Regulation of calcium homeostasis and flux between the endoplasmic reticulum and the cytosol.

The concentration of Ca2+ in the endoplasmic reticulum (ER) is critically important for maintaining its oxidizing environment as well as for maintaining luminal ATP levels required for chaperone activity. Therefore, local luminal Ca2+ concentrations and the dynamic Ca2+ flux between the different subcellular compartments are tightly controlled. Influx of Ca2+ into the ER is enabled by a reductive shift, which opens the sarcoendoplasmic reticulum calcium transport ATPase pump, building the Ca2+ gradient across the ER membrane required for ATP import. Meanwhile, Ca2+ leakage from the ER has been reported to occur via the Sec61 translocon following protein translocation. In this review, we provide an overview of the complex regulation of Ca2+ homeostasis, Ca2+ flux between subcellular compartments, and the cellular stress response (the unfolded protein response) induced upon dysregulated luminal Ca2+ metabolism. We also provide insight into the structure and gating mechanism at the Sec61 translocon and examine the role of ER-resident cochaperones in assisting the central ER-resident chaperone BiP in the control of luminal Ca2+ concentrations.

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.

The function of the co-chaperone ERdj4 in diverse (patho-)physiological conditions.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces a well-orchestrated cellular response to reduce the protein burden within the ER. This unfolded protein response (UPR) is controlled primarily by three transmembrane proteins, IRE1α, ATF6, and PERK, the activity of which is controlled by BiP, the ER-resident Hsp70 protein. Binding of BiP to co-chaperones via their highly conserved J-domains stimulates the intrinsic ATPase activity of BiP, thereby providing the energy necessary for (re-)folding of proteins, or for targeting of misfolded proteins to the degradation pathway, processes specified and controlled by the respective co-chaperone. In this review, our aim is to elucidate the function of the co-chaperone ERDJ4, also known as MDG1, MDJ7, or DNAJB9. Knockout and knockin experiments clearly point to the central role of ERDJ4 in controlling lipogenesis and protein synthesis by promoting degradation of SREBP1c and the assembly of the protein complex mTORC2. Accumulating data reveal that ERDJ4 controls epithelial-to-mesenchymal transition, a central process during embryogenesis, in wound healing, and tumor development. Overexpression of ERdj4 has been shown to improve engraftment of transplanted human stem cells, possibly due to its ability to promote cellular survival in stressed cells. High ERDJ4-plasma levels are specific for fibrillary glomerulonephritis and serve as a diagnostic marker. As outlined in this review, the functions of ERDJ4 are manifold, depending on the cellular (patho-) physiological state, the cellular protein repertoire, and the subcellular localization of ERDJ4.

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.

Function and mutual interaction of BiP-, PERK-, and IRE1α-dependent signalling pathways in vascular tumours.

Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1α levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1α/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1α, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1α and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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.

Communication between distant epithelial cells by filopodia-like protrusions during embryonic development.

Long-range intercellular communication is essential for the regulation of embryonic development. Apart from simple diffusion, various modes of signal transfer have been described in the literature. Here, we describe a novel type of cellular extensions found in epithelial cells of the somites in chicken embryos. These filopodia-like protrusions span the subectodermal space overlying the dorsal surface of the somites and contact the ectoderm. We show that these protrusions are actin- and tubulin-positive and require Rac1 for their formation. The presence of glycophosphatidylinositol-anchored proteins and net retrograde trafficking of the transmembrane Wnt-receptor Frizzled-7 along the protrusions indicate their role in signal transport and distribution. Taken together, our data suggest a role of filopodia-like protrusions in mediating signaling events between distant epithelial cells during embryonic development.

Signaling filopodia in vertebrate embryonic development.

Next to classical diffusion-based models, filopodia-like cellular protrusions have been proposed to mediate long range signaling events and morphogen gradient formation during communication between distant cells. An increasing wealth of data indicates that in spite of variable characteristics of signaling filopodia in different biological contexts, they represent a paradigm of intercellular crosstalk which is presently being unraveled in a growing literature. Here, we summarize recent advances in investigating the morphology, cellular basis and function of signaling filopodia, with focus on their role during embryonic development in vertebrates.

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.

Aromatase Expression in the Hippocampus of AD Patients and 5xFAD Mice.

Numerous studies show that 17ß-estradiol (E2) protects against Alzheimer's disease (AD) induced neurodegeneration. The E2-synthesizing enzyme aromatase is expressed in healthy hippocampi, but although the hippocampus is severely affected in AD, little is known about the expression of hippocampal aromatase in AD. To better understand the role of hippocampal aromatase in AD, we studied its expression in postmortem material from patients with AD and in a mouse model for AD (5xFAD mice). In human hippocampi, aromatase-immunoreactivity was observed in the vast majority of principal neurons and signal quantification revealed higher expression of aromatase protein in AD patients compared to age- and sex-matched controls. The tissue-specific first exons of aromatase I.f, PII, I.3, and I.6 were detected in hippocampi of controls and AD patients by RT-PCR. In contrast, 3-month-old, female 5xFAD mice showed lower expression of aromatase mRNA and protein (measured by qRT-PCR and semiquantitative immunohistochemistry) than WT controls; no such differences were observed in male mice. Our findings stress the importance of hippocampal aromatase expression in neurodegenerative diseases.

