Early Embryonic Stages Research Articles

ScMultiome analysis identifies a single caudal hindbrain compartment in the developing zebrafish nervous system

BackgroundA key step in nervous system development involves the coordinated control of neural progenitor specification and positioning. A long-standing model for the vertebrate CNS postulates that transient anatomical compartments – known as neuromeres – function to position neural progenitors along the embryonic anteroposterior neuraxis. Such neuromeres are apparent in the embryonic hindbrain – that contains six rhombomeres with morphologically apparent boundaries – but other neuromeres lack clear morphological boundaries and have instead been defined by different criteria, such as differences in gene expression patterns and the outcomes of transplantation experiments. Accordingly, the caudal hindbrain (CHB) posterior to rhombomere (r) 6 has been variably proposed to contain from two to five ‘pseudo-rhombomeres’, but the lack of comprehensive molecular data has precluded a detailed definition of such structures.MethodsWe used single-cell Multiome analysis, which allows simultaneous characterization of gene expression and chromatin state of individual cell nuclei, to identify and characterize CHB progenitors in the developing zebrafish CNS.ResultsWe identified CHB progenitors as a transcriptionally distinct population, that also possesses a unique profile of accessible transcription factor binding motifs, relative to both r6 and the spinal cord. This CHB population can be subdivided along its dorsoventral axis based on molecular characteristics, but we do not find any molecular evidence that it contains multiple pseudo-rhombomeres. We further observe that the CHB is closely related to r6 at the earliest embryonic stages, but becomes more divergent over time, and that it is defined by a unique gene regulatory network.ConclusionsWe conclude that the early CHB represents a single neuromere compartment that cannot be molecularly subdivided into pseudo-rhombomeres and that it may share an embryonic origin with r6.

Sonic hedgehog (shh) gene from Pseudopleuronectes yokohamae (Teleostei: Pleuronectidae): Molecular cloning, characterization, and expression profile during early embryonic, juvenile, and adult stages.

The hedgehog signaling pathway plays an important role in early development and growth of most vertebrates. Sonic hedgehog (shh) gene is a critical regulator of embryonic development in many species, including humans. However, it is not clear what roles shh can play in the development of fish. In this paper, shh gene was cloned from Pseudopleuronectes yokohamae. The full-length complementary DNA (cDNA) of P. yokohamae sonic hedgehog gene (Pyshh) comprises 3194 bp, with a 1317-bp open reading frame (ORF) that encodes a polypeptide of 438 amino acids with a typical HH-signal domain and Hint-N domain. The conserved sequences of the protein among species were predicted by using multiple sequence comparison. The phylogenetic tree construction showed that PySHH is clustered in a branch of Pleuronectidae. To explore the expression of Pyshh gene in various tissues of P. yokohamae, we used real-time fluorescence quantitative PCR technology to detect it. The results showed that Pyshh gene is widely distributed in various tissues of P. yokohamae juveniles, different tissues of adult males and females, and is particularly expressed in immune organs. The Pyshh gene expression was higher in the muscle and brain of juvenile fish, and higher in bone, gill, and skin of male fish than that of female fish, suggesting that Pyshh might be involved in the formation of immune organs of P. yokohamae. The expression of Pyshh gene significantly upregulated from the gastrula stage to the hatching stage. Western blotting of the expression levels of PySHH during different embryonic development stages revealed that PySHH levels increased gradually during development stages from oosperm stage to hatching stage. These results indicate that Pyshh is highly conserved among species and plays a critical role in the complex process of embryonic development. Its precise regulation is essential for the proper formation of many organs and tissues in the body, and disruptions in its function may have serious consequences for the formation of immune organs in fish.

