Developmental Stage-specific Changes Research Articles

Age-dependent genetic architectures of chicken body weight explored by multidimensional GWAS and molQTL analyses

Chicken body weight (BW) is a critical trait in breeding. Although genetic variants associated with BW have been investigated by genome-wide association studies (GWAS), the contributions of causal variants and their molecular mechanisms remain largely unclear in chickens. In this study, we construct a comprehensive genetic atlas of chicken BW by an integrative analysis of 30 age points and 5 quantitative trait loci (QTL) across 27 tissues. We find that chicken growth is a cumulative non-linear process, which can be divided into three distinct stages. Our GWAS analysis reveals that BW-related genetic variations show ordered patterns in these three stages. Genetic variations in chromosome 1 may regulate the overall growth process, likely by modulating the hypothalamus-specific expression of SLC25A30 and retina-specific expression of NEK3. Moreover, genetic variations in chromosome 4 and chromosome 27 may play dominant roles in regulating BW during stage Ⅱ (8-22 weeks) and stage Ⅲ (23-72 weeks), respectively. In summary, our study presents a comprehensive genetic atlas regulating developmental stage-specific changes in chicken BW, thus providing important resources for genomic selection in breeding programs.

P–258 Ubiquitin, in the human embryo secretome, is a biomarker for embryo viability: a potential predictor of live-births, post embryo transfer

Abstract Study question Whether embryo-secreted ubiquitin could serve as a predictive biomarker for embryo development and viability for assessing pregnancy outcome? Summary answer Embryo-secreted ubiquitin concentrations showed positive correlations with (a) developing embryonic stages, (b) implantation rates, (iii) live-birth rates. Their altered levels were associated with miscarriages. What is known already Human infertility affects 15–20% couple and is mitigated by ART approaches. Poor biological-viability of in vitro developed embryos contributes to implantation failure and low birth rates(LBR). The current morphology-based embryo selection approach has shortcomings in identifying biologically-viable embryos capable of producing live-births. Earlier studies have identified ubiquitin as a biomarker for embryo developmental competence. However, there have been no studies on estimations of ubiquitin in embryo-spent medium samples (E-SMs) and their correlative analysis with embryo-quality score and pregnancy outcome. Hence, such studies are required to establish whether or not ubiquitin could be a biomarker predicting pregnancy outcome. Study design, size, duration This was a retrospective, multi-centric study performed between July 2018 and September 2020. A total of 574 E-SMs (from 574 individual embryos), from 325 infertile women, were analysed for ubiquitin levels. Frozen E-SMs post-thaw were subjected to sandwich ELISA (Mybiosource, USA). Correlation analysis was performed on ubiquitin levels with developing embryonic stages and their scores, implantation rates (IRs) and pregnancy outcomes in terms of LBR. Participants/materials, setting, methods We measured ubiquitin levels in E-SMs obtained from three embryonic stages i.e., cleavage-stage (2–10-cells; n = 182), morulae (n = 102) and blastocysts (n = 290). Ubiquitin concentrations among three developmental stages were compared and analysed using the Student’s t-test/ANOVA (P ≤ 0.05), followed by Tukey posthoc test. Levels of ubiquitin were correlated (using Pearson/Spearman analysis) with (a) developing embryonic stages, (b) embryo morphology, (c) IRs, and (d) pregnancy outcomes in terms of LBR. Main results and the role of chance Of 574 E-SMs analysed, 540 (94.07%) had detectable ubiquitin levels (pg/ml) and they varied in an increasing manner across developing embryonic stages and, across the three clinics. We observed a significantly different (p < 0.0001) levels of ubiquitin in three sets of secretors i.e low (153.1 ± 5.4; n = 219), medium (498.9 ± 15.7) & high (1615 ± 46.5) secretors. Levels of ubiquitin among three developmental stages were significantly (p < 0.05) different under FET, but not with fresh-ET categories. Ubiquitin levels were independent of cleavage-stage morphology score but showed a positive correlation with blastocyst grades. Also, we observed a significant (p < 0.05) positive correlation of ubiquitin levels with implantation rates. Importantly, ubiquitin levels were higher in E-SMs of embryos which gave live-births vis-à-vis those with no-births. Moreover, altered levels (very high low) were associated with those embryos which resulted in miscarriages. This is the first report which measured ubiquitin in individual hE-SMs from three developing embryos and showed a development stage-wise positive correlations as well as a significant association (p < 0.0001) of ubiquitin levels with implantation and live-birth rates. Limitations, reasons for caution Observed variations in levels of ubiquitin across clinics could be attributed to (i) oocyte/sperm donors’ variation and their infertility status (i) IVF-ET protocol differences. A large multi-centric cohort studies are required to establish the predictive value of ubiquitin for assessing embryo-viability and pregnancy outcome in term of live-births. Wider implications of the findings: For the first time, our multi-centric study showed developmental stage-specific changes in ubiquitin levels. It could be a valuable biomarker of embryo-viability and to predict IR and live-births. Ubiquitin, as a biomarker, could be a valuable adjunct to currently practicing embryo score system for selecting transferable quality embryos. Trial registration number Not applicable