A thymosin beta15-like peptide promotes intersegmental myotome extension in the chicken embryo.

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors.

The peripheral astrocyte process (PAP) preferentially associates with the synapse. The PAP, which is not found around every synapse, extends to or withdraws from it in an activity-dependent manner. Although the pre- and postsynaptic elements have been described in great molecular detail, relatively little is known about the PAP because of its difficult access for electrophysiology or light microscopy, as they are smaller than microscopic resolution. We investigated possible stimuli and mechanisms of PAP plasticity. Immunocytochemistry on rat brain sections demonstrates that the actin-binding protein ezrin and the metabotropic glutamate receptors (mGluRs) 3 and 5 are compartmentalized to the PAP but not to the GFAP-containing stem process. Further experiments applying ezrin siRNA or dominant-negative ezrin in primary astrocytes indicate that filopodia formation and motility require ezrin in the membrane/cytoskeleton bound (i.e., T567-phosphorylated) form. Glial processes around synapses in situ consistently display this ezrin form. Possible motility stimuli of perisynaptic glial processes were studied in culture, based on their similarity with filopodia. Glutamate and glutamate analogues reveal that rapid (5 min), glutamate-induced filopodia motility is mediated by mGluRs 3 and 5. Ultrastructurally, these mGluR subtypes were also localized in astrocytes in the rat hippocampus, preferentially in their fine PAPs. In vivo, changes in glutamatergic circadian activity in the hamster suprachiasmatic nucleus are accompanied by changes of ezrin immunoreactivity in the suprachiasmatic nucleus, in line with transmitter-induced perisynaptic glial motility. The data suggest that (i) ezrin is required for the structural plasticity of PAPs and (ii) mGluRs can stimulate PAP plasticity.

Dual topology of co-chaperones at the membrane of the endoplasmic reticulum.

Dual topologies of proteins at the ER membrane are known for a variety of proteins allowing the same protein to exert different functions according to the topology adopted. A dual topology of the co-chaperone ERdj4, which resides in the endoplasmic reticulum (ER), was proposed recently, a thesis that we found to align all published data and existing controversies into one whole picture. The aim of this review is to reassess all primary data available in the literature on ER-resident Hsp40 co-chaperones with respect to their topology. After careful and critical analyses of all experimental data published so far, we identified, next to ERdj4, two other co-chaperones, ERdj3 and ERdj6, that also display features of a dual topology at the ER membrane. We assume that during cellular stress subpools of some ER-resident J protein can alter their topology so that these proteins can exert different functions in order to adapt to cellular stress.

When and why was the phrenicoabdominal branch of the left phrenic nerve placed into the esophageal hiatus in German textbooks of anatomy? An anatomical study on 400 specimens reevaluating its course through the diaphragm.

BACKGROUND: The phrenicoabdominal branch of the left phrenic nerve passes between muscle fiber bundles within the costal part of the diaphragm near the pericardium. In most German textbooks of anatomy, however, its passage is described to be found in the esophageal hiatus. The aim of this study was to reevaluate its topography relative to the diaphragm in a multicentric study and to identify the initiation of this description. METHODS: In this multicentric study, the most dorsomedial branch of the left phrenic nerve was identified as the phrenicoabdominal branch in 400 embalmed anatomic specimens of Caucasian origin. The distance between its passage and the apex of the pericardium, the left border of the esophageal hiatus, and the inner aspect of the left sixth rib was measured on the cranial aspect of the diaphragm. Textbooks on human anatomy published in German language between 1700 and 2018 were reviewed for their description of the passage of the left phrenicoabdominal branch through the diaphragm. RESULTS: The first statement on the passage of the left phrenicoabdominal branch through the esophageal hiatus was given in 1791 by Sömmering. Since then, in German textbooks of anatomy, a duality in the description of the passage of the left phrenicoabdominal branch persists. In none of the individuals examined in this study, the left phrenicoabdominal branch passed through the esophageal hiatus. In 99.5% of all cases, it pierced the costal part of the diaphragm dorsal to or at the same level as the apex of the pericardium. The mean distances (standard deviations) were 3.4 (±1.5) cm to the apex of the pericardium, 5.8 (±2.2) cm to the esophageal hiatus, and 5.5 (±1.6) cm to the inner aspect of the left sixth rib. CONCLUSION: The findings on the position of the left phrenicoabdominal branch relative to the diaphragm help to improve topographical knowledge and prevent inadvertent nerve injury during surgical interventions on or near the diaphragm. Further to this, these results may form a substantial basis to adopt the correct description of the passage of the left phrenicoabdominal branch to anatomical textbook knowledge.

