Spatiotemporal distribution analyses of Wnt5a ligand and its receptors Ror2, Frizzled2, and Frizzled5 during tongue muscle development in prenatal mice.

BACKGROUND: The mammalian tongue is a highly specialized muscular organ. The Wnt5a ligand regulates muscle development by mediating the activation of several noncanonical Wnt signaling pathways in a receptor context-dependent fashion. However, there is poor information on the expression and behavior of Wnt5a proteins during muscle development of the embryonic tongue. METHODS: The spatiotemporal distribution profiles of the Wnt5a ligand and its receptors, receptor tyrosine kinase-like orphan receptor 2 (Ror2), Frizzled2 (Fzd2), and Frizzled5 (Fzd5), in the developing tongue muscles of prenatal mice from embryonic day 12.5-18.5 were analyzed using immunofluorescence (IF) double staining of a target protein and desmin, a marker protein of myogenic cells. Immunolabeling images were subjected to digital detection analysis using the WinROOF 2018 version 4.19.0 image processing software when needed. RESULTS: IF signals of the Wnt5a ligand protein and its receptors Ror2 and Fzd2 were detected in developing myoblasts and myotubes of the embryonic tongue, but they were undetectable in mature myofibers equipped with sarcomere structures. Fzd2 expression was specific for desmin-positive developing muscle cells, whereas those of Ror2 and the Wnt5a ligand were widespread and nonselective for desmin-positive cells and that of Fzd5 was predominant in desmin-negative cells of the epithelium and subepithelial mesenchyme. CONCLUSION: Developing muscle cells but not mature myofibers of the mouse embryonic tongue express the Wnt5a ligand and its receptors Ror2 and Fzd2, which may mediate Wnt5a signaling in the development processes of tongue muscle fibers.

Tbx15/18/22 shares a binding site with Tbx6-r.b to maintain expression of a muscle structural gene in ascidian late embryos.

The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c, has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis-regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf, which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c, and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b.

Inner Ear and Muscle Developmental Defects in Smpx-Deficient Zebrafish Embryos.

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.

Marf-mediated mitochondrial fusion is imperative for the development and functioning of indirect flight muscles (IFMs) in drosophila.

Dynamic changes in mitochondrial shape and size are vital for mitochondrial health and for tissue development and function. Adult Drosophila indirect flight muscles contain densely packed mitochondria. We show here that mitochondrial fusion is critical during early muscle development (in pupa) and that silencing of the outer mitochondrial membrane fusion gene, Marf, in muscles results in smaller mitochondria that are functionally defective. This leads to abnormal muscle development resulting in muscle dysfunction in adult flies. However, post-developmental silencing of Marf has no obvious effects on mitochondrial and muscle phenotype in adult flies, indicating the importance of mitochondrial fusion during early muscle development.

A postlarval instar of Phoxichilidium femoratum (Pycnogonida, Phoxichilidiidae) with an exceptional malformation.

Individuals of the marine chelicerate lineage Pycnogonida (sea spiders) show considerable regenerative capabilities after appendage injury or loss. In their natural habitats, especially the long legs of sea spiders are commonly lost and regenerated, as is evidenced by the frequent encounter of specimens with missing or miniature legs. In contrast to this, the collection of individuals with abnormally developed appendages or trunk regions is comparably rare. Here, we studied a remarkable malformation in a postlarval instar of the species Phoxichilidium femoratum (Rathke, 1799) and describe the external morphology and internal organization of the specimen using a combination of fluorescent histochemistry and scanning electron microscopy. The individual completely lacks the last trunk segment with leg pair 4 and the normally penultimate trunk segment bears only a single aberrant appendage resembling an extension of the anteroposterior body axis. Externally, the proximal units of the articulated appendage are unpaired, but further distally a bifurcation into two equally developed leg-like branches is found. Three-dimensional reconstruction of the musculature reveals components of two regular leg muscle sets in several of the proximal articles. This confirms interpretation of the entire appendage as a malformed leg and reveals an externally hidden paired organization along its entire proximodistal axis. To explain the origin of this unique malformation, early pioneering studies on the regenerative potential of pycnogonids are evaluated and (a) an injury-induced partial fusion of the developing limb buds of leg pair 3, as well as (b) irregular leg regeneration following near complete loss of trunk segments 3 and 4 are discussed. Which of the two hypotheses is more realistic remains to be tested by dedicated experimental approaches. These will have to rely on pycnogonid species with established laboratory husbandry in order to overcome the limitations of the few short-term regeneration studies performed to date.