Developmental Stage-Specific Changes in Protein Synthesis Differentially Sensitize Hematopoietic Stem Cells and Erythroid Progenitors to Impaired Ribosome Biogenesis.

SummaryAdult hematopoietic stem cell (HSC) self-renewal requires precise control of protein synthesis, but fetal and adult HSCs have distinct self-renewal mechanisms and lineage outputs. This raises the question of whether protein synthesis rates change with age. Here, we show that protein synthesis rates decline during HSC ontogeny, yet erythroid protein synthesis rates increase. A ribosomal mutation that impairs ribosome biogenesis (Rpl24Bst/+) disrupts both fetal and adult HSC self-renewal. However, the Rpl24Bst/+ mutation selectively impairs fetal erythropoiesis at differentiation stages that exhibit fetal-specific attenuation of protein synthesis. Developmental changes in protein synthesis thus differentially sensitize hematopoietic stem and progenitor cells to impaired ribosome biogenesis.

Developmental stage-specific changes in immunological biomarkers in male C3H/HeN mice after early life toluene exposure

We investigated the effects of low-level toluene exposure on immunological biomarkers during different developmental stages in infant mice. Fetuses in utero in the pregnant mice on gestational day (GD) 14 as well as male pups on postnatal day (PND) 2 and PND 8 were exposed either to filtered air (control; 0 ppm), or to 5 ppm or to 50 ppm of toluene for 5 consecutive days in the whole body exposure chamber. Immunological biomarkers in the blood and spleen of PND 21 male mice were examined by ELISA, real-time RT-PCR, flow cytometry, and histological analysis. Plasma total IgG1 levels were markedly reduced in PND 21 mice exposed to 5 ppm of toluene during all developmental stages. Splenic T lymphocyte subsets and mRNA expression levels of Th1 cytokine IL-12, transcription factor T-bet, and Foxp3 were significantly suppressed in PND 21 male mice exposed to toluene during PNDs 8–12. There were changes in some immunological biomarkers in PND 42 mice exposed to toluene during PNDs 8–12. These findings indicate developmental stage-specific changes in non-specific immunological biomarkers in mice after early life toluene exposure.

Ernest Everett Just, Johannes Holtfreter, and the origin of certain concepts in embryo morphogenesis.

Ernest E. Just (1883-1941) is best known for his discovery of the "wave of negativity" that sweeps of the sea urchin egg during fertilization, and his elucidation of what are known as the fast and slow blocks to polyspermy. Just's contemporary Johannes Holtfreter (1901-1992) is known for his pioneering work in amphibian morphogenesis, which helped to lay the foundation for modern vertebrate developmental biology. This paper, after briefly describing the life and scientific contributions of Just, argues that his work and ideas strongly influenced two of the concepts for which Holtfreter is best known: tissue affinity and autoneuralization (or autoinduction). Specifically, this paper argues that, first, Just's experiments demonstrating developmental stage-specific changes in the adhesiveness of the blastomeres of cleavage embryos helped lay the foundation for Holtfreter's concept of tissue affinity and, second, Just's notion of the intrinsic irritability of the egg cell, which is evident in experimental parthenogenesis, strongly informed Holtfreter's concept of the nonspecific induction of neural tissue formation in amphibian gastrula ectoderm explants, a phenomenon known as autoinduction. Acknowledgment of these contributions by Just in no way diminishes the importance of Holtfreter's groundbreaking work. It does, however, extend the impact of Just's work into the area of embryo morphogenesis. It connects Just to Holtfreter and positions his work as an antecedent to embryo research that continues to this day.

