Smoc1 and Smoc2, members of the SPARC family of genes, encode signaling molecules downstream of growth factors such as the TGF-β, FGF, and PDGF families. Smoc1 has been implicated in playing a crucial role in microphthalmia with limb anomalies in humans and mice, while Smoc2 deficiency causes dental developmental defects. Although developmental cytokines/growth factors including TGF-β superfamily have been shown to play critical roles in postnatal spermatogenesis, there are no reports analyzing the spatial and temporal expression of Smoc1 and Smoc2 in the postnatal testis. In this study, we investigated the mRNA and protein expression of Smoc1 and Smoc2 in neonatal, juvenile, and adult mouse testes by RNA in situ hybridization, immunofluorescence, and single-cell RNA-seq analysis. We show that Smoc1 and Smoc2 have distinct expression patterns in male germ cells: Smoc1 is more highly expressed than Smoc2 in the germline. In contrast, Smoc2 is highly expressed in testicular somatic cells from neonatal to juvenile stages. The Smoc2-expressing cells then switch from somatic cells to germ cells in adults. Thus, although SMOC1 and SMOC2 proteins are structurally very similar, their spatial and temporal expression patterns in the postnatal testis differ significantly, suggesting their distinct roles in reproduction.

The Hippo pathway is a critical regulator of animal development. Activation of the Hippo pathway causes a cascade of phosphorylation events that culminate in the phosphorylation of the transcriptional co-factors YAP and TAZ, which limits their entry into the nucleus. When the Hippo pathway is 'off', however, YAP and TAZ can enter the nucleus, where they interact with the transcription factors of the TEA Domain (TEAD) family to regulate transcriptional activity. Despite the importance of the Hippo pathway for development, including within the nervous system, the expression of the TEAD family remains poorly defined in mammals. Here, we mapped the expression of TEAD1 in the developing mouse brain. We find that TEAD1 expression is confined to progenitor cells during embryonic development, namely radial glia and intermediate progenitor cells. TEAD1 expression is not evident in post-mitotic neurons of the cortical plate. We also identify expression of TEAD1 in developing and mature ependymal cells of the lateral and third ventricle, including within the subcommissural organ, as well as by cells within the choroid plexuses and the forebrain neurogenic niches. Finally, we find that adult mice conditionally heterozygous for Tead1 in the central nervous system exhibit a significantly smaller brain. Collectively, these findings reveal a specific pattern of expression for TEAD1 during telencephalic development and implicate this factor in regulating neural progenitor cell proliferation.

Wnt signal is crucial to correctly regenerate tissues along the original axis in many animals. Lizards are able to regenerate their tails spontaneously, while the anterior-posterior axis information required for the successful regeneration is still elusive. In this study, we investigated the expression pattern of Wnt ligands and HOX genes during regeneration. The results of in situ hybridization revealed that Wnt6 and Wnt10A mRNA levels are higher in wound epithelium (WE) than that in blastema during regeneration. In addition, we showed that Wnt agonist positively regulated the expression of HOXA13 in cultured blastema cells, while did not show similar effect on that of HOXB13, HOXC13 and HOXD13. Finally, we found that HOXA13 showed a gradient level along the anterior-posterior axis of regenerated blastema, with higher level at the caudal end. These data proposed that Wnt6, Wnt10A and HOXA13 might play an important role in establishing distal position for regeneration.

ROBO4 involves in the stabilization of blood vessel and mediates the migration of hematopoietic stem cell and newborn neuron. However, the patterns of expression and regulation are not quite clear. To resolve this, we analyzed the single cell sequence data, and confirmed that Robo4 mainly expresses in various endothelial cells, but also in epithelial cells, pericytes, and stem or progenitor cells of bone marrow, fibroblast cells/mesenchymal stem cell of adipose tissues, muscle cells and neuron. Robo4 expressions in endothelial cells derived from capillary vessel, tip/stalk/activated endothelial cells were higher than that in artery and large vein (matured endothelial cells). On the other hand, via mining the gene expression data deposited in the NCBI Gene Expression Omnibus database as well as National Genomics Data Center (NGDC), we uncovered that the expression of Robo4 were regulated by different stimulus and variable in diseases' condition.Moreover, we constructed enhanced GFP (eGFP) transgene mouse controlled by Robo4 promoter using CRISPR/CAS9 system. We found GFP signals in many cell types from the embryonic section, confirming a widely expression of Robo4. Together, Robo4 widely and dynamically express in multiple cell types, and can be regulated by diverse factors.