P-159 Single-cell high-throughput methylation and transcriptomic analysis indicates variations in sensitivity to Folate-mediated one-carbon metabolism in mouse preimplantation embryos

Abstract Study question Do Folate-mediated one-carbon metabolism (FOCM) supplements impose methylating conditions that influence methylation and transcriptomic profiles during preimplantation period of mouse embryos? Summary answer Subtle differences in embryo methylation profiles were induced by FOCM supplements, despite transcriptomic signatures remained stable and selective genes related to metabolism were activated. What is known already Previous human studies of populations affected by seasonal variations in diet including folate availability have shown methylation changes in specific loci known as metastable epialleles (MEs). MEs exhibit stability across tissues, indicating these could be potential biosensors for heightened sensitivity to external methylating influences in early embryonic stages. Despite significant progress in IVF embryo culture conditions, the sensitivity to the outer metabolic environment during the extensive genomic transformation of embryo reprogramming remains inadequately understood. Study design, size, duration This is a prospective experimental study conducted from January 2023 to December 2023. A total of 111 fresh mouse zygotes (F1 hybrid (B6/CBA)) were subjected embryo culture conditions with different FOCM supplies of 5-MTHF and betaine up to expanded blastocyst stage (105 hours). At different timepoints, individual embryos were collected and disaggregated in individual cells for downstream single-cell analysis. Participants/materials, setting, methods Mouse zygotes were collected from the oviduct, washed and culture in KSOM supplemented with 5-MTHF and betaine at respectively concentrations of 50μM and 50μg/mL (THF/bet(50)), 10μM and 10μg/mL (THF/bet(10)) and no supplement till blastocyst stage at 37 °C, 6%CO2 and 5%O2. Individual embryos were collected at 2 cells, 4 cells, 8 cells, Morula and Blastocyst stage and disaggregated into single cells for subsequent single-cell RNAseq (SmartSeq2) and single-cell methylome analysis through Splinted Ligation Adapter Tagging (scSPLAT). Main results and the role of chance The methylation analysis conducted though scSPLAT revealed a comprehensive reduction in global methylation levels throughout developmental stages. Notably, zygotes exhibited a maximal proportion of methylated cytosines at CpG sites, while blastocysts displayed minimal methylation levels, with sporadic increases in isolated cells. Quantitative profiling indicated lower methylation intensities in treated embryos from zygote to 4 cells, followed by a reversal trend from 8 cells to blastocyst, suggestive of an enhanced assimilation of one-carbon metabolism (FOCM) supplements. Transcriptomic profiles obtained through scRNAseq exhibited correlation with developmental trajectories from zygote to blastocyst. However, no discernible variations were attributed to the combined exposure of Betaine and 5-MTHF. Gene enrichment analysis of differentially expressed genes at various timepoints highlighted specific metabolic responses, including mitochondrial oxidative stress and variations in glutathione metabolism. This suggests that the folate forms utilized in this study may possess a protective role as antioxidants, contributing to the observed effects on gene expression during embryonic development. Limitations, reasons for caution The methylation analysis shown in this study was a pilot carried out with low coverage of sequencing deepness, further analysis are ongoing to increase reads coverage. Wider implications of the findings Exposure of mouse embryos to FOCM enhanced global methylation during cell division from 8 cells to blastocyst, indicating heightened assimilation during DNA synthesis. While the transcriptomic signature remained stable, increased expression of genes linked to mitochondrial oxidative stress and glutathione metabolism suggests FOCM’s potential antioxidant role. Trial registration number not applicable

Atypical Course of the Habenulo-Interpeduncular Tract in Chick Embryos.

Classical studies of the avian diencephalon hardly mention the habenulo-interpeduncular tract (a.k.a. retroflex tract), although both the habenula (HB) (its origin) and the interpeduncular nuclear complex (its target) are present. Retroflex tract fibers were described at early embryonic stages but seem absent in the adult in routine stains. However, this tract is a salient diencephalic landmark in all other vertebrate lineages. It typically emerges out of the caudal HB, courses dorsoventrally across thalamic alar and basal plates just in front of the thalamo-pretectal boundary, and then sharply bends 90° caudalwards at paramedian basal plate levels (this is the "retroflexion"), to approach longitudinally via paramedian pretectum and midbrain the rostralmost hindbrain, specifically the prepontine median interpeduncular complex across isthmus and rhombomere 1. We systematize this habenulo-interpeduncular course into four parts named subhabenular, retrothalamic, tegmental, and interpeduncular. We reexamined the chicken habenulo-interpeduncular fibers at stages HH30 and HH35 (6.5- and 9-day incubation) by mapping them specifically with immunoreaction for BEN protein, a well-known marker. We found that only a small fraction of the stained retroflex tract fibers approaches the basal plate by coursing along the standard dorsoventral pathway in front of the thalamo-pretectal boundary. Many other habenular fibers instead diverge into atypical dispersed courses across the thalamic cell mass (implying alteration of the first subhabenular part of the standard course) before reaching the basal plate; this dispersion explains their invisibility. A significant number of such transthalamic habenular fibers cross orthogonally the zona limitans (ZLI) (the rostral thalamic boundary) and invade the caudal alar prethalamus. Here, they immediately descend dorsoventrally, just rostrally to the ZLI, until reaching the prethalamic basal plate, where they bend (retroflex) caudalwards, entering the thalamic basal paramedian area. These atypical fibers gradually fasciculate with the other groups of habenular efferent fibers in their final longitudinal approach to the hindbrain interpeduncular complex. We conclude that the poor visibility of this tract in birds is due to its dispersion into a diversity of atypical alternative routes, though all components eventually reach the interpeduncular complex. This case merits further analysis of the diverse permissive versus nonpermissive guidance mechanisms called into action, which partially correlate distinctly with successive diencephalic, mesencephalic, and hindbrain neuromeric fields and their boundaries.