A novel SULF1 splice variant inhibits Wnt signalling but enhances angiogenesis by opposing SULF1 activity

The importance of SULF1 in modulating the activities of multiple signalling molecules is now well established. Several studies, however, reported little or no effect of Sulf1 null mutations, questioning the relevance of this gene to in vivo development. The failure of SULF1 deletion to influence development may be predicted if one considers the involvement of a naturally occurring SULF1 antagonist, generated by alternative splicing of the same gene. We demonstrate that while the previously described SULF1 (SULF1A) enhances Wnt signalling, the novel shorter isoform (SULF1B) inhibits Wnt signalling. Our studies show developmental stage specific changes in the proportions of SULF1A and SULF1B isoforms at both the mRNA and protein levels in many developing tissues, with particularly pronounced changes in developing and adult blood vessels. Unlike SULF1A, SULF1B promotes angiogenesis and is highly expressed in endothelial cells during early blood vessel development while SULF1A predominates in mature endothelial cells. We propose that the balance of two naturally occurring SULF1 variants, with opposing functional activities, may regulate the overall net activities of multiple secreted factors and the associated signalling cascades essential for normal development and maintenance of most tissues.

Developmental Profiles of Neuroendocrine Gene Expression in the Preoptic Area of Male Rats

Reproductive function is controlled by GnRH cells and their steroid-sensitive regulatory inputs. The proper maturation of this system is critical to sexual development and maintenance of adult function. However, the molecular mechanisms underlying these developmental changes, and the potential roles of gonadal hormones in sculpting these processes, have not been fully explored. We performed a developmental profile from postnatal day (P) 1 through P60 of a network of five genes in the preoptic area (POA) that are critical to reproduction in male Sprague Dawley rats. GnRH, estrogen receptors-alpha, and -beta, androgen receptor (AR), and progesterone receptor (PR) mRNAs in the POA were assayed, and serum hormones were measured, in developing male rats. We also used a Taqman low-density array to identify candidate genes that may be important in development. Of the five targeted genes, only AR and PR changed robustly (7- and 3- to 4-fold increases, respectively) during development. All of the gonadal serum hormones changed markedly and with very different patterns from their receptor mRNAs: testosterone decreased from P1 to P30 and then increased to P60; progesterone peaked on P30; and estradiol decreased from P1 to P30. Using the Taqman low-density array, we identified several genes that changed dramatically in the POA with development, particularly G protein-coupled receptor 30, IGF-I, vitamin D receptor, estrogen-related receptor-alpha, and thyroid receptor-alpha. Our data demonstrate developmental stage-specific changes in neuroendocrine genes, particularly AR and PR. Moreover, the relationships between hormones and their corresponding receptors undergo dynamic changes across development in male rats.

Tissue- and developmental stage-specific changes in the subcellular localization of the 26S proteasome in the ovary of Drosophila melanogaster

The intracellular localization of the 26S proteasome in the different ovarian cell types of Drosophila melanogaster was studied by means of immunofluorescence staining and laser scanning microscopy, with the use of antibodies specific for regulatory complex subunits or the catalytic core of the 26S proteasome. During the previtellogenic phase of oogenesis (stages 1–6), strong cytoplasmic staining was observed in the nurse cells and follicular epithelial cells, but the proteasome was not detected in the nuclei of these cell types. The subcellular distribution of the 26S proteasome was completely different in the oocyte. Besides a constant, very faint cytoplasmic staining, there was a gradual nuclear accumulation of proteasomes during the previtellogenic phase of oogenesis. A characteristic subcellular redistribution of the 26S proteasome occurred in the ovarian cells during the vitellogenic phase of oogenesis. There was a gradual decline in the concentration of the 26S proteasome in the nucleus of the oocyte, and in the stage 10 oocyte the proteasome could barely be detected in the nucleus. This was accompanied by a massive nuclear accumulation of proteasomes in the follicular epithelial cells. These results demonstrate that the subcellular distribution of the 26S proteasome in higher eukaryotes is strictly tissue- and developmental stage-specific.

