The pattern of protein and glycoprotein synthesis in presumptive lens and non-lens ectoderm of the chicken embryo

Summary Lens induction is a classic example of the tissue interactions that lead to cell specialization during early vertebrate development. Previous studies have shown that a large region of head ectoderm, but not trunk ectoderm, of 36 h (stage 10) chicken embryos retains the potential to form lenses and synthesize the protein δ-crystallin under some conditions. We have used polyacrylamide gel electrophoresis and fluorography to examine protein and glycoprotein synthesis in presumptive lens ectoderm and presumptive dorsal (trunk) epidermis to look for differentiation markers for these two regions prior to the appearance of δ-crystallin at 50 h. Although nearly all of the proteins incorporating³H-leucine were shared by presumptive lens ectoderm and trunk ectoderm, these two regions showed more dramatic differences in the incorporation of³H-sugars into glycoproteins. when non-lens head ectoderm that has a capacity for lens formation in vitro was labeled, a hybrid pattern of glycoprotein synthesis was discovered: glycoproteins found in either presumptive lens ectoderm or trunk ectoderm were oftentimes also found in other head ectoderm. Therefore, molecular markers have been identified for three regions of ectoderm committed to different fates (lens and skin), well before features of terminal differentiation begin to appear in the lens.     

Mesodermal patterning during avian gastrulation and neurulation: Experimental induction of notochord from non-notochordal precursor cells

The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 μm and a mediolateral extent of less than 250 μm, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate. © 1995 Wiley-Liss, Inc.     

Cell junctions in the rapidly moving edge of chick and quail blastoderms during gastrulation

Summary The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.     

Calretinin and calbindin in the retina of the developing chick

Summary Calretinin and calbindin-D28k are two calcium-binding proteins that are present in largely different sets of nerve cells in the central nervous system. Their appearance during development of the chick retina was studied by immunohistochemistry and Western blots. The patterns are mature one day before hatching. Each cell type acquires its characteristic calcium-binding protein several days after its differentiation has started, but in most cases before morphological maturation is complete. There is also an early phase of calbindin immunoreactivity in many immature amacrine cells, and of calretinin immunoreactivity in the presumptive photoreceptor layer, suggesting that these proteins may have distinct functions in differentiating cells.Abbreviations CR+ Immunoreactive for calretinin only - CB+ immunoreactive for calbindin only - CR+CB+ immunoreactive for both antisera - IPL inner plexiform layer - OPL outer plexiform layer     

Measurement of intra-embryonic pH during the early stages of development in the chick embryo

Summary Measurements have been made of the pH in the extracellular space, adjacent to the neural tube, in 73 isolated chick embryos in vitro at stages from 4–22 somites. A pH of 7.8–8.4 was observed in the segmented region, while caudally, in the segmental plate, the pH was consistently lower falling by as much as 0.5 pH units at the regressing primitive streak. Variations were noted in the pH of embryos of the same age but the regional variation in pH was a consistent finding in all of the embryos examined. The buffering capacity of the extracellular space was found to be 12.9 mequiv/pH unit/1 in the segmented region and 13.9 mequiv/pH unit/1 in the segmental plate. Thus it is unlikely that the regional variations in pH result from local variations in the buffering power of the extracellular space. Varying the K⁺, Cl^-, Mg²⁺ or HCO ₃ ^- ion concentrations in the bathing medium caused little change in the intra-embryonic pH, while reducing the concentrations of Na⁺ or Ca²⁺ caused a small acidification. This suggests that the ectoderm and endoderm form an effective barrier between the embryo and the external environment. Exposure of the embryo to KCN reduced the intra-embryonic pH suggesting that the alkaline environment is maintained by active processes.     

Neuromuscular synapses mediate motor axon branching and motoneuron survival during the embryonic period of programmed cell death

The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.     

The human placenta is a hematopoietic organ during the embryonic and fetal periods of development

We studied the potential role of the human placenta as a hematopoietic organ during embryonic and fetal development. Placental samples contained two cell populations—CD34⁺⁺CD45^low and CD34⁺CD45^low—that were found in chorionic villi and in the chorioamniotic membrane. CD34⁺⁺CD45^low cells express many cell surface antigens found on multipotent primitive hematopoietic progenitors and hematopoietic stem cells. CD34⁺⁺CD45^low cells contained colony-forming units culture (CFU-C) with myeloid and erythroid potential in clonogenic in vitro assays, and they generated CD56⁺ natural killer cells and CD19⁺CD20⁺sIgM⁺ B cells in polyclonal liquid cultures. CD34⁺CD45^low cells mostly comprised erythroid- and myeloid-committed progenitors, while CD34⁻ cells lacked CFU-C. The placenta-derived precursors were fetal in origin, as demonstrated by FISH using repeat-sequence chromosome-specific probes for X and Y. The number of CD34⁺⁺CD45^low cells increased with gestational age, but their density (cells per gram of tissue) peaked at 5-8 wk, decreasing more than sevenfold at the onset of the fetal phase (9 wk of gestation). In addition to multipotent progenitors, the placenta contained myeloid- and erythroid-committed progenitors indicative of active in situ hematopoiesis. These data suggest that the human placenta is an important hematopoietic organ, raising the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.     