Ultra-high static magnetic fields altered the embryonic division and development in Caenorhabditis elegans via multipolar spindles

IntroductionUltra-high static magnetic fields (SMFs) have unique advantages in improving medical and academic research. However, the research on the early embryo exposure of ultra-high SMFs is minimal, extensive exploration is indispensable in living organisms. ObjectivesThe present study was aimed to study the effects of ultra-high SMFs on the early embryonic division and development of Caenorhabditis elegans (C. elegans). MethodsEarly adult parents containing fertilized eggs in vivo were exposed to SMFs at intensities ranging from 4 T to 27 T. The number of mitotic cells in the reproductive glands of the P0 worms, early embryonic cell spindle localization, embryo hatching and the reproductive as well as developmental indicators of F1 and F2 nematodes were examined as endpoints. ResultsOur results indicated that ultra-high SMFs has no obvious effect on the germ cell cycle, while 14 T and 27 T SMFs significantly increased the proportion of multi-polar spindle formation in early embryonic cells, and reduced the developmental rate and lifespan of C. elegans exposed at the embryonic stage. Spindle abnormalities of early embryonic cells, as well as the down-regulation of genes related to asymmetric embryonic division and the abnormal expression of the non-muscle myosin NMY-2 in the division grooves played a critical role in the slowing down of embryonic development induced by ultra-high SMFs. ConclusionsThis study provided novel information and a new sight for evaluating the biosafety assessment by exposure to ultra-high SMFs at the early embryonic stage in vivo.

Netrin-1 Is an Important Mediator in Microglia Migration.

Microglia migrate to the cerebral cortex during early embryonic stages. However, the precise mechanisms underlying microglia migration remain incompletely understood. As an extracellular matrix protein, Netrin-1 is involved in modulating the motility of diverse cells. In this paper, we found that Netrin-1 promoted microglial BV2 cell migration in vitro. Mechanism studies indicated that the activation of GSK3β activity contributed to Netrin-1-mediated microglia migration. Furthermore, Integrin α6/β1 might be the relevant receptor. Single-cell data analysis revealed the higher expression of Integrin α6 subunit and β1 subunit in microglia in comparison with classical receptors, including Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam. Microscale thermophoresis (MST) measurement confirmed the high binding affinity between Integrin α6/β1 and Netrin-1. Importantly, activation of Integrin α6/β1 with IKVAV peptides mirrored the microglia migration and GSK3 activation induced by Netrin-1. Finally, conditional knockout (CKO) of Netrin-1 in radial glial cells and their progeny led to a reduction in microglia population in the cerebral cortex at early developmental stages. Together, our findings highlight the role of Netrin-1 in microglia migration and underscore its therapeutic potential in microglia-related brain diseases.

Gene regulatory landscape of cerebral cortex folding.