A serpin is required for ectomesoderm, a hallmark of spiralian development.

Among animals, diploblasts contain two germ layers, endoderm and ectoderm, while triploblasts have a distinct third germ layer called the mesoderm. Spiralians are a group of triploblast animals that have highly conserved development: they share the distinctive spiralian cleavage pattern as well as a unique source of mesoderm, the ectomesoderm. This population of mesoderm is distinct from endomesoderm and is considered a hallmark of spiralian development, but the regulatory network that drives its development is unknown. Here we identified ectomesoderm-specific genes in the mollusc Tritia (aka Ilyanassa) obsoleta through differential gene expression analyses comparing control and ectomesoderm-ablated embryos, followed by in situ hybridization of identified transcripts. We identified a Tritia serpin gene (ToSerpin1) that appears to be specifically expressed in the ectomesoderm of the posterior and head. Ablation of the 3a and 3b cells, which make most of the ectomesoderm, abolishes ToSerpin1 expression, consistent with its expression in these cells. Morpholino knockdown of ToSerpin1 causes ectomesoderm defects, most prominently in the muscle system of the larval head. This is the first gene identified that is specifically implicated in spiralian ectomesoderm development.

Lineage-Resolved Enhancer and Promoter Usage during a Time Course of Embryogenesis.

Enhancers are essential drivers of cell states, yet the relationship between accessibility, regulatory activity, and in vivo lineage commitment during embryogenesis remains poorly understood. Here, we measure chromatin accessibility in isolated neural and mesodermal lineages across a time course of Drosophila embryogenesis. Promoters, including tissue-specific genes, are often constitutively open, even in contexts where the gene is not expressed. In contrast, the majority of distal elements have dynamic, tissue-specific accessibility. Enhancer priming appears rarely within a lineage, perhaps reflecting the speed of Drosophila embryogenesis. However, many tissue-specific enhancers are accessible in other lineages early on and become progressively closed as embryogenesis proceeds. We demonstrate the usefulness of this tissue- and time-resolved resource to definitively identify single-cell clusters, to uncover predictive motifs, and to identify many regulators of tissue development. For one such predicted neural regulator, l(3)neo38, we generate a loss-of-function mutant and uncover an essential role for neuromuscular junction and brain development.

Deficiency and overexpression of Rtl1 in the mouse cause distinct muscle abnormalities related to Temple and Kagami-Ogata syndromes.

Temple and Kagami-Ogata syndromes are genomic imprinting diseases caused by maternal and paternal duplication of human chromosome 14, respectively. They exhibit different postnatal muscle-related symptoms as well as prenatal placental problems. Using the mouse models for these syndromes, it has been demonstrated that retrotransposon gag like 1 [Rtl1, also known as paternally expressed 11 (Peg11)] located in the mouse orthologous imprinted region is responsible for the prenatal placental problems because it is an essential placental gene for maintenance of fetal capillary network during gestation. However, the causative imprinted gene for the postnatal muscle-related symptoms remains unknown. Here, we demonstrate that Rtl1 also plays an important role in fetal/neonatal skeletal muscle development: its deletion and overproduction in mice lead to neonatal lethality associated with severe but distinct skeletal muscle defects, similar to those of Temple and Kagami-Ogata syndromes, respectively. Thus, it is strongly suggested that RTL1 is the major gene responsible for the muscle defects in addition to the placental defects in these two genomic imprinting diseases. This is the first example of an LTR retrotransposon-derived gene specific to eutherians contributing to eutherian skeletal muscle development.

Prediction of the Secretome and the Surfaceome: A Strategy to Decipher the Crosstalk between Adipose Tissue and Muscle during Fetal Growth.