Pleiotrophin is expressed in avian somites and tendon anlagen.

Pleiotrophin (Ptn) is a secreted, developmentally regulated growth factor associated with the extracellular matrix. During mammalian embryogenesis, Ptn has been suggested to play a role in the development of various embryonic structures including nervous system and skeleton. In the avian embryo, Ptn has been proposed to be involved in limb cartilage development, but embryonic Ptn expression has not been comprehensively studied. We isolated a cDNA fragment containing the full-length coding sequence of chick Ptn and studied the expression of Ptn in detail until embryonic day 10. We, furthermore, isolated a 6,385-bp phage clone containing the Ptn cDNA of 2,551 bp and additional 3,787 bp downstream of the published Ptn cDNA sequence classifying a yet Ptn-unrelated chEST clone as the 3' untranslated region of Ptn. Our studies revealed novel expression domains in developing somites and during limb formation. We found prominent expression in the somitocoel cells of epithelial somites, and in a sclerotomal subcompartment, the syndetome, which gives rise to the axial tendons in the vertebral motion segment. In the limbs, Ptn was markedly expressed in tendon anlagen and in phalangeal joints. Our results introduce Ptn as a novel marker gene in avian somite and tendon development.

Cystathionine gamma-lyase expression during avian embryogenesis.

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulphuration pathway for the biosynthesis of cysteine from methionine and catalyses the hydrolysis of cystathionine into cysteine. It has been reported to be expressed in mammalian liver and kidney but so far no comprehensive developmental expression analysis of CSE has been available. We cloned a 600 bp fragment of chick CSE cDNA and analysed its expression pattern during avian embryonic development until embryonic day 13. We found CSE expression in various developing organs including the notochord, eye, neural tube, limb bud mesenchyme and sclerotomal compartment of the somites. Notably, prominent expression was found in renal epithelia throughout kidney development, i.e. in the tubular structures of pronephros, mesonephros and metanephros. Our data introduce CSE as a novel marker gene to study avian kidney development.

Reelin and aromatase cooperate in ovarian follicle development.

Reelin plays an important role in cerebral cortex development and synaptogenesis. In the hippocampus, the neurosteroid estrogen affects reelin expression. In this study we tested a potential crosstalk between estradiol and reelin, thus the possibility of a reelin-induced activation of the estradiol synthesizing enzyme aromatase. As a model system, we used ovaries, which express reelin and are a major source of estradiol. We found that in wild-type mice, reelin and aromatase are expressed in granulosa cells of growing follicles. The expression of reelin varies with the estrus cycle and is highest shortly before ovulation, when estradiol serum levels are at their maximum. In ovaries of reelin-deficient reeler mice, aromatase mRNA and protein are significantly reduced, as evidenced by real-time PCR, western blot analysis, and quantitative immunohistochemistry in granulosa cells of preovulatory follicles. In line with reduced estradiol synthesis, ovarian estrus cycle length is prolonged in reeler mice. Most importantly, treating cultured granulosa cells with recombinant reelin results in significant upregulation of aromatase mRNA and protein and increased secretion of estradiol into the supernatant. Our data provide evidence of a local increase of aromatase expression by reelin. Regarding reproduction, this crosstalk may contribute to follicular stability and counteract luteinization in ovaries.

Induction of cellular stress and chaperone activation in organotypic slice cultures of hippocampus.

Neurodegenerative diseases are often associated with the occurrence of misfolded proteins preceding neuronal cell death. Accumulation of misfolded proteins in the endoplasmic reticulum induces ER stress, which in consequence enhances chaperone expression to restore protein homeostasis. Here we used organotypic hippocampal slice cultures to analyze the time course of chaperone expression and neuronal death after induction of ER stress by tunicamycin treatment. Shortly after explantation many cells stain positive for Fluoro Jade B demonstrating neuronal cell death. While in control cultures the number of Fluoro Jade B labeled cells remarkably decrease over the total period of cultivation, neuronal death remains elevated in ER-stressed slice cultures. Caspase-3 staining revealed that neuronal death is primarily due to apoptosis in tunicamycin-treated slice cultures. The chaperone GRP78/BiP is expressed at low levels in control sections. Its expression is largely restricted to hippocampal neurons. Tunicamycin treatment resulted in upregulation of GRP78/BiP in the neuronal cells. Double-immunolabeling for GFAP shows a concomitant de novo expression of GRP78/BiP in astrocytes. The astrocytic GRP78/BiP upregulation might reflect an early, neuroprotective response. The increase of GRP78/BiP in neurons and astrocytes show successful induction of the ER stress response. The hippocampal slice cultures are, thus, a useful tool to examine the process of neurodegeneration and to investigate neuroprotective devices in an ER stress paradigm.