Folding of the cerebral cortex is a key aspect of mammalian brain development and evolution, and defects are linked to severe neurological disorders. Primary folding occurs in highly stereotyped patterns that are predefined in the cortical germinal zones by a transcriptomic protomap. The gene regulatory landscape governing the emergence of this folding protomap remains unknown. We characterized the spatiotemporal dynamics of gene expression and active epigenetic landscape (H3K27ac) across prospective folds and fissures in ferret. Our results show that the transcriptomic protomap begins to emerge at early embryonic stages, and it involves cell-fate signaling pathways. The H3K27ac landscape reveals developmental cell-fate restriction and engages known developmental regulators, including the transcription factor Cux2. Manipulating Cux2 expression in cortical progenitors changed their proliferation and the folding pattern in ferret, caused by selective transcriptional changes as revealed by single-cell RNA sequencing analyses. Our findings highlight the key relevance of epigenetic mechanisms in defining the patterns of cerebral cortex folding.

Functional characterization of irf3 against viral hemorrhagic septicemia virus infection using a CRISPR/Cas9-mediated zebrafish knockout model

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.

Multigenerational Effect of Heat Stress on the Drosophila melanogaster Sperm Proteome.

The effect of the parental environment on offspring through non-DNA sequence-based mechanisms, such as DNA methylation, chromatin modifications, noncoding RNAs, and proteins, could only be established after the conception of "epigenetics". These effects are now broadly referred to as multigenerational epigenetic effects. Despite accumulating evidence of male gamete-mediated multigenerational epigenetic inheritance, little is known about the factors that underlie heat stress-induced multigenerational epigenetic inheritance via the male germline in Drosophila. In this study, we address this gap by utilizing an established heat stress paradigm in Drosophila and investigating its multigenerational effect on the sperm proteome. Our findings indicate that multigenerational heat stress during the early embryonic stage significantly influences proteins in the sperm associated with translation, chromatin organization, microtubule-based processes, and the generation of metabolites and energy. Assessment of life-history traits revealed that reproductive fitness and stress tolerance remained unaffected by multigenerational heat stress. Our study offers initial insights into the chromatin-based epigenetic mechanisms as a plausible means of transmitting heat stress memory through the male germline in Drosophila. Furthermore, it sheds light on the repercussions of early embryonic heat stress on male reproductive potential. The data sets from this study are available at the ProteomeXchange Consortium under the identifier PXD037488.

Abstract 1123: The Functional Roles Of Cxcr4 In Lymphangiogenesis

Introduction: The lymphatic vessels play important roles in maintaining tissue fluid homeostasis, intestinal lipid uptake and immune surveillance. Malformation or dysfunction of these vessels are associated with a number of diseases, including lymphedema, inflammation, fibrosis, myocardial infarction and atherosclerosis etc. Recent studies have shown that stimulation of lymphatic vessel growth could prevent lymphedema and improve many disease pathologies, highlighting targeting lymphatic growth as a novel therapeutic approach for lymphatic related disease. Hypothesis: Lymphatic vessels grow by active sprouting, and subsequently mature into a vascular complex including lymphatic capillaries and collecting vessels that ensure fluid transport. However, the mechanisms regulate lymphatic sprouting and patterning are largely unknown. Here we have identified one of the key regulators and signaling pathways involved in lymphangiogenesis and function during development and disease especially CXCL12(SDF-1)/Cxcr4, a novel chemokine signaling, that plays critical roles in regulating lymphatic sprouting and morphogenesis during development. Results: We showed that Cxcr4 is highly expressed in migrating and sprouting lymphatic endothelial cells (LECs) during early embryonic stages. Importantly, LEC specific CXCR4 deficient embryos develop severe edema, with impaired lymphatic sprouting and migration. In addition, LEC specific Cxcr4 deficient embryos show reduced cell surface VEGFR3 but increased intracellular VEGFR3 levels in lymphatic vessels.In vitro studies showed that the blockade of Cxcr4 activity affects VEGFC-mediated LEC function. SDF-1 treatment stimulated LEC sprouting, migration and tube formation, and these effects were greatly inhibited by AMD3100 treatment which is Cxcr4 antagonist. Conclusions: These data suggest Cxcr4 has a novel role in regulating lymphangiogenesis, and identifying the molecular mechanism that pave the way for novel therapeutic strategies in lymphatic related disorders and cardiovascular diseases.