Crosstalk between adipose and muscular tissues is hypothesized to regulate the number of muscular and adipose cells during fetal growth, with post-natal consequences on lean and fat masses. Such crosstalk largely remains, however, to be described. We hypothesized that a characterization of the proteomes of adipose and muscular tissues from bovine fetuses may enhance the understanding of the crosstalk between these tissues through the prediction of their secretomes and surfaceomes. Proteomic experiments have identified 751 and 514 proteins in fetal adipose tissue and muscle. These are mainly involved in the regulation of cell proliferation or differentiation, but also in pathways such as apoptosis, Wnt signalling, or cytokine-mediated signalling. Of the identified proteins, 51 adipokines, 11 myokines, and 37 adipomyokines were predicted, together with 26 adipose and 13 muscular cell surface proteins. Analysis of protein-protein interactions suggested 13 links between secreted and cell surface proteins that may contribute to the adipose-muscular crosstalk. Of these, an interaction between the adipokine plasminogen and the muscular cell surface alpha-enolase may regulate the fetal myogenesis. The in silico secretome and surfaceome analyzed herein exemplify a powerful strategy to enhance the elucidation of the crosstalk between cell types or tissues.

Polyunsaturated Fatty Acid Content in Muscle Tissue Is Associated with the Duration of Embryo Development in Salmonoid Fishes (Salmonoidei).

A hypothesis was advanced and grounded that the total content of eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3) acids in fish muscle tissue is associated with the species-specific (taxon-specific) duration of embryo development. A meta-analysis of the original and published data was performed using fishes of the families Coregonidae and Salmonidae as an example. Fishes with longer embryo development times, which are observed at lower temperatures, were found to have significantly higher EPA + DHA contents in muscles as compared with the species that belong to the same families but have shorter embryo development times. This association was explained by the fact that an embryo forms more cells per unit tissue volume at lower temperatures, which requires a greater specific amount of cell membranes and, therefore, greater amounts of EPA and DHA to produce them.

Embryonic development and secondary axis induction in the Brazilian white knee tarantula Acanthoscurria geniculata, C. L. Koch, 1841 (Araneae; Mygalomorphae; Theraphosidae).

Tarantulas represent some of the heaviest and most famous spiders. However, there is little information about the embryonic development of these spiders or their relatives (infraorder Mygalomorphae) and time-lapse recording of the embryonic development is entirely missing. I here describe the complete development of the Brazilian white knee tarantula, Acanthoscurria geniculata, in fixed and live embryos. The establishment of the blastoderm, the formation, migration and signalling of the cumulus and the shape changes that occur in the segment addition zone are analysed in detail. In addition, I show that there might be differences in the contraction process of early embryos of different theraphosid spider species. A new embryonic reference transcriptome was generated for this study and was used to clone and analyse the expression of several important developmental genes. Finally, I show that embryos of A. geniculata are amenable to tissue transplantation and bead insertion experiments. Using these functional approaches, I induced axis duplication in embryos via cumulus transplantation and ectopic activation of BMP signalling. Overall, the mygalomorph spider A. geniculata is a useful laboratory system to analyse evolutionary developmental questions, and the availability of such a system will help understanding conserved and divergent aspects of spider/chelicerate development.

Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles.

Despites several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, we provide evidence that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so-far-ignored AMP role involves their filopodia-based interactions with nerve growth cones. In parallel, we report the previously undetected expression of the guidance molecule-encoding genes sidestep and side IV in AMPs. Altogether, our data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is crucial for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent, as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs.

Development of the chick wing and leg neuromuscular systems and their plasticity in response to changes in digit numbers.

