Building a vertebra: Development of the amniote sclerotome.

In embryonic development, the vertebral column arises from the sclerotomal compartment of the somites. The sclerotome is a mesenchymal cell mass which can be subdivided into several subpopulations specified by different regulatory mechanisms and giving rise to different parts of the vertebrae like vertebral body, vertebral arch, ribs, and vertebral joints. This review gives a short overview on the molecular and cellular basis of the formation of sclerotomal subdomains and the morphogenesis of their vertebral derivatives.

Development of ribs and intercostal muscles in the chicken embryo.

In the thorax of higher vertebrates, ribs and intercostal muscles play a decisive role in stability and respiratory movements of the body wall. They are derivatives of the somites, the ribs originating in the sclerotome and the intercostal muscles originating in the myotome. During thorax development, ribs and intercostal muscles extend into the lateral plate mesoderm and eventually contact the sternum during ventral closure. Here, we give a detailed description of the morphogenesis of ribs and thoracic muscles in the chicken embryo (Gallus gallus). Using Alcian blue staining as well as Sox9 and Desmin whole-mount immunohistochemistry, we monitor synchronously the development of rib cartilage and intercostal muscle anlagen. We show that the muscle anlagen precede the rib anlagen during ventrolateral extension, which is in line with the inductive role of the myotome in rib differentiation. Our studies furthermore reveal the temporary formation of a previously unknown eighth rib in the chicken embryonic thorax.

Regulation of Translation, Translocation, and Degradation of Proteins at the Membrane of the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) of mammalian cells is the central organelle for the maturation and folding of transmembrane proteins and for proteins destined to be secreted into the extracellular space. The proper folding of target proteins is achieved and supervised by a complex endogenous chaperone machinery. BiP, a member of the Hsp70 protein family, is the central chaperone in the ER. The chaperoning activity of BiP is assisted by ER-resident DnaJ (ERdj) proteins due to their ability to stimulate the low, intrinsic ATPase activity of BiP. Besides their co-chaperoning activity, ERdj proteins also regulate and tightly control the translation, translocation, and degradation of proteins. Disturbances in the luminal homeostasis result in the accumulation of unfolded proteins, thereby eliciting a stress response, the so-called unfolded protein response (UPR). Accumulated proteins are either deleterious due to the functional loss of the respective protein and/or due to their deposition as intra- or extracellular protein aggregates. A variety of metabolic diseases are known to date, which are associated with the dysfunction of components of the chaperone machinery. In this review, we will delineate the impact of ERdj proteins in controlling protein synthesis and translocation under physiological and under stress conditions. A second aspect of this review is dedicated to the role of ERdj proteins in the ER-associated degradation pathway, by which unfolded or misfolded proteins are discharged from the ER. We will refer to some of the most prominent diseases known to be based on the dysfunction of ERdj proteins.

Protein expression pattern of the molecular chaperone Mdg1/ERdj4 during embryonic development.

The vertebrate-specific co-chaperone Mdg1/ERdj4, which is localized in the endoplasmic reticulum, controls the folding and degradation of proteins. We characterized its protein pattern during chick embryonic development. During early development, Mdg1/ERdj4 protein is present in mesenchymal and epithelial cells. In mesenchymal cells, it has a salt and pepper pattern. In contrast, during epithelial tissue differentiation, Mdg1/ERdj4 marks the basal and/or apical compartment of epithelial linings. The distinct protein pattern in epithelial tissue might point to its role in organizing and maintaining the epithelial structure. This could be achieved, e.g. by controlling folding and secretion of membrane-bound receptors or by inhibiting the IRE1α-Xbp1s-SNAI1/2-induced mesenchymalization. High Mdg1/ERdj4 protein levels are maintained in tissue with sustained secretory activity as in ependymal cells or enterocytes, substantiating its important role for secretion. We conclude that the transient elevation of Mdg1/ERdj4 protein levels controls the differentiation of epithelial linings while constitutive high levels are closely linked to secretory activity.

Somite development in the avian tail.