HH5 double-carrier embryos fail to progress through early conceptus elongation

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause preterm mortality. Holstein haplotype 5 (HH5) is a deleterious haplotype present in the Holstein Friesian population that involves the ablation of the transcription factor B1 mitochondrial (TFB1M) gene. The developmental stage at which HH5 double-carrier (DC, homozygous) embryos or fetuses die remains unknown and this is a relevant information to estimate the economic losses associated with the inadvertent cross between carriers. To determine whether HH5 DC survive to maternal recognition of pregnancy, embryonic day (E) 14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. Double-carrier E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic reduction of their extra-embryonic membranes, which were >26-fold shorter than those of carrier or noncarrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos-functionally equivalent to DC embryos-were generated by clustered regularly interspaced short palindromic repeats (CRISPR) technology and cultured to the blastocyst stage, in vitro culture day (D) 8, and to the early embryonic disc stage, D12. No significant effect of TFB1M ablation was observed on the differentiation and proliferation of embryonic lineages and relative mitochondrial DNA (mtDNA) content up to D12. In conclusion, HH5 DC embryos are able to develop to early embryonic disc stage but fail to undergo early conceptus elongation, which is required for pregnancy recognition.

The biological modeling of autism spectrum disorders

IntroductionAutism Spectrum Disorders (ASD) are heterogeneous pathological conditions characterized by difficulties in establishing social contacts and the manifestation of repetitive behavior. An atypical trajectory of brain maturation, impaired neurogenesis, synaptogenesis, and an imbalance in the excitatory and inhibitory systems of the CNS form the morphofunctional basis of the ASDObjectivesscientific publicationsMethodsscientific analysisResultsThese pathological changes appear at different stages of brain maturation They are the result of multifactorial environmental influences. To understand the functioning of this complexly organized system in time and space, a three-dimensional model is needed. The closest in vitro model of the human brain from early embryonic stages to aging is brain organoids. Human brain organoids are self-organizing three-dimensional cell aggregates derived from pluripotent stem cells. Organoids summarize neurogenesis, gliogenesis, synaptogenesis, cell migration and cell differentiation, gyrification of the cerebral cortex, reflect the connections of brain regions. The use of a 3D brain model makes it possible to simulate diseases, reactions to drugs in cells obtained from patients. The use of telencephalon organoids in the ASD model revealed that neuronal migration deficiency, acceleration and disruption of cell cycle synchronization, aberrant cell proliferation, abundant synaptogenesis, temporary deviations in the development of the cortex, increased branching of neurons, unbalanced inhibitory differentiation of neurons, high activity of ion channels are the result of impaired activity FOXG1. FOXG1 is responsible for the overproduction of GABAergic neurons. The shift towards GABAergic neurons induced by FOXG1 is positively correlated with the severity of ASD symptoms and is seen as a precursor to the future of ASDConclusionsThus, ASD as a socially significant disease with a heterogeneous type of inheritance, multi-link pathogenesis, realized in different periods of ontogenesis and involving different brain loci, requires special attention of researchers for the personification of diagnosis and therapy. The hiPSCs can provide insight into the cellular mechanisms underlying ASD as a neuropsychiatric disorder, providing access to the development of platforms for in vitro drug screening and patient-tailored therapy.Disclosure of InterestNone Declared

Exploring Altitude's Influence: Contrasting Embryonic Development of Hyla annectans Jerdon in High and Low Altitude Environments

The study investigated the embryonic development of Hyla annectans Jerdon, a frog species, at high and low altitude breeding sites of Meghalaya, North East India. Results revealed significant differences in the duration of embryonic development and hatching between the two altitudes. Embryos at low altitude sites exhibited a faster development, hatching approximately four days earlier than those at high altitude sites. Early embryonic stages displayed similar characteristics in size between the two sites, but distinct differences emerged during later stages, notably in stages associated with heart development, muscular response, and gill circulation. Furthermore, metamorphosis completion varied between altitudes, with a shorter duration observed at low altitude compared to high altitude. Environmental conditions, particularly water properties, also differed significantly between the two sites. Water temperature and free carbon dioxide levels were higher at low altitude, while pH and dissolved oxygen levels were higher at high altitude. These findings suggest a potential influence of altitudinal gradients as environmental factors on embryonic development in H. annectans.

Spatial organization of astrocyte clones: The role of developmental progenitor timing.