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation. During limb morphogenesis, a number of distinct tissue types are patterned and subsequently must be integrated to form coherent functional units. For example, the musculoskeletal apparatus of the limb requires the coordinated development of the skeletal elements, connective tissues, muscles and nerves. Here, using light-sheet microscopy and 3D-reconstructions, we concomitantly follow the developmental emergence of nerve and muscle patterns in chicken wings and legs, two appendages with highly specialized locomotor outputs. Despite a comparable flexor/extensor-arrangement of their embryonic muscles, wings and legs show a rotated innervation pattern for their three main motor nerve branches. To test the functional implications of these distinct neuromuscular topologies, we challenge their ability to adapt and connect to an experimentally altered skeletal pattern in the distal limb, the autopod. Our results show that, unlike autopod muscle groups, motor nerves are unable to fully adjust to a changed peripheral organisation, potentially constrained by their original projection routes. As the autopod has undergone substantial morphological diversifications over the course of tetrapod evolution, our results have implications for the coordinated modification of the distal limb musculoskeletal apparatus, as well as for our understanding of the varying degrees of motor functionality associated with human hand and foot malformations.

A systems biology approach reveals neuronal and muscle developmental defects after chronic exposure to ionising radiation in zebrafish.

Contamination of the environment after the Chernobyl and Fukushima Daiichi nuclear power plant (NPP) disasters led to the exposure of a large number of humans and wild animals to radioactive substances. However, the sub-lethal consequences induced by these absorbed radiological doses remain understudied and the long-term biological impacts largely unknown. We assessed the biological effects of chronic exposure to ionizing radiation (IR) on embryonic development by exposing zebrafish embryo from fertilization and up to 120 hours post-fertilization (hpf) at dose rates of 0.5 mGy/h, 5 mGy/h and 50 mGy/h, thereby encompassing the field of low dose rates defined at 6 mGy/h. Chronic exposure to IR altered larval behaviour in a light-dark locomotor test and affected cardiac activity at a dose rate as low as 0.5 mGy/h. The multi-omics analysis of transcriptome, proteome and transcription factor binding sites in the promoters of the deregulated genes, collectively points towards perturbations of neurogenesis, muscle development, and retinoic acid (RA) signaling after chronic exposure to IR. Whole-mount RNA in situ hybridization confirmed the impaired expression of the transcription factors her4.4 in the central nervous system and myogenin in the developing muscles of exposed embryos. At the organ level, the assessment of muscle histology by transmission electron microscopy (TEM) demonstrated myofibers disruption and altered neuromuscular junctions in exposed larvae at 5 mGy/h and 50 mGy/h. The integration of these multi-level data demonstrates that chronic exposure to low dose rates of IR has an impact on neuronal and muscle progenitor cells, that could lead to motility defects in free swimming larvae at 120 hpf. The mechanistic understanding of these effects allows us to propose a model where deregulation of RA signaling by chronic exposure to IR has pleiotropic effects on neurogenesis and muscle development.

Shaping the zebrafish myotome by intertissue friction and active stress.

Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During zebrafish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to aid swimming. Myotome shape can be altered by perturbing muscle cell differentiation or the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the future myotome and the surrounding tissues appears to spatially vary, effectively resulting in spatially heterogeneous friction. Using a vertex model combined with experimental validation, we show that the interplay of tissue spreading and friction is sufficient to drive the initial phase of chevron shape formation. However, local anisotropic stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the chevron in wild-type embryos. Finally, tissue plasticity is required for formation and maintenance of the chevron shape, which is mediated by orientated cellular rearrangements. Our work sheds light on how a spatiotemporal sequence of local cellular events can have a nonlocal and irreversible mechanical impact at the tissue scale, leading to robust organ shaping.

Matrix metalloproteinase 1 modulates invasive behavior of tracheal branches during entry into Drosophila flight muscles.

Tubular networks like the vasculature extend branches throughout animal bodies, but how developing vessels interact with and invade tissues is not well understood. We investigated the underlying mechanisms using the developing tracheal tube network of Drosophila indirect flight muscles (IFMs) as a model. Live imaging revealed that tracheal sprouts invade IFMs directionally with growth-cone-like structures at branch tips. Ramification inside IFMs proceeds until tracheal branches fill the myotube. However, individual tracheal cells occupy largely separate territories, possibly mediated by cell-cell repulsion. Matrix metalloproteinase 1 (MMP1) is required in tracheal cells for normal invasion speed and for the dynamic organization of growth-cone-like branch tips. MMP1 remodels the CollagenIV-containing matrix around branch tips, which show differential matrix composition with low CollagenIV levels, while Laminin is present along tracheal branches. Thus, tracheal-derived MMP1 sustains branch invasion by modulating the dynamic behavior of sprouting branches as well as properties of the surrounding matrix.

