fgf signalling revealing a conserved mechanism for specifying head muscle identity. We present evidence that another key gene important in the development of mouse head muscles, Tbx1, is also critical for specification of mandibular arch muscle identity and that this is independent of Fgf signalling. These data imply that dorsal mandibular arch muscle identity in fish, chick and mouse is specified by a highly conserved molecular process despite differing functions of these muscles in different lineages.     

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss‐of‐function disorders such as Hirschsprung disease.     

Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion

Myoblast fusion is a highly regulated process that is key for forming skeletal muscle during development and regeneration in mammals. Much remains to be understood about the molecular regulation of myoblast fusion. Some molecules that influence mammalian muscle fusion display specific cellular localization during myogenesis. Such molecules can be localized to the contact region between two fusing cells either in both cells or only in one of the cells. How distinct localization of molecules contributes to fusion is not clear. Further complexity exists as other molecules are functionally restricted to myoblasts at later stages of myogenesis to regulate their fusion with multinucleated myotubes. This review examines these three categories of molecules and discusses how spatial and functional restriction may contribute to the formation of a multinucleated cell. Understanding how and why molecules become restricted in location or function is likely to provide further insights into the mechanisms regulating mammalian muscle fusion.     

Src-dependent autophagic degradation of Ret in FAK-signalling-defective cancer cells

We have recently described that autophagic targeting of Src maintains cancer cell viability when FAK signalling is defective. Here, we show that the Ret tyrosine kinase is also degraded by autophagy in cancer cells with altered/reduced FAK signalling, preventing its binding to FAK at integrin adhesions. Inhibition of autophagy restores Ret localization to focal adhesions. Importantly, Src kinase activity is required to target Ret to autophagosomes and enhance Ret degradation. Src is thus a general mediator of selective autophagic targeting of adhesion-linked kinases, and Ret a second FAK-binding tyrosine kinase degraded through autophagy in cancer cells under adhesion stress. Src-by controlling not only its own degradation but also that of other FAK-binding partners-allows cancer cell survival, suggesting a new therapeutic strategy.     

The Caenorhabditis elegans presenilin sel-12 is required for mesodermal patterning and muscle function

Mutations in presenilin genes impair Notch signalling and, in humans, have been implicated in the development of familial Alzheimer's disease. We show here that a reduction of the activity of the Caenorhabditis elegans presenilin sel-12 results in a late defect during sex muscle development. The morphological abnormalities and functional deficits in the sex muscles contribute to the egg-laying defects seen in sel-12 hermaphrodites and to the severely reduced mating efficiency of sel-12 males. Both defects can be rescued by expressing sel-12 from the hlh-8 promoter that is active during the development of the sex muscle-specific M lineage, but not by expressing sel-12 from late muscle-specific promoters. Both weak and strong sel-12 mutations cause defects in the sex muscles that resemble the defects we found in lin-12 hypomorphic alleles, suggesting a previously uncharacterised LIN-12 signalling event late in postembryonic mesoderm development. Together with a previous study indicating a role of lin-12 and sel-12 during the specification of the pi cell lineage required for proper vulva-uterine connection, our data suggest that the failure of sel-12 animals to lay eggs properly is caused by defects in at least two independent signalling events in different tissues during development.     

Homeotic gene function in the muscles of Drosophila larvae

The segmental musculature of Drosophila melanogaster larvae consists of 24-30 muscles per segment. Unique patterns of muscles are found in the three thoracic segments and the first and last abdominal segments; the remaining abdominal segments share the same pattern. Mutations in Ultrabithorax (Ubx) cause partial transformation of the muscle pattern of larval abdominal segments towards metathorax. The muscles of the thorax are not affected. In the first two abdominal segments the changes include the loss of at least 11 `abdominal' muscles and the gain of 11 `thoracic' muscles. Less extensive transformations are seen in more posterior abdominal segments. Anterobithorax, bithorax, postbithorax and bithoraxoid mutations also induce transformations of the larval musculature. Each allelic group affects a domain that is a subset of the entire Ubx domain but these domains are not restricted to compartments or segments and may extend through as many as five segments. In the muscles the segmental distribution of Ubx antigen correlates with the segments affected by Ubx mutations. The different domains of Ubx in mesoderm and ectoderm argue that the segmental diversity of the muscle pattern is not simply induced by the overlying epidermis and that Ubx function in the mesoderm is required for the correct development of abdominal segments.     

Skeletal muscle leucine incorporation and testosterone uptake in exercised guinea pigs.

We examined the changes induced by daily treadmill exercise on body weights, plantaris muscle weights, plantaris protein concentrations, and L-leucine-4,5-3H incorporation into plantaris muscles of normal and castrated young male guinea pigs and of castrated animals receiving testosterone replacement therapy, and compared the testosterone-1,2-3H uptake by plantaris muscles of trained normal guinea pigs to that of untrained animals. Trained animals exhibited significantly lower body and muscle weights and greater labeled leucine incorporation into sarcoplasmic and myofibrillar proteins but did not show significant changes in protein concentrations or labeled testosterone uptake. The level of physical activity of the young animals studied appeared to be more important than gonadal endocrine function in altering protein metabolism and muscle and body weights. Because hypertrophy did not occur in the trained plantaris muscles, which had elevated rates of labeled leucine incorporation, it appears that the trained animals had a higher muscle protein turnover rate. It seems unlikely that testosterone plays an important role in these activity-related phenomena.     

