The development of thoracic and abdominal muscle depends on SDF1 and CXCR4.

In vertebrates, the lateral body wall muscle formation is thought to be initiated by direct outgrowth of the dermomyotomes resulting in the elongation of the hypaxial myotomes. This contrasts with the formation of the muscles of the girdle, limbs and intrinsic tongue muscles, which originate from long-range migrating progenitors. Previous work shows that the migration of these progenitors requires CXCR4 which is specifically expressed in the migrating cells, but not in the dermomyotome. Here, we show that cells in the ventrolateral-lip (VLL) of the dermomyotome at the flank level express CXCR4 in a pattern consistent with that of Pax3 and MyoR. In ovo gain-of-function experiments using electroporation of SDF-1 constructs into the VLL resulted in increased expression of c-Met, Pax3 and MyoD. In contrast, a loss-of-function approach by implantation of CXCR4-inhibitor beads into the VLL of the flank region caused a reduction in the expression of these markers. These data show that CXCR4 is expressed in the VLL, and by experimentally manipulating the CXCR4/SDF-1 signaling, we demonstrate the importance of this axis in body wall muscle development.

How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells.

Cell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18-HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

Chicken Second Branchial Arch Progenitor Cells Contribute to Heart Musculature in vitro and in vivo.

In the past, the heart muscle was thought to originate from a single source of myocardial progenitor cells. More recently, however, an additional source of myocardial progenitors has been revealed to be the second heart field, and chicken embryos were important for establishing this concept. However, there have been few studies in chicken on how this field contributes to heart muscles in vitro. We have developed an ex vivo experimental system from chicken embryos between stages HH17-20 to investigate how mesodermal progenitors in the second branchial arch (BA2) differentiate into cardiac muscles. Using this method, we presented evidence that the progenitor cells within the BA2 arch differentiated into beating cardiomyocytes in vitro. The beating explant cells were positive for cardiac actin, Nkx2.5, and ventricular myosin heavy chain. In addition, we performed a time course for the expression of second heart field markers (Isl1 and Nkx2.5) in the BA2 from stage HH16 to stage HH21 using in situ hybridization. Accordingly, using EGFP-based cell labeling techniques and quail-chicken cell injection, we demonstrated that mesodermal cells from the BA2 contributed to the outflow tract and ventricular myocardium in vivo. Thus, our findings highlight the cardiogenic potential of chicken BA2 mesodermal cells in vitro and in vivo.

Chicken embryo as a model in second heart field development.

Previously, a single source of progenitor cells was thought to be responsible for the formation of the cardiac muscle. However, the second heart field has recently been identified as an additional source of myocardial progenitor cells. The chicken embryo, which develops in the egg, outside the mother can easily be manipulated in vivo and in vitro. Hence, it was an excellent model for establishing the concept of the second heart field. Here, our review will focus on the chicken model, specifically its role in understanding the second heart field. In addition to discussing historical aspects, we provide an overview of recent findings that have helped to define the chicken second heart field progenitor cells. A better understanding of the second heart field development will provide important insights into the congenital malformations affecting cardiac muscle formation and function.

Rethinking Learning Experience: How Generally Perceived Life Stress Influences Students' Course Perceptions in Different Learning Environments.

Previous research work has already demonstrated that both the form of teaching as well as different teaching methods directly influence students' learning experience along with their psychobiological responses at the endocrine and autonomic level. Aiming to gain deeper insights into the constitution of the learning experience, this study examined the influence of external factors such as generally perceived life stress and self-efficacy on the immediate learning experience in different learning environments. Therefore, a randomized experimental field study was conducted in which both psychological constructs and physiological data (heart rate variability) were collected from healthy first-year medical students (n = 101) during the COVID-19 pandemic. In an effort to determine the consistency of the effects across various teaching formats, the same content of a practical histology course was carried out in a face-to-face setting as well as in passive and active online teaching. While self-efficacy was a strong predictor for positive course perceptions in all learning conditions (Pearson's r = 0.41-0.58), generally perceived worries correlated with higher anxiety during passive online learning and face-to-face learning (Pearson's r = 0.21-0.44), a finding supported by the negative correlation between the level of perceived life demands and enjoyment during the learning unit (Pearson's r = -0.40--0.43). Here, we additionally report initial evidence pointing towards the role of reduced general life stress as a resilience factor for the expression of physiological stress parameters in an academic context (small-sized effect; Pearson's r = 0.18). The data gathered in this study illustrate the relevance of emerging emotional manifestations-either aversive; negative effect or positive; protective effect-for the immediate learning process and thus establish a connection between medical education and the importance of mental health and wellbeing-especially discussed against the background of current social and political challenges in increasingly complex societal structures.