Astrocytes represent a diverse and morphologically complex group of glial cells critical for shaping and maintaining nervous system homeostasis, as well as responding to injuries. Understanding the origins of astroglial heterogeneity, originated from a limited number of progenitors, has been the focus of many studies. Most of these investigations have centered on protoplasmic and pial astrocytes, while the clonal relationship of fibrous astrocytes or juxtavascular astrocytes has remained relatively unexplored. In this study, we sought to elucidate the morphological diversity and clonal distribution of astrocytes across adult cortical layers, with particular emphasis on their ontogenetic origins. Using the StarTrack lineage tracing tool, we explored the characteristics of adult astroglial clones derived from single and specific progenitors at various embryonic stages. Our results revealed a heterogeneous spatial distribution of astroglial clones, characterized by variations in location, clonal size, and rostro-caudal dispersion. While a considerable proportion of clones were confined within specific cortical layers, others displayed sibling cells crossing layer boundaries. Notably, we observed a correlation between clone location and developmental stage at earlier embryonic stages, although this relationship diminished in later stages. Fibrous astrocyte clones were exclusively confined to the corpus callosum. In contrast, protoplasmic or juxtavascular clones were located in either the upper or lower cortical layers, with certain clones displayed sibling cells distributed across both regions. Our findings underscore the developmental origins and spatial distribution of astroglial clones within cortical layers, providing new insights into the interplay between their morphology, clonal sizes, and progenitor heterogeneity.

Extraction of Innate Immune Genes in Dairy Cattle and the Regulation of Their Expression in Early Embryos.

As more and more of the available genomic data have been published, several databases have been developed for deciphering early mammalian embryogenesis; however, less research has been conducted on the regulation of the expression of natural immunity genes during early embryonic development in dairy cows. To this end, we explored the regulatory mechanism of innate immunity genes at the whole-genome level. Based on comparative genomics, 1473 innate immunity genes in cattle were obtained by collecting the latest reports on human innate immunity genes and updated bovine genome data for comparison, and a preliminary database of bovine innate immunity genes was constructed. In order to determine the regulatory mechanism of innate immune genes in dairy cattle early embryos, we conducted weighted co-expression network analysis of the innate immune genes at different developmental stages of dairy cattle early embryos. The results showed that specific module-related genes were significantly enriched in the MAPK signaling pathway. Protein-protein interaction (PPI) analysis showed gene interactions in each specific module, and 10 of the highest connectivity genes were chosen as potential hub genes. Finally, combined with the results for differential expressed genes (DEGs), ATF3, IL6, CD8A, CD69, CD86, HCK, ERBB3, LCK, ITGB2, LYN, and ERBB2 were identified as the key genes of innate immunity in dairy cattle early embryos. In conclusion, the bovine innate immunity gene set was determined and the co-expression network of innate immunity genes in the early embryonic stage of dairy cattle was constructed by comparing and analyzing the whole genome of bovines and humans. The findings in this study provide the basis for exploring the involvement and regulation of innate immune genes in the early embryonic development of dairy cattle.

What can we learn about fish neutrophil and macrophage response to immune challenge from studies in zebrafish

Fish rely, to a high degree, on the innate immune system to protect them against the constant exposure to potential pathogenic invasion from the surrounding water during homeostasis and injury. Zebrafish larvae have emerged as an outstanding model organism for immunity. The cellular component of zebrafish innate immunity is similar to the mammalian innate immune system and has a high degree of sophistication due to the needs of living in an aquatic environment from early embryonic stages of life. Innate immune cells (leukocytes), including neutrophils and macrophages, have major roles in protecting zebrafish against pathogens, as well as being essential for proper wound healing and regeneration. Zebrafish larvae are visually transparent, with unprecedented in vivo microscopy opportunities that, in combination with transgenic immune reporter lines, have permitted visualisation of the functions of these cells when zebrafish are exposed to bacterial, viral and parasitic infections, as well as during injury and healing. Recent findings indicate that leukocytes are even more complex than previously anticipated and are essential for inflammation, infection control, and subsequent wound healing and regeneration.

Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

Regulation of early spermatogenesis in the giant prawn Macrobrachium rosenbergii by a GCL homolog†.