Expression of sialyl Lewis(x) and Lewis(x) defines distinct stages of chicken B cell maturation.

Commitment of cells to the B lineage in chickens occurs only during a brief period of embryogenesis. B cell progenitors then progress through discrete developmental stages resulting in the production of mature B cells that are competent to form a functioning humoral immune system in the adult bird. During embryogenesis, chicken B cell precursors undergo tissue and developmental stage-specific changes in cell-surface glycosylation. Immature B cell progenitors that migrate to the bursa of Fabricius express the carbohydrate epitope sialyl Lewis(x). Such cells undergo initial clonal expansion within the bursa without undergoing gene conversion. Beginning between days 15 and 17 of embryogenesis, B cells in the bursa undergo a tissue specific change in surface glycosylation that results in the loss of sialyl Lewis(x) expression and the acquisition of the related carbohydrate structure Lewis(x). Expression of Lewis(x) identifies B lymphocytes that have begun to undergo gene conversion. Before emigration from the bursa, bursal lymphocytes undergo another phenotypic switch in glycosylation and down-regulate Lewis(x) expression. Therefore, developmental switches in glycosylation allow us to distinguish three populations of B cells in the bursa of Fabricius at defined stages of development and suggest that regulation of cell-surface glycosylation plays a role in B cell development in the chicken.

Generation of a Monoclonal Antibody Against Avian Small Dermatan Sulfate Proteoglycan: Immunolocalization and Tissue Distribution of PG-II (Decorin) in Embryonic Tissues

Chick embryonic skeletal muscle synthesizes three major types of proteoglycans: large chondroitin sulfate proteoglycans, small dermatan sulfate proteoglycans and small heparan sulfate proteoglycans. A monoclonal antibody has been raised which recognizes the small dermatan sulfate proteoglycan. Immunblot analysis of a partially purified preparation of skeletal muscle proteoglycans indicates that the antibody reacts with a molecule which migrates with an estimated Mr of 100000. Prior treatment of the proteoglycans with chondroitinase results in immunostaining of a species of estimated Mr 45 000. These values for the intact proteoglycan and its core protein suggest that the antibody is directed against a proteoglycan of the PG-II or decorin class. Immunohistochemistry indicates a widespread distribution of the proteoglycan, which is localized in connective tissue septa of skeletal and cardiac muscle, dermis, tendon, bone, perichondrium and cornea. Immunoblot analysis of the proteoglycan core proteins from these tissues demonstrates that the antibody recognizes the same 45 000-dalton band in each tissue. The widespread tissue distribution is also consistent with the antibody being directed against an epitope of PG-11. Neither the glycosaminoglycan chains nor N-linked oligosaccharides are required for reactivity and the antibody cross-reacts with other avian material, but not mammalian. This antibody, which has been designated CB-1, reveals developmental stage-specific changes in the deposition of PG-II in embryonic limb bud and skeletal muscle.

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.

Neuron-glia interactions of rat hippocampal cells in vitro: glial- guided neuronal migration and neuronal regulation of glial differentiation