Implementing a multi-colour genetic marker analysis technique for embryology education.

Embryology belongs to the basic sciences and is usually an integral part of the anatomy. The subject is traditionally taught by visual inspection of embryonic tissue slides stained with Haematoxylin and Eosin (H&E) to expose the dynamics of tissue histology as development proceeds. While combining in situ hybridization for gene expression analysis and immunostaining for protein expression analysis is an established technique for embryology research, the implementation of this tool in embryology teaching has not been described. The present study was conducted to assess the use of an online multi-colour gene expression analysis technique, alongside histological sections and diagrams, to improve students' understanding of embryology. The participants of this study were bachelor's students of Veterinary Medicine at the University of Khartoum. The method was also evaluated by distributing questionnaire items to Veterinary students via Google forms; subsequently, their responses were analysed qualitatively. The majority of students stated that the new technique was beneficial for their learning of embryology. The multi-colour images proved a more effective means for learning embryology than the traditional H&E image. Results from the students strengthen the belief in applying the multi-colour technique for better embryology course learning.

The morphological postnatal development of the testis of the Nubian bucks.

This study was designed to monitor the morphological development of the reproductive tract of the Nubian bucks in relation to puberty. Thirty-two Nubain male kids were used in the study. The animals were slaughtered at intervals of 2 weeks starting from 1 day old up to 24 weeks of age. Tissue samples were obtained from the testes and processed for ultrastructural studies. The boundary tissue of the newly forming seminiferous tubule adhered closely to the basal lamina. It consisted of a single continuous layer of myoid cells, the outer surface of which was covered by scattered fibroblasts. The ultrastructural study of the boundary of the seminiferous tubule revealed that it consisted of three layers; inner fibrous, middle and outer cellular. The seminiferous tubules at week one were lined by two layers of epithelia; spermatogonia and Sertoli cells in the basal layer, and primary spermatocytes in the second layer. A gradual increase in the diameter of the tubules and epithelial height continued to increase with age. Furthermore, spermatocytes number showed an increase with age. In conclusion, based on the appearance of spermatozoa in the lumina of the seminiferous tubules, puberty age was achieved between weeks 18 and 20.

New Insights into the Diversity of Branchiomeric Muscle Development: Genetic Programs and Differentiation.

Branchiomeric skeletal muscles are a subset of head muscles originating from skeletal muscle progenitor cells in the mesodermal core of pharyngeal arches. These muscles are involved in facial expression, mastication, and function of the larynx and pharynx. Branchiomeric muscles have been the focus of many studies over the years due to their distinct developmental programs and common origin with the heart muscle. A prerequisite for investigating these muscles' properties and therapeutic potential is understanding their genetic program and differentiation. In contrast to our understanding of how branchiomeric muscles are formed, less is known about their differentiation. This review focuses on the differentiation of branchiomeric muscles in mouse embryos. Furthermore, the relationship between branchiomeric muscle progenitor and neural crest cells in the pharyngeal arches of chicken embryos is also discussed. Additionally, we summarize recent studies into the genetic networks that distinguish between first arch-derived muscles and other pharyngeal arch muscles.

The Emergence of Embryonic Myosin Heavy Chain during Branchiomeric Muscle Development.

A prerequisite for discovering the properties and therapeutic potential of branchiomeric muscles is an understanding of their fate determination, pattering and differentiation. Although the expression of differentiation markers such as myosin heavy chain (MyHC) during trunk myogenesis has been more intensively studied, little is known about its expression in the developing branchiomeric muscle anlagen. To shed light on this, we traced the onset of MyHC expression in the facial and neck muscle anlagen by using the whole-mount in situ hybridization between embryonic days E9.5 and E15.5 in the mouse. Unlike trunk muscle, the facial and neck muscle anlagen express MyHC at late stages. Within the branchiomeric muscles, our results showed variation in the emergence of MyHC expression. MyHC was first detected in the first arch-derived muscle anlagen, while its expression in the second arch-derived muscle and non-somitic neck muscle began at a later time point. Additionally, we show that non-ectomesenchymal neural crest invasion of the second branchial arch is delayed compared with that of the first brachial arch in chicken embryos. Thus, our findings reflect the timing underlying branchiomeric muscle differentiation.