The germ cell-less gene is crucial for gonad development in various organisms. Early interventions in its expression suggested a regulatory role at the mitotic stages of spermatogenesis, and its early knockout resulted in complete sterility in Drosophila. Genomic and transcriptomic data available for the catadromous giant prawn Macrobrachium rosenbergii enabled the identification of a germ cell-less homolog for this species, which we termed MroGCL (mRNA accession number OQ533056). An open reading frame containing 494 amino acids and a typical evolutionarily conserved BTB/POZ domain suggests possible protein-protein interaction functions in keeping with the Drosophila germ cell-less protein. Genomic mapping of MroGCL showed a full length of 120896 bases. Analysis of the temporal expression of MroGCL showed constant expression in early prawn embryonic and larval stages, but a significant increase 10days after metamorphosis when crucial sexual differentiation processes occur in prawns. In adult animals, high expression was detected in the gonads compared to the somatic tissues. RNAi-based knock-down experiments showed that both the silenced and control groups reached advanced spermatogenic stages, but that there was a significant decrease in the yield of spermatozoa in about half of the silenced animals. This finding supports our hypothesis that MroGCL is crucial for mitosis during early stage spermatogenesis. In conclusion, this study contributes to the understanding of crustacean gonad development and provides a stepping stone in the development of environmentally valuable sterile crustacean populations.

Acidosis defense mechanisms in the preimplantation stages of embryos in BALB/c strain mice

Regulation of intracellular pH (pHi) is an important homeostatic function of cells. There are three major pHi regulatory mechanisms: the HCO3−/Cl− exchanger (AE), which alleviates alkalosis, and the Na+/H+ exchanger (NHE) and Na+,HCO3−/Cl− exchanger (NDBCE), both of which counteract acidosis. NHE activity, which is high at the germinal vesicle stage of oocyte, is inhibited during meiotic maturation, while this inhibition is abolished when the oocyte reaches the pronuclear (PN) stage of the zygote. On the other hand, we have previously found that NDBCE performs complementary regulation against acidosis during meiotic maturation. Additionally, we found that AE activity, which is a defense mechanism against alkalosis, gradually decreases during preimplantation period of embryonic development. Considering that NHE activity is inhibited during meiotic maturation and AE activity gradually decreases during embryonic development stages, we investigated whether NHE and NDBCE activities, both of which act against acidosis, functionally change from the PN zygote to the blastocyst stage of the embryo and identified these pH-regulating proteins at the molecular level in mice of the Balb/c strain. PN zygotes, two-cell (2-c), four-cell (4-c), morula and blastocyst stage embryos were obtained from 5-8-week-old, sexually mature female Balb/c mice by using the classical superovulation procedure. pHi was recorded by using the microspectrofluorometric technique on zygotes and embryos simultaneously loaded with the pH-sensitive fluorophore, 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The activities of NHE and NDBCE were determined from the recovery curve of induced-acidosis in bicarbonate-free and bicarbonate-containing media, respectively. Specific inhibitors such as cariporide (1 μM), S3226 (1 and 10 μM), EIPA (1, 5, and 25 μM), and amiloride (1 mM) were used to functionally identify NHE isoforms, and the nonspecific inhibitor 4,4′–diisocyanatostilbene-2,2′ disulphonic acid, disodium salt (DIDS) was used to confirm NDBCE activity. The isoforms of the pHi-regulatory proteins were also identified by molecular biology using real-time PCR. We found that NHE activity was high at all embryonic stages, and differences between stages were not significant. Functional and molecular findings indicated that isoforms of NHE 1 and 5 are present in the blastocyst, whereas isoforms of NHE 1, 3, and 4 are functional at earlier embryonic stages. Although the contribution of NDBCE activity to recovery from induced-acidosis was detected at all embryonic stages, it was significant only in the PN zygote and the 2-c embryo. This finding was confirmed by molecular analysis, which detected the expression of SLC4A8 encoding NDBCE at all embryonic stages. In conclusion, NHE is an active and important defense mechanism against acidosis and is encoded by at least two protein isoforms in all stages of the Balb/c strain of mice. NDBCE has a supportive function in all embryonic stages, especially in the PN zygote and the 2-c embryo. Preimplantation stage embryos have effective mechanisms to defend against acidosis in response to their metabolic end products (increased acid load) and the acidic environment in utero.