Keratinization and lipogenesis in epidermal derivatives of the zebrafinch, Taeniopygia guttata castanotis (Aves, Passeriformes, Ploecidae) during embryonic development

Little is known of the lipid content of beta-keratin-producing cells such as those of feathers, scutate scales, and beak. The sequence of epidermal layers in some apteria and in interfollicular epidermis in the zebrafinch embryo (Taeniopygia guttata castanotis) was studied. Also, the production of beta-keratin in natal down feathers and beak was ultrastructurally analyzed in embryos from 3-4 to 17-18 days postdeposition, before hatching. Two layers of periderm initially cover the embryo, but there are eventually 6-8 over the epidermis of the beak. In the beak and sheath cells of feathers, peridermal granules are numerous at 12-14 days postdeposition but they are less frequent in apteria. These granules swell and disappear during sheath or peridermal degeneration at 15-17 days postdeposition. A thin beta-keratin layer forms under the periderm among feather germs of pterylous areas but is discontinuous or disappears in apteria. In differentiating cells of barbs, barbules, and calamus cells of natal down, electron-dense beta-keratin filaments form bundles oriented along the main axis of these cells. Cells of the pulp epidermis and collar, at the base of the follicle, contain lipids and bundles of alpha-keratin filaments. Degenerating pulp cells show vacuolization and nuclear pycnosis. During beta-keratin packing, keratin bundles turn electron-pale, perhaps due to the addition of lipids to produce the final, homogenous beta-keratin matrix. In contrast to the situation in feathers, in the cells of beak beta-keratin packets are irregularly oriented. In both feather and beak epidermal cells the Golgi apparatus and smooth endoplasmic reticulum produce vesicles containing lipid-like material which is also found among forming beta-keratin. The contribution of lipids or lipoprotein to the initial aggregation of beta-keratin molecules is discussed.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.     

Inhibition of polyamine synthesis reduces the growth rate and delays the expression of differentiated phenotypes in primary cultures of embryonic mesoderm from chick

Summary Inhibition of polyamine synthesis in early chick embryos blocks their development at gastrulation. Analyses of arrested embryos show that mesodermal outgrowth and differentiation are drastically impaired. To study these effects in greater detail, we have used primary cultures of embryonic mesoderm from chick. The cultures were treated with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, the first and rate-limiting enzyme in polyamine synthesis. In control culture medium, mesodermal cells retained their in ovo outgrowth behavior and differentiation pattern. Addition of 10 mM DFMO to the culture medium, however, retarded attachment and outgrowth, and reduced the rate of proliferation of the mesodermal cells. Furthermore, the expression of differentiated phenotypes, such as beating heart tissue, erythroid cells, and adipocyte-like cells, was delayed. Simultaneous addition of 100 M putrescine prevented or reduced the effects of DFMO, showing that these were indeed caused by polyamine deficiency. In the DFMO-treated mesoderm, DNA synthesis was markedly suppressed by the first day. Similar effects on RNA and protein synthesis developed at a later time. Our data suggest that a reduction in the concentrations of the polyamines decreases the rate of mesodermal cell proliferation, and as a conseqence delays the expression of differentiated phenotypes.     

Localization of high-affinity GABA uptake and GABA content in the rat duodenum during development

Summary The localization of high-affinity uptake sites for ³H-aminobutyric acid (³H-GABA) was investigated in the rat duodenum during ontogenesis and also at the adult stage (from 15.5 days of fetal life up to 105 days post natum) by means of low- and high-resolution autoradiography. At all stages studied, specific endocrine cell types of the epithelium were labelled and an intense uptake was detected in the nervous tissue, especially in glial cells but also in scarce neurones. When the incubation medium was supplemented with -alanine (1 mM), a blocker of the glial uptake for GABA, the labelling persisted only in endocrine cells and in few neurones. The intensity and the frequency of the labelling decreased at later periods compared to the earlier developmental stages. The GABA content of the duodenum as measured by a new ion-exchange column chromatography-HPLC-coupled method was higher in the early postnatal period compared to later stages. These observations suggest that GABA, in addition to being a neurotransmitter, may play an important role during development of the duodenum.     

Therapeutic approaches for the treatment of head and neck squamous cell carcinoma–An update on clinical trials

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common non-skin cancer with a tobacco consumption and infection with high-risk human papillomavirus (HPV) being major risk factors. Despite advances in numerous therapy modalities, survival rates for HNSCC have not improved considerably; a vast number of clinical outcomes have demonstrated that a combination strategy (the most well-known docetaxel, cisplatin, and 5-fluorouracil) is the most effective treatment choice. Immunotherapy that targets immunological checkpoints is being tested in a number of clinical trials, either alone or in conjunction with chemotherapeutic or targeted therapeutic drugs. Various monoclonal antibodies, such as cetuximab and bevacizumab, which target the EGFR and VEGFR, respectively, as well as other signaling pathway inhibitors, such as temsirolimus and rapamycin, are also being studied for the treatment of HNSCC. We have reviewed the primary targets in active clinical studies in this study, with a particular focus on the medications and drug targets used.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.