213 SAGE ANALYSIS OF TRANSITIONS IN THE PORCINE CONCEPTUS TRANSCRIPTOME DURING TROPHECTODERM ELONGATION

Rapid elongation of the pre-implantation conceptus trophectoderm between gestational days 11 and 12 represents a critical developmental period in swine. Serial analysis of gene expression (