To examine neuron-glia interactions of hippocampal cells, including glial-guided neuronal migration, glial organization of neuronal positioning and neuronal regulation of astroglial differentiation, rat hippocampal tissue, harvested between embryonic day 16 (E16) and postnatal day 3 (P3), was dissociated into a single cell suspension and plated in glass coverslip microcultures (Hatten and Liem, 1981; Hatten et al., 1984). Immunostaining the cells with antibodies against the glial filament protein (AbGFP) revealed developmental stage-specific changes in the number and extent of morphological differentiation of hippocampal astroglial cells. At E16-E18, fewer than 5% of the cells were AbGFP-positive; stained cells were immature, bearing very short processes. By E19-E20, the number of stained cells increased to 15% of the total cell population. Three forms of differentiated glial cells predominated, a bipolar form bearing processes 30-50 microns, an elongated form which resembled the radial glia of hippocampus, bearing processes 120 microns in length, and a stellate form with 3 or more processes 30-50 microns in length. At P0-P3, glial morphological differentiation varied with the culture substratum; differentiated forms resembling those seen at E20 occurred on Matrigel, but not on polylysine. Quantitation of the distribution of neurons relative to AbGFP-stained glial processes revealed developmental stage-specific changes in glial organization of neuronal positioning in the cultures. In cultures of E16-E18 hippocampal cells, the neurons did not preferentially associate with astroglial cells. By E19-E20, extensive neuron-glia interactions occurred, with 80-90% of the neurons being located within 5-10 microns of a glial process. In addition to their organization of neuronal positioning, E20 hippocampal astroglial cells supported extensive neuronal migration. Migrating hippocampal neurons displayed a cytology and neuron-glia cell apposition identical to that described for migrating cerebellar granule cells in vitro (Edmondson and Hatten, 1987), closely apposing their cell soma against the hippocampal glial process and moving along the glial arm by extending a thickened, leading process. Migration was seen only along highly elongated glial profiles resembling radial glial seen in vivo. The morphological differentiation of hippocampal glial cells in vitro was dependent on cell-cell interactions with neurons. In the absence of neurons, purified hippocampal astroglia had flat, undifferentiated profiles and proliferated rapidly. The addition of hippocampal neurons rapidly arrested glial growth and induced glial process extension.

Developmentally regulated proteolytic processing of a yolk glycoprotein complex in embryos of the sea urchin, Strongylocentrotus purpuratus

We have isolated a yolk glycoprotein complex from eggs and early embryos of the sea urchin, Strongylocentrotus purpuratus. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of these complexes and peptide mapping of their individual glycoprotein components indicate that developmental stage-specific changes in molecular composition of the complex are due to proteolytic processing events. Our data revealed that a 180 kDa glycoprotein of the egg complex is separated by a single proteolytic cleavage into intermediate glycoproteins of 115 and 76 kDa early in development. By the hatched blastula stage, each of these intermediate glycoproteins has been further processed to lower molecular weight forms: the 115 kDa protein is proteolytically clipped to a 84 kDa form, perhaps through 110 and 105 kDa intermediaries, while the 76 kDa molecule is directly processed to a 65 kDa form.

Developmental stage-specific changes in lectin binding to mouse cerebellar cells in vitro

Eleven fluorescein isothiocyanate-conjugated (FITC) lectins, each with distinct carbohydrate-binding properties, were used to assess cell surface glycoconjugates of embryonic and early postnatal cerebellar cells in vitro. Fluorescence staining of embryonic day 13 (E13) cerebellar cells with FITC Ricinus communis agglutinin diminished markedly between 24 and 72 hr in vitro. No staining of postnatal day 0 (P0) or postnatal day 7 (P7) cells was observed with FITC Ricinus communis agglutinin. A similar, but less pronounced decrease in FITC concanavalin A, FITC Lens culinaris, and FITC wheat germ agglutinin was observed between embryonic day 13 and birth. No specific staining of E13, P0, or P7 cultures was observed with FITC peanut agglutinin, FITC Dolichos bifloris agglutinin, FITC soybean agglutinin, FITC Wistaria floribundis agglutinin, FITC Phaseolus vulgaris agglutinin, FITC Limulus polyphemus agglutinin, or FITC Ulex europaeusI agglutinin. Similar results were obtained with 125I-lectin binding assays. Ricinus communis 125-I-agglutinin binding decreased dramatically between embryonic day 13 and birth. Less pronounced decreases were observed in 125I-concanavalin A and wheat germ 125I-agglutinin binding. Very low levels of soybean 125I-agglutinin or Ulex europaeusI 125I-agglutinin were bound by either embryonic or early postnatal cerebellar cells in vitro.