Somites are epithelial segments of the paraxial mesoderm. Shortly after their formation, the epithelial somites undergo extensive cellular rearrangements and form specific somite compartments, including the sclerotome and the myotome, which give rise to the axial skeleton and to striated musculature, respectively. The dynamics of somite development varies along the body axis, but most research has focused on somite development at thoracolumbar levels. The development of tail somites has not yet been thoroughly characterized, even though vertebrate tail development has been intensely studied recently with respect to the termination of segmentation and the limitation of body length in evolution. Here, we provide a detailed description of the somites in the avian tail from the beginning of tail formation at HH-stage 20 to the onset of degeneration of tail segments at HH-stage 27. We characterize the formation of somite compartment formation in the tail region with respect to morphology and the expression patterns of the sclerotomal marker gene paired-box gene 1 (Pax1) and the myotomal marker genes MyoD and myogenic factor 5 (Myf5). Our study gives insight into the development of the very last segments formed in the avian embryo, and provides a basis for further research on the development of tail somite derivatives such as tail vertebrae, pygostyle and tail musculature.

Double electroporation in two adjacent tissues in chicken embryos.

In ovo electroporation is a well established method to introduce transgenes into a number of tissues in chicken embryos, e.g., neural tissue, limb mesenchyme, and somites. This method has been widely used to investigate cell lineage, cell morphology, and molecular pathways by localized expression of fluorescent reporter constructs. Furthermore gain- and loss-of-function experiments can be performed by electroporating transgenes or gene-silencing constructs. We have developed a new technique to electroporate tissues positioned opposite to each other with different plasmids using an electroporation chamber. As proof of principle, we electroporated the dorsal surface ectoderm with a reporter construct expressing mCherry and the subjacent somites with a reporter construct expressing EGFP. This double-electroporation technique allows investigation of the localization of two different proteins of interest in two adjacent tissues and will be useful to examine the cellular and molecular interaction of neighboring structures during embryonic development. Developmental Dynamics 247:1211-1216, 2018. © 2018 Wiley Periodicals, Inc.

Developmental dynamics of occipital and cervical somites.

Development of somites leading to somite compartments, sclerotome, dermomyotome and myotome, has been intensely investigated. Most knowledge on somite development, including the commonly used somite maturation stages, is based on data from somites at thoracic and lumbar levels. Potential regional differences in somite maturation dynamics have been indicated by a number of studies, but have not yet been comprehensively examined. Here, we present an overview on the developmental dynamics of somites at occipital and cervical levels in the chicken embryo. We show that in these regions, the onset of sclerotomal and myotomal compartment formation is later than at thoracolumbar levels, and is initiated simultaneously in multiple somites, which is in contrast to the serial cranial- to- caudal progression of somite maturation in the trunk. Our data suggest a variant spatiotemporal regulation of somite development in occipitocervical somites.

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells.

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc(-)) DT40 cells arrest in G2 phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc(-) cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G2 checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G2 arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G2 phase.

Regulation of the Rac GTPase pathway by the multifunctional Rho GEF Pebble is essential for mesoderm migration in the Drosophila gastrula.

The Drosophila guanine nucleotide exchange factor Pebble (Pbl) is essential for cytokinesis and cell migration during gastrulation. In dividing cells, Pbl promotes Rho1 activation at the cell cortex, leading to formation of the contractile actin-myosin ring. The role of Pbl in fibroblast growth factor-triggered mesoderm spreading during gastrulation is less well understood and its targets and subcellular localization are unknown. To address these issues we performed a domain-function study in the embryo. We show that Pbl is localized to the nucleus and the cell cortex in migrating mesoderm cells and found that, in addition to the PH domain, the conserved C-terminal tail of the protein is crucial for cortical localization. Moreover, we show that the Rac pathway plays an essential role during mesoderm migration. Genetic and biochemical interactions indicate that during mesoderm migration, Pbl functions by activating a Rac-dependent pathway. Furthermore, gain-of-function and rescue experiments suggest an important regulatory role of the C-terminal tail of Pbl for the selective activation of Rho1-versus Rac-dependent pathways.