Recombination activating genes (RAGs) mediates the process of rearrangement and somatic recombination (V(D)J) to generate different antibody repertoire. Studies on the expression pattern of adaptive immune genes during ontogenic development are crucial for the formulation of fish immunization strategy. In the present study, Nile tilapia was taken to explore the relative expression profile of RAG genes during their developmental stages. The developmental stages of Nile tilapia, i.e., unfertilized egg, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 days post-hatch (dph) and kidney, blood, gill, liver and spleen tissues from adult fish were collected and the cDNA synthesis was carried out. Gene specific primers for RAG-1 and RAG-2 of Nile tilapia were designed and their annealing temperature (Tm) was optimized by gradient PCR. Consequently, PCR was performed to confirm the specific amplification of RAG-1 and RAG-2 genes. Quantitative real-time PCR (qRT-PCR) gene expression of RAG-1 and RAG-2 were noticed in all the developmental stages; however, a significant increase was observed after 12 dph and peaked at 24 dph, followed by a gradual decrease until 30 dph. Tissue-specific gene expression profiling revealed that the highest expression of RAG-1 and RAG-2 was observed in the kidney, followed by spleen, gill, liver and blood. The findings of the study explored the suitable timing of lymphoid maturation that could be technically used for the adoption of strategies to improve disease resistance of fish larvae for mitigating larval mortality.

Transmembrane 9 superfamily proteins (TM9SFs) define a highly conserved protein family, each member of which is characterized by a variable extracellular domain and presumably nine transmembrane domains. Although previous studies have delineated the potential cytological roles of TM9SFs like autophagy and secretory pathway, their functions during development are largely unknown. To establish the basis for dissecting the functions of TM9SFs in vivo, we employed the open-source database, structure prediction, immunofluorescence and Western blot to describe the gene and protein expression patterns of TM9SFs in human and mouse. While TM9SFs are ubiquitously and homogeneously expressed in all tissues in human with RNA sequencing and proteomics analysis, we found that all mice Tm9sf proteins are preferentially expressed in lung except Tm9sf1 which is enriched in brain although they all distributed in various tissues we examined. In addition, we further explored their expression patterns in the mice central nervous system (CNS) and its extension tissue retina. Interestingly, we could show that Tm9sf1is developmentally up-regulated in brain. In addition, we also detected all Tm9sf proteins are located in neurons and microglia instead of astrocytes. Importantly, Tm9sf3 is localized in the nuclei which is distinct from the other members that are dominantly targeted to the plasma membrane/cytoplasm as expected. Finally, we also found that Tm9sf family members are broadly expressed in the layers of INL, OPL, and GCL of retina and likely targeted to the plasma membrane of retinal cells. Thus, our data provided a comprehensive overview of TM9SFs expression patterns, illustrating their ubiquitous roles in different organs, implying the possible roles of Tm9sf2/3/4 in lung functions and Tm9sf1 in neurodevelopment, and highlighting a unique cell biological functions of TM9SF3 in neuronal and microglia.