A Neuro-Mechanical Model of a Single Leg Joint Highlighting the Basic Physiological Role of Fast and Slow Muscle Fibres of an Insect Muscle System

In legged animals, the muscle system has a dual function: to produce forces and torques necessary to move the limbs in a systematic way, and to maintain the body in a static position. These two functions are performed by the contribution of specialized motor units, i.e. motoneurons driving sets of specialized muscle fibres. With reference to their overall contraction and metabolic properties they are called fast and slow muscle fibres and can be found ubiquitously in skeletal muscles. Both fibre types are active during stepping, but only the slow ones maintain the posture of the body. From these findings, the general hypothesis on a functional segregation between both fibre types and their neuronal control has arisen. Earlier muscle models did not fully take this aspect into account. They either focused on certain aspects of muscular function or were developed to describe specific behaviours only. By contrast, our neuro-mechanical model is more general as it allows functionally to differentiate between static and dynamic aspects of movement control. It does so by including both muscle fibre types and separate motoneuron drives. Our model helps to gain a deeper insight into how the nervous system might combine neuronal control of locomotion and posture. It predicts that (1) positioning the leg at a specific retraction angle in steady state is most likely due to the extent of recruitment of slow muscle fibres and not to the force developed in the individual fibres of the antagonistic muscles; (2) the fast muscle fibres of antagonistic muscles contract alternately during stepping, while co-contraction of the slow muscle fibres takes place during steady state; (3) there are several possible ways of transition between movement and steady state of the leg achieved by varying the time course of recruitment of the fibres in the participating muscles.     

A critical period for formation of secondary myotubes defined by prenatal undernourishment in rats

Rats fed a restricted diet during gestation and lactation gave birth to pups with about 60% the normal birthweight. Maintaining the undernutrition after birth reduced the rate of growth of the pups so that their body weights were only 40% of control at PN7. Soleus and lumbrical muscles in these animals had reduced numbers of muscle fibres, and quantitative examination of embryonic muscles revealed that this was due solely to a decreased formation of secondary myotubes; the number of primary myotubes remained normal. Undernutrition did not affect the number of motoneurones surviving normal developmental death. Restoration of normal dietary intake on E21, one day before birth, did not correct the deficit in muscle fibre numbers in soleus muscles examined when the animals reached one month of age. Development of the lumbrical muscle lags behind the soleus and unrestricted feeding from E21 onwards allowed a normal number of fibres to develop from this time on, although the initial deficit was never restored. These experiments define a critical period in muscle development during which the potential maximum number of secondary myotubes is determined.     

beta-thymosin is required for axonal tract formation in developing zebrafish brain

beta-Thymosins are polypeptides that bind monomeric actin and thereby function as actin buffers in many cells. We show that during zebrafish development, &bgr;-thymosin expression is tightly correlated with neuronal growth and differentiation. It is transiently expressed in a subset of axon-extending neurons, essentially primary neurons that extend long axons, glia and muscle. Non-neuronal expression in the brain is restricted to a subset of glia surrounding newly forming axonal tracts. Skeletal muscle cells in somites, jaw and fin express beta-thymosin during differentiation, coinciding with the time of innervation. Injection of beta-thymosin antisense RNA into zebrafish embryos results in brain defects and impairment of the development of beta-thymosin-associated axon tracts. Furthermore, irregularities in somite formation can be seen in a subset of embryos. Compared to wild-type, antisense-injected embryos show slightly weaker and more diffuse engrailed staining at the midbrain-hindbrain boundary and a strong reduction of Isl-1 labeling in Rohon Beard and trigeminal neurons. The decreased expression is not based on a loss of neurons indicating that beta-thymosin may be involved in the maintenance of the expression of molecules necessary for neuronal differentiation. Taken together, our results strongly indicate that beta-thymosin is an important regulator of development.     

Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development

Background

In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/Principal Findings

Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1^−/− mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/Significance

This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.     

Notch Signalling Is Required for the Formation of Structurally Stable Muscle Fibres in Zebrafish

Background

Accurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown.

Methodology/Principal Findings

Here we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis.

Conclusions/Significance

We propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin.     

Ret isoform function and marker gene expression in the enteric nervous system is conserved across diverse vertebrate species

The enteric nervous system (ENS) derives from migratory neural crest cells that colonize the developing gut tube, giving rise to an integrated network of neurons and glial cells, which together regulate important aspects of gut function, including coordinating the smooth muscle contractions of the gut wall. The absence of enteric neurons in portions of the gut (aganglionosis) is the defining feature of Hirschsprung’s disease (HSCR) and has been replicated in a number of mouse models. Mutations in the RET tyrosine kinase account for over half of familial cases of HSCR and mice mutant for Ret exhibit aganglionosis. RET exists in two main isoforms, RET9 and RET51 and studies in mouse have shown that RET9 is sufficient to allow normal development of the ENS. In the last several years, zebrafish has emerged as a model of vertebrate ENS development, having been supported by a number of demonstrations of conservation of gene function between zebrafish, mouse and human. In this study we further analyse the potential similarities and differences between ENS development in zebrafish, mouse and human. We demonstrate that zebrafish Ret is required in a dose-dependent manner to regulate colonization of the gut by neural crest derivatives, as in human. Additionally, we show that as in mouse and human, zebrafish ret is produced as two isoforms, ret9 and ret51. Moreover, we show that, as in mouse, the Ret9 isoform is sufficient to support colonization of the gut by enteric neurons. Finally, we identify zebrafish orthologues of genes previously identified to be expressed in the mouse ENS and demonstrate that these genes are expressed in the developing zebrafish ENS, thereby identifying useful ENS markers in this model organism. These studies reveal that the similarities between gene expression and gene function across vertebrate species is more extensive than previously appreciated, thus supporting the use of zebrafish as a general model for vertebrate ENS development and the use of zebrafish genetic screens as a way to identify candidate genes mutated in HSCR cases.