Drosophila Myoblast Fusion: Invasion and Resistance for the Ultimate Union.

Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, Drosophila myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell-cell fusion in vivo. Many evolutionarily conserved fusion-promoting factors have been identified and so has a surprising and conserved cellular mechanism. In this review, we revisit key findings in Drosophila myoblast fusion and highlight the critical roles of cellular invasion and resistance in driving cell membrane fusion.

Normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, with special reference to nose leaf formation.

The order Chiroptera (bats) is the second largest group of mammals, composed of more than 1,300 species. Although powered flight and echolocation in bats have attracted many biologists, diversity in bat facial morphology has been almost neglected. Some bat species have a "nose leaf," a leaf-like epithelial appendage around their nostrils. The nose leaf appears to have been acquired at least three times independently in bat evolution, and its morphology is highly diverse among bats species. Internal tissue morphology of nose-leaves has been investigated through histological analyses of late-stage fetuses of some bat species possessing the nose leaf. However, the proximate factors that bring about chiropteran nose-leaves have not been identified. As an initial step to address the question above, we describe the normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, and examine development of the tissues associated with their nose leaf during embryogenesis through histological analyses. We found that the nose leaf of R. ferrumequinum is formed through two phases. First, the primordium of the nose leaf appears as two tissue bulges aligned top and bottom on the face at embryonic stages 15-16. Second, the sub-regions of the nose leaf are differentiated through ingrowth as well as outgrowth of the epithelium at stage 17. In embryogenesis of Carollia perspicillata, a phyllostomid species with a nose leaf, the nose leaf primordium is formed as a small tissue bulge on the nostril at stage 17. This tissue bulge grows into a dorsally projected thin epithelial structure. Such differences in the nose leaf developmental process between chiropteran lineages may suggest that distinct developmental mechanisms have been employed in each lineage's nose leaf evolution.

Mother-embryo isotope fractionation in the pygmy devilray Mobula kuhlii cf. eregoodootenkee.

We determined stable-isotope ratios for replicate muscle tissues in 13 gravid Mobula kuhlii cf. eregoodootenkee (110.4-120.4 cm disc width; WD ) and their embryos (7.0-42.3 cm WD ) and also yolks and histrotroph, to assess the potential implications for juvenile nutrition and habitat use. Irrespective of their development in the uterus, embryos had similar δ13 C values in their muscle tissue as the mothers and both had greater values than in the histotroph. During gestation, δ13 C values increased across all sample types. However, while embryo muscle tissue and the histotroph were associated with increasing 15 N levels during embryonic development, this was depleted in the mothers' muscle tissue and yolk. Although speculative, the observed variation in stable-isotope ratios might imply a dietary shift among gravid females during their early gestation. Irrespective of the underlying mechanisms, the results indicate neonates will have relatively greater δ15 N values than post-partum females, which would probably confound juvenile foraging-ecology estimates.

Effects of hyperthyroidism in the development of the appendicular skeleton and muscles of zebrafish, with notes on evolutionary developmental pathology (Evo-Devo-Path).

The hypothalamus-pituitary-thyroid (HPT) axis plays a crucial role in the metabolism, homeostasis, somatic growth and development of teleostean fishes. Thyroid hormones regulate essential biological functions such as growth and development, regulation of stress, energy expenditure, tissue compound, and psychological processes. Teleost thyroid follicles produce the same thyroid hormones as in other vertebrates: thyroxin (T4) and triiodothyronine (T3), making the zebrafish a very useful model to study hypo- and hyperthyroidism in other vertebrate taxa, including humans. Here we investigate morphological changes in T3 hyperthyroid cases in the zebrafish to better understand malformations provoked by alterations of T3 levels. In particular, we describe musculoskeletal abnormalities during the development of the zebrafish appendicular skeleton and muscles, compare our observations with those recently done by us on the normal developmental of the zebrafish, and discuss these comparisons within the context of evolutionary developmental pathology (Evo-Devo-Path), including human pathologies.