Protein synthesis in embryonic tissues during mouse postimplantation development

Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (M(r)) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.     

The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.     

A protein that accumulates during starvation in Tetrahymena nuclei

Tetrahymena pyriformis was starved in 50 mM Tris-HCl, pH 7.5, at 28 degrees C. The number of cells did not change appreciably under the starvation conditions. Nuclear proteins of unstarved cells and cells starved for 1, 2, 4, and 7 d were analyzed by SDS-polyacrylamide gel electrophoresis. Most of the large amount of nonhistone proteins present in the unstarved cell nucleus disappeared with the starvation time. However, the relative amounts of the high mobility group protein and histones did not change appreciably. On the other hand, a protein with a molecular weight of ca. 16,000 gradually accumulated in the nucleus on starvation. This protein was extracted with 0.25 M HCl, but was not soluble in 0.5 M perchloric acid. The amino acid composition and molecular weight of this protein were similar to those of HMG protein LG-2 of T. thermophila. Some lysyl endopeptidase peptides of this protein were found to have amino acid sequences present in LG-2, thus we tentatively named it an LG-2-like protein.     

Aortic carboxypeptidase-like protein is expressed in collagen-rich tissues during mouse embryonic development

Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis.     

Polycystin expression during embryonic development of human kidney in adult tissues and ADPKD tissue

Normal renal tissue, ranging from 8 weeks' gestation to full term to adult, was probed with polyclonal antibodies raised to peptide epitopes within the translated PKD1 gene sequence. Three antibodies were studied, all of which gave similar results. Renal tissue from patients with autosomal dominant polycystic kidney disease (ADPKD) and samples from normal adult liver, heart, brain, skeletal muscle and lymph node were also studied. Tissue staining demonstrated that the pattern of polycystin expression changed with gestational age in normal kidney. Whereas the precursors to the renal excretory unit were stained at 12 weeks, and the proximal and distal convoluted tubules stained to differing degrees through out development, the glomeruli were poorly stained until full term and also in the adult. Extrarenal tissue stained in both adult and juvenile samples, with the exception of lymph node, which remained unstained. The intensity of polycystin staining increased in ADPKD renal tissue. The widespread distribution of polycystin was consistent with the systemic nature of ADPKD and the role of epithelial cells in the disease This revised version was published online in November 2006 with corrections to the Cover Date.     

Evidence that free radical generation occurs during scorpion envenomation

Although it is well established that symptomatology, morbidity and death following scorpion envenomation are due to increases in neurotransmitter release secondary to toxins binding to voltage-sensitive sodium channels, the mechanism by which venom action is involved in damaging heart, liver, lungs and kidneys remains unclear. We hypothesized that scorpion toxins could induce the generation of high levels of free radicals responsible for membrane damage in organs targeted by venom action. We have investigated lipid peroxidation in different organs, through the evaluation of thiobarbituric acid reactive substances (TBARS), after experimental envenomation of rats by toxic fractions of Androctonus australis Hector venom. We have shown that scorpion toxins cause considerable lipid peroxidation in most vital organs. We also evaluated the protective effects of antioxidants in mice injected with lethal doses of toxins. Among the drugs tested, N-acetylcysteine (NAC) was effective in protecting the mice when injected prior to toxin application. However, the free radical scavenging properties of NAC seem less implicated in these protective effects than its ability to increase the fluidity of bronchial secretions. We therefore conclude that free radical generation only plays a minor role in the toxicity of scorpion venom.     

Calcium signalling during embryonic development

Consider a hypothetical design specification for an integrated communication-control system within an embryo. It would require short-range (subcellular) and long-range (pan-embryonic) abilities, it would have to be flexible and, at the same time, robust enough to operate in a dynamically changing environment without information being lost or misinterpreted. Although many signalling elements appear, disappear and sometimes reappear during development, it is becoming clear that embryos also depend on a ubiquitous, persistent and highly versatile signalling system that is based around a single messenger, Ca2+.     

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response

The storage disaccharide, trehalose, is accumulated in yeast during a temperature shift from 30 to 45 degrees C. The response peaks at 90 min and is transient since levels of trehalose decline rapidly in cells returned to 30 degrees C. Storage of trehalose is inhibited when cells are incubated in the presence of acridine orange or ethidium bromide prior to and during temperature shift, suggesting a requirement for de novo RNA synthesis. Accumulation of trehalose occurs when cells are exposed to either ethanol, copper sulphate or hydrogen peroxide at 30 degrees C, indicating that the phenomenon may be a general response to physiological stress. Parallels are drawn between the trehalose accumulation response and the heat shock response in yeast.     

Ecdysteroids in Carausius eggs during embryonic development

Peaks of ecdysteroids were observed during the different phases of embryonic development of intact Carausius eggs or eggs precociously deprived of their exochorion and cultivated under paraffin oil. Several groups of ecdysteroids were separated and analyzed by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) combined with radioimmunoassay. Ecdysteroids were similar in the two categories of eggs, including high-polarity products (essentially conjugates hydrolyzable by Helix pomatia digestive juice, or alkaline phosphatase), possible ecdysonoic acids (unhydrolyzable polar substances), free hormones, and nonpolar ecdysteroids. Four ecdysteroids were identified by co-elution during HPLC with reference compounds of 20,26-dihydroxyecdysone, 20-hydroxyecdysone, ecdysone, and 2-deoxy-20-hydroxyecdysone. Concentrations of these substances (free and conjugated forms) were studied during the different stages of embryonic development: 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone were the major free ecdysteroids. They showed parallel variations with large peaks at stages VI₈ and VII₆ whereas ecdysone titers were consistently low. Injected labelled ecdysone was converted efficiently into 20-hydroxyecdysone, and both compounds underwent 26-hydroxylation and/or conjugation to polar or apolar metabolites.     