The CXCR4/SDF-1 Axis in the Development of Facial Expression and Non-somitic Neck Muscles.

Trunk and head muscles originate from distinct embryonic regions: while the trunk muscles derive from the paraxial mesoderm that becomes segmented into somites, the majority of head muscles develops from the unsegmented cranial paraxial mesoderm. Differences in the molecular control of trunk versus head and neck muscles have been discovered about 25 years ago; interestingly, differences in satellite cell subpopulations were also described more recently. Specifically, the satellite cells of the facial expression muscles share properties with heart muscle. In adult vertebrates, neck muscles span the transition zone between head and trunk. Mastication and facial expression muscles derive from the mesodermal progenitor cells that are located in the first and second branchial arches, respectively. The cucullaris muscle (non-somitic neck muscle) originates from the posterior-most branchial arches. Like other subclasses within the chemokines and chemokine receptors, CXCR4 and SDF-1 play essential roles in the migration of cells within a number of various tissues during development. CXCR4 as receptor together with its ligand SDF-1 have mainly been described to regulate the migration of the trunk muscle progenitor cells. This review first underlines our recent understanding of the development of the facial expression (second arch-derived) muscles, focusing on new insights into the migration event and how this embryonic process is different from the development of mastication (first arch-derived) muscles. Other muscles associated with the head, such as non-somitic neck muscles derived from muscle progenitor cells located in the posterior branchial arches, are also in the focus of this review. Implications on human muscle dystrophies affecting the muscles of face and neck are also discussed.

In ovo technique for cell injection in the CPM followed by bead implantation in the BA2 of chicken embryos.

The major advantage of chicken embryos model is their accessibility for microsurgical manipulations and the dissection of tissues for ex vivo explant culture. Branchial arches are embryonic structure located next to the top of developing heart. Each arch is made of surface ectoderm, endoderm, myogenic mesoderm cells and cranial neural crest cells. The myogenic mesoderm originates from cranial paraxial mesoderm (CPM), which is transiently migrated to branchial arches (BAs). The first branchial arch (BA1) mesoderm contributes to formation of mastication muscles. The second branchial arch (BA2) mesoderm gives rise to facial expression muscles. This article focuses on cell injection in the CPM and bead implantation (gain of function approaches) in the BA2. In order to follow the migration of mesoderm progenitor cells from CPM to BA2, we injected quail cells in the CPM of stage HH10-11 embryos, followed by implantation of SDF-1 bead at stage HH15-16. Later the attraction of quail cells (CXCR4+) towards the SDF-1 source has been observed, using whole-mount immunostaining of a specific quail antibody (QCPN) at stage HH19-22. •Our method, which involves bead implantation followed by quail cell injection, provides useful tools for tracing migratory mesodermal cells in vivo.•The proposed method does not require any commercial kits and can be used for various developmental process.•It does not employ any complicated methods such as genetically engineered permanent cell labeling, multiplicity of fluorescent markers or clonal analysis.

Cxcr4 and Sdf-1 are critically involved in the formation of facial and non-somitic neck muscles.

The present study shows that the CXCR4/SDF-1 axis regulates the migration of second branchial arch-derived muscles as well as non-somitic neck muscles. Cxcr4 is expressed by skeletal muscle progenitor cells in the second branchial arch (BA2). Muscles derived from the second branchial arch, but not from the first, fail to form in Cxcr4 mutants at embryonic days E13.5 and E14.5. Cxcr4 is also required for the development of non-somitic neck muscles. In Cxcr4 mutants, non-somitic neck muscle development is severely perturbed. In vivo experiments in chicken by means of loss-of-function approach based on the application of beads loaded with the CXCR4 inhibitor AMD3100 into the cranial paraxial mesoderm resulted in decreased expression of Tbx1 in the BA2. Furthermore, disrupting this chemokine signal at a later stage by implanting these beads into the BA2 caused a reduction in MyoR, Myf5 and MyoD expression. In contrast, gain-of-function experiments based on the implantation of SDF-1 beads into BA2 resulted in an attraction of myogenic progenitor cells, which was reflected in an expansion of the expression domain of these myogenic markers towards the SDF-1 source. Thus, Cxcr4 is required for the formation of the BA2 derived muscles and non-somitic neck muscles.