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.

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.

Atonal homolog 8/Math6 regulates differentiation and maintenance of skeletal muscle.

Atonal Homolog 8 (Atoh8) belongs to a large superfamily of transcriptional regulators called basic helix-loop-helix (bHLH) transcription factors. Atoh8 (murine homolog "Math6") has been shown to be involved in organogenesis during murine embryonic development. We have previously identified the expression of Atoh8 during skeletal myogenesis in chicken where we described its involvement in hypaxial myotome formation suggesting a regulatory role of Atoh8 in skeletal muscle development. Within the current study, we analyzed the effect of the loss of function of Atoh8 in murine primary myoblasts and during differentiation of pluripotent stem cells into myotubes, and the effect of its gain of function in C2C12 cells. Based on the observed results, we conclude that Atoh8 regulates myoblast proliferation via modulating myostatin signaling. Further, our data revealed a reduced muscle mass, strength and fiber size with significant changes to the muscle fiber type suggesting atrophy in skeletal muscle of Atoh8 mutants. We further report that Atoh8 knockout mice suffer from a condition similar to ambient hypoxia which may be the primary cause of the phenotype. Altogether, this study shows the significance of Atoh8 not only in myogenesis but also in the maintenance of skeletal muscle.

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.

Murine transcription factor Math6 is a regulator of placenta development.

The murine basic helix-loop-helix transcription (bHLH) factor mouse atonal homolog 6 (Math6) is expressed in numerous organs and supposed to be involved in several developmental processes. However, so far neither all aspects nor the molecular mechanisms of Math6 function have been explored exhaustively. To analyze the in vivo function of Math6 in detail, we generated a constitutive knockout (KO) mouse (Math6-/-) and performed an initial histological and molecular biological investigation of its main phenotype. Pregnant Math6-/- females suffer from a disturbed early placental development leading to the death of the majority of embryos independent of the embryonic Math6 genotype. A few placentas and fetuses survive the severe uterine hemorrhagic events at late mid-gestation (E13.5) and subsequently develop regularly. However, these fetuses could not be born due to obstructions within the gravid uterus, which hinder the birth process. Characterization of the endogenous spatiotemporal Math6 expression during placenta development reveals that Math6 is essential for an ordered decidualization and an important regulator of the maternal-fetal endocrine crosstalk regulating endometrial trophoblast invasion and differentiation. The strongly disturbed vascularization observed in the maternal placenta appears as an additional consequence of the altered endocrine status and as the main cause for the general hemorrhagic crisis.

GGNBP2 is necessary for testis morphology and sperm development.

Gametogenetin Binding Protein 2 (GGNBP2) was identified as a tumor suppressor and verified as such by several studies. GGNBP2 has also been reported to be essential for pregnancy maintenance via regulation of trophoblast stem cells. Gametogenetin (GGN) is a testicular germ cell-specific gene expressed in adult testes. As a potential GGN1-interacting protein, the role of GGNBP2 in spermatogenesis has not yet been clarified. We generated heterozygous GGNBP2 knockout mice and bred them by intercrossing. We found that among the offspring, homozygous GGNBP2 knockout (KO) mice were present in severely reduced numbers. The GGNBP2 KO pups developed normally, but the male siblings showed dramatically reduced fertility. In these male homozygous GGNBP2 KO mice, the only pathological finding was abnormal morphology of the testes and absence of spermatozoa. In addition, increased apoptosis was observed in the testes of GGNBP2 KO mice. SOX9 staining revealed that SOX9-positive Sertoli cells were absent in the seminiferous tubules. In homozygous mice, proliferating cell nuclear antigen (PCNA)-positive cells were localized in the lumen of the convoluted seminiferous tubules. These results suggest that GGNBP2 plays a key role in spermatogenesis by affecting the morphology and function of SOX9-positive Sertoli cells.

Expression Pattern of Axin2 During Chicken Development.

Canonical Wnt-signalling is well understood and has been extensively described in many developmental processes. The regulation of this signalling pathway is of outstanding relevance for proper development of the vertebrate and invertebrate embryo. Axin2 provides a negative-feedback-loop in the canonical Wnt-pathway, being a target gene and a negative regulator. Here we provide a detailed analysis of the expression pattern in the development of the chicken embryo. By performing in-situ hybridization on chicken embryos from stage HH 04+ to HH 32 we detected a temporally and spatially restricted dynamic expression of Axin2. In particular, data about the expression of Axin2 mRNA in early embryogenesis, somites, neural tube, limbs, kidney and eyes was obtained.

Rapid impact of progesterone on the neuronal growth cone.

In the last two decades, sensory neurons and Schwann cells in the dorsal root ganglia (DRG) were shown to express the rate-limiting enzyme of the steroid synthesis, cytochrome P450 side-chain cleavage enzyme (P450scc), as well as the key enzyme of progesterone synthesis, 3ß-hydroxysteroid dehydrogenase (3ß-HSD). Thus, it was well justified to consider that DRG neurons similarly are able to synthesize progesterone de novo from cholesterol. Because direct progesterone effects on axonal outgrowth in peripheral neurons have not been investigated up to now, the present study provides the first insights into the impact of exogenous progesterone on axonal outgrowth in DRG neurons. Our studies including microinjection and laser scanning microscopy demonstrate morphological changes especially in the neuronal growth cones after progesterone treatment. Furthermore, we were able to detect a distinctly enhanced motility only a few minutes after the start of progesterone treatment using time-lapse imaging. Investigation of the cytoskeletal distribution in the neuronal growth cone before, during, and after progesterone incubation revealed a rapid reorganization of actin filaments. To get a closer idea of the underlying receptor mechanisms, we further studied the expression of progesterone receptors in DRG neurons using RT-PCR and immunohistochemistry. Thus, we could demonstrate for the first time that classical progesterone receptor (PR) A and B and the recently described progesterone receptor membrane component 1 (PGRMC1) are expressed in DRG neurons. Antagonism of the classical progesterone receptors by mifepristone revealed that the observed progesterone effects are transmitted through PR-A and PR-B.