Amur common carp (Cyprinus carpio haematopterus), is a commercially important fish species that has been genetically improved over the years through selective breeding. Despite its significance in aquaculture, limited knowledge exists regarding its embryogenesis and immune genes associated with its early stages of life. This article represents a detailed study of the embryogenesis and innate immune gene expression analysis of the Amur common carp during its ontogenic developments. The entire embryonic developmental process of ∼44 h could be divided into eight periods, beginning with the formation of the zygote, followed by cleavage, morula, blastula, segmentation, pharyngula, and hatching. The segmentation period, which lasted for ∼ 6 h, exhibited the most significant changes, such as muscle contraction, rudimentary heart formation, increased somites number, and the initiation of blood circulation throughout the yolk. The expression of immune-related genes, namely toll-like receptor (TLR)4, nucleotide-binding oligomerization domain (NOD)1, NOD2 and interleukin (IL)-8 showed stage-specific patterns with varying levels of expression across the developmental stages. The TLR4 gene exhibited the highest expression during the neurella stage, while NOD1 and NOD2 peaked during hatching and IL-8 reached its maximum level during the gastrula stage. This is the first report of the innate immune gene expression during the embryogenesis of Amur common carp.

Except the addition of TBL1Y in human, transducing beta like 1 (TBL1) family mainly consists of two members TBL1X and TBL1XR1, taking part in multiple intracellular signaling pathways such as Wnt/β-catenin and NF-κB in cancer progression. However, the gene expression patterns of this family during embryonic development remain largely unknown. Here we took advantage of zebrafish model to characterize the spatial and temporal expression patterns of TBL1 family genes including tbl1x, tbl1xr1a and tbl1xr1b. The in situ hybridization studies of gene expression showed robust expressions of tbl1x and tbl1xr1b as maternal transcripts except tbl1xr1a. As the embryo develops, zygotic expressions of all TBL1 family members occur and have a redundant and broad pattern including in brain, neural retina, pharyngeal arches, otic vesicles, and pectoral fins. Ubiquitous expression of all family members were ranked from the strongest to the weakest: tbl1xr1a, tbl1x, and tbl1xr1b. In addition, one tbl1xr1a transcript tbl1xr1a202 showed unique and rich expression in the developing heart and lateral line neuromasts. Overall, all members of zebrafish TBL1 family shared numerous similarities and exhibited certain distinctions in the expression patterns, indicating that they might have redundant and exclusive functions to be further explored.

It can be observed from aminoglycoside-induced hair cell damage that the cochlea basal turn is more susceptible to trauma than the apex. Drug-induced hearing loss is closely related to oxidative damage. The basilar membrane directly exposed to these ototoxic drugs exhibits differences in damage, indicating that there is an inherent difference in the sensitivity to oxidative damage from the apex to the base of the cochlea. It has been reported that the morphology and characteristics of the cochlea vary from the apex to the base. Therefore, we investigated oxidative stress-related gene expression profiles in the apical, middle, and basal turns of the cochlea. The Oxidative Stress RT Profiler™ PCR Array revealed that three of the 84 genes (Mb, Mpo, and Ncf1) were upregulated in the middle turn compared to their level in the apical turn. Moreover, eight genes (Mb, Duox1, Ncf1, Ngb, Fmo2, Gpx3, Mpo, and Gstk1) were upregulated in the basal turn compared to their level in the apical turn. The qPCR verification data were similar to that of the PCR Array. We found that MPO was expressed in the rat cochlea and protected against gentamicin-induced hair cell death. This study summarized the data for the gradient of expression of oxidative stress-related genes in the cochlea and found potential candidate targets for prevention of ototoxic deafness, which may provide new insights for cochlear pathology.

In most mammals the major site of sex hormone-binding globulin (SHBG) synthesis is the liver wherefrom it is secreted into the bloodstream and is the primary determinant of sex steroid access to target tissues. The minor site of SHBG synthesis is the testis and in lower mammals testicular SHBG has long been known to be synthesized and secreted by Sertoli cells. However, human testicular SHBG is expressed in developing germ cells from an upstream alternative promoter (altP-SHBG). Transcripts arising from this region comprise an alternative first exon (1A) with the resultant protein confined to the acrosomal compartment of the mature spermatozoa. I have dissected the regulatory components of the alternative SHBG promoter and identified motifs that are required for optimal transcriptional activity from this region. Transcriptional activity is driven by two CACCC elements that appear to be functionally redundant. The transcription factor KLF4 interacts with promoter the region spanning these elements in vivo. Knockdown of Klf4 results in decreased altP-SHBG activity, while Klf4 overexpression relieves the effects of knockdown. Based on their shared patterns of expression in vivo, I conclude that KLF4 is a transcriptional regulator of SHBG in male germ cells.