Neurobiology of mammalian olfactory learning that occurs during sensitive periods

This review examines the organizational principles underlying olfactory learning in three specialized contexts that occur during sensitive periods of enhanced neural plasticity and emphasizes some of their common features. All three forms of olfactory learning are associated with neural changes in the olfactory bulb (OB) at the first stage of sensory processing. These changes require the association of the olfactory and somatosensory signals in the OB. They all depend on somatosensory stimulation -induced r...     

Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development

The distribution of dividing cells is described for embryos and larvae of amphioxus (Branchiostoma floridae) pulse labeled with bromodeoxyuridine. Because cell division is assessed for all of the developing tissues, this is the first comprehensive study of developmental cell proliferation for an animal lacking a stereotyped cell lineage. In amphioxus, cell divisions are virtually synchronous during cleavage, but become asynchronous at the blastula stage. Starting at the neurula stage, after the origin of the mesoderm, the proportion of dividing cells progressively declines in the somitic mesoderm and notochord. Other tissues, however, deviate from this pattern. For example, in the mid-neurula, there is a brief, intense burst of mitosis at the anterior end of the neural plate. Also, from the neurula through the early larval stage, all of the ectoderm cells cease dividing and develop cilia that propel the animal through the water; subsequently, in the epidermis of later larvae, mitosis resumes and the proportion of ciliated cells declines as muscular undulation gradually replaces ciliation for swimming. Finally, in the early larvae, there is a terminal arrest of cell division in three cell types that differentiate early to participate in feeding as soon as the mouth opens-namely the ciliated pharyngeal cells that produce the feeding current and the secretory cells of the club-shaped gland and endostyle that export food-trapping mucus into the pharynx. In sum, these stage- and tissue-specific changes in cell proliferation intensity illustrate how the requirements of embryonic and larval natural history can shape developmental programs.     

XK endo B is preferentially expressed in several induced embryonic tissues during the development of Xenopus laevis

XK endo B is a type I keratin that was originally identified by its preferential expression in the embryonic notochord of the amphibian Xenopus laevis. A peptide identical to a short region of its predicted amino acid sequence was used to generate antibodies against the XK endo B protein. This paper reports an immunocytochemical study of the spatial expression pattern of XK endo B during development. The protein was observed in the notochord and endoderm as predicted from previous RNA analysis. In addition, XK endo B was detected in the cement gland, in the pituitary, olfactory and pharyngeal pouch rudiments, and in a nonuniform distribution in the neural tube as well as the inner sensorial layer of the ectoderm. XK endo B expression is not limited to any germ layer or any particular cell type, but is nevertheless highly restricted in its distribution in the embryo. Its expression in several different embryonic tissues requiring inductive interactions for differentiation makes XK endo B a valuable tool with which to study the regulation of induced gene expression during embryogenesis.     

Calcium signalling during zebrafish embryonic development

Calcium signals appear throughout the first 24 hours of zebrafish development. These begin at egg activation, then continue to be generated throughout the subsequent zygote, cleavage, blastula, gastrula, and segmentation periods. They are thus associated with the major phases of pattern formation: cell proliferation, cell differentiation, axis determination, the generation of primary germ layers, the emergence of rudimentary organ systems, and therefore the establishment of the basic vertebrate body plan. When signals need to be transmitted across significant distances they take the form of waves, either intracellular waves when the cell size is large, or later in development when the cell size is reduced, intercellular waves. We will consider both types of calcium signals and their integration into signalling networks, and discuss their possible functions and developmental significance with regard to pattern formation. BioEssays 22:113-123, 2000.     

CTGF expression during mouse embryonic development

Connective tissue growth factor (CTGF) is a potent fibroblast mitogen and angiogenic factor which plays an important role in wound healing, cancerogenesis and fibrotic and vascular disease. Here we explored the regulation and the cellular site of the mRNA synthesis for this growth factor in the developing mouse embryo by in situ hybridisation. Strong and persistent CTGF gene expression was limited to three types of tissue: the vascular endothelium, particularly the high-pressure part of the cardiovascular system, condensed connective tissue around bone and cartilage, and maturing layer VII neurons in the cerebral cortex. With few exceptions (late tooth bud, neuroepithelium) epithelial tissue was negative. Very transient but strong expression was observed early during formation of cartilage, in late stages during perichondral ossification, on cerebral neuroepithelium, and in several discrete stages of tooth formation, on mesenchymal precursors of odontoblasts condensing on inner dental epithelium, and later on apposing regions of ameloblast and odontoblast epithelium. Altogether, the current study suggests that CTGF performs a dual role: a continuous function in the cardiovascular system, bone and cartilage-associated mesenchyme and maturing layer VII neurons, but also a more transient function associated with the formation of cartilage, bone, tooth and cerebral nerve cells.