Exosome Complex Components 1 and 2 (EXOSC1 and 2) are two proteins in the RNA Exosome complex whose main function is 5' → 3' RNA degradation and processing. The RNA exosome complex is comprised of nine subunits that form two separate components: the S1/KH cap and the PH-core. EXOSC1 and 2 are both part of the S1/KH cap and are involved in binding nascent RNA. As part of a systemic characterization of early lethal alleles produced by the Knockout Mouse Project, we have examined Exosc1 and Exosc2 homozygous null (mutant) embryos to determine developmental and molecular phenotypes of embryos lacking their functions. Our studies reveal that Exosc1 null embryos implant and form an egg cylinder but are developmentally delayed and fail to initiate gastrulation by embryonic day 7.5. In contrast, Exosc2 null embryos are lethal during peri-implantation stages, and while they do form a morphologically normal blastocyst at E3.5, they cannot be recovered at post-implantation stages. We show the absence of stage-specific developmental and altered lineage-specification in both Exosc1 and Exosc2 mutant embryos and conclude that these genes are essential for the successful progression through early mammalian development.

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment markers (REX1, CDX2, GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFβ inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2-7 post-insemination in goat. Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG, OCT4 and GATA4, while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4, while the combination of the three molecules significantly decreases the expression of NANOG, SOX2 and CDX2. According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4, NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat.

Plasmalemma vesicle associated protein (PLVAP) is commonly considered to be specifically expressed in endothelial cells in which it localized to diaphragms of caveolae, fenestrae, and transendothelial channels. PLVAP is reported to be an important regulator of heart development and a novel target to promote cardiac repair in the ischemic heart. However, the dynamics of plvap expression in heart development, homeostasis and pathology have not been comprehensively described. In this study, we analyzed the temporal and spatial expression of plvap in mouse heart under different conditions. We found that, during embryonic and neonatal stages, PLVAP was detected in endocardial endothelial cells, epicardial mesothelial cells, and a small amount of coronary vascular endothelial cells. In adult heart, PLVAP was also identified in endocardial cells and a few coronary vascular endothelial cells. However, epicardial expression of PLVAP was lost during postnatal heart development and cannot be detected in mouse heart by immunostaining since 3-week-old. We also analyzed the expression of plvap in a model of cardiac hypertrophy and failure induced by transverse aortic constriction surgery, and identified expression of PLVAP in endocardial cells and coronary vascular endothelial cells in the injured heart. This study provides new evidence to better understand the role of plvap in mouse heart development and injury.

Tissue fusion is a critical process that is repeated in multiple contexts during embryonic development and shares common attributes to processes such as wound healing and metastasis. Ocular coloboma is a developmental eye disorder that presents as a physical gap in the ventral eye, and is a major cause of childhood blindness. Coloboma results from fusion failure between opposing ventral retinal epithelia, but there are major knowledge gaps in our understanding of this process at the molecular and cell behavioural levels. Here we catalogue the expression of cell adhesion proteins: N-cadherin, E-cadherin, R-cadherin, ZO-1, and the EMT transcriptional activator and cadherin regulator SNAI2, in the developing chicken embryonic eye. We find that fusion pioneer cells at the edges of the fusing optic fissure have unique and dynamic expression profiles for N-cad, E-cad and ZO-1, and that these are temporally preceded by expression of SNAI2. This highlights the unique properties of these cells and indicates that regulation of cell adhesion factors may be a critical process in optic fissure closure.

Peroxidase genes (Prdx) encode a family of antioxidant proteins, which can protect cells from oxidative damage by reducing various cellular peroxides. This study investigated the spatiotemporal expression patterns of gene members in this family during the early development of Xenopus tropicalis. Real-time quantitative PCR showed that all members of this gene family have a distinct temporal expression pattern during the early development of X. tropicalis embryos. Additionally, whole mount in situ hybridization revealed that individual prdx genes display differential expression patterns, with overlapping expression in lymphatic vessels, pronephros, proximal tubule, and branchial arches. This study provides a basis for further study of the function of the prdx gene family.

Major intrinsic protein (MIP) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. The pathogenic mechanism behind the loss of MIP function in the lens, which leads to degraded optical quality and cataract formation, is still unclear. In this study, a zebrafish model with the mipb mutant was produced. The expression of mipb mRNA and protein was dramatically reduced in the mutant. Immunological analysis reveals that loss function of mip leads to the diffuse distribution of ZL-1 in the mutant lens. Furthermore, in situ hybridization reveals that mip knockout results in a decrease in the transcripts of beaded filament structural protein 2 (Bfsp2) in the lens. Histology study shows that lens fibers in the mutants are less uniform in shape and the fiber arrangement is disrupted. The presented data provides evidence for the essential role of mipb in the development of lens fibers. The absence of mipb during lens formation is likely to result in aberrant lens fiber formation and impaired lens function.

ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.

Development of the mammalian telencephalon, which is the most complex region of the central nervous system, is precisely orchestrated by many signaling molecules. Wnt signaling derived from the cortical hem, a signaling center, is crucial for telencephalic development including cortical patterning and the induction of hippocampal development. Secreted protein R-spondin (Rspo) 1-4 and their receptors, leucine-rich repeat-containing G-protein-coupled receptor (Lgr) 4-6, act as activators of Wnt signaling. Although Rspo expression in the hem during the early stages of cortical development has been reported, comparative expression analysis of Rspos and Lgr4-6 has not been performed. In this study, we examined the detailed spatiotemporal expression patterns of Rspo1-4 and Lgr4-6 in the embryonic and postnatal telencephalon to elucidate their functions. In the embryonic day (E) 10.5-14.5 telencephalon, Rspo1-3 were prominently expressed in the cortical hem. Among their receptors, Lgr4 was observed in the ventral telencephalon, and Lgr6 was highly expressed throughout the telencephalon at the same stages. This suggests that Rspo1-3 and Lgr4 initially regulate telencephalic development in restricted regions, whereas Lgr6 functions broadly. From the late embryonic stage, the expression areas of Rspo1-3 and Lgr4-6 dramatically expanded; their expression was found in the neocortex and limbic system, such as the hippocampus, amygdala, and striatum. Increased Rspo and Lgr expression from the late embryonic stages suggests broad roles of Rspo signaling in telencephalic development. Furthermore, the Lgr regions were located far from the Rspo regions, especially in the E10.5-14.5 ventral telencephalon, suggesting that Lgrs act via a Rspo-independent pathway.

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a conserved RNA binding protein (RBP) that plays an important role in the alternative splicing of gene transcripts, and thus in the generation of specific protein isoforms. Global deficiency in hnRNPL in mice results in preimplantation embryonic lethality at embryonic day (E) 3.5. To begin to understand the contribution of hnRNPL-regulated pathways in the normal development of the embryo and placenta, we determined hnRNPL expression profile and subcellular localization throughout development. Proteome and Western blot analyses were employed to determine hnRNPL abundance between E3.5 and E17.5. Histological analyses supported that the embryo and implantation site display distinct hnRNPL localization patterns. In the fully developed mouse placenta, nuclear hnRNPL was observed broadly in trophoblasts, whereas within the implantation site a discrete subset of cells showed hnRNPL outside the nucleus. In the first-trimester human placenta, hnRNPL was detected in the undifferentiated cytotrophoblasts, suggesting a role for this factor in trophoblast progenitors. Parallel in vitro studies utilizing Htr8 and Jeg3 cell lines confirmed expression of hnRNPL in cellular models of human trophoblasts. These studies [support] coordinated regulation of hnRNPL during the normal developmental program in the mammalian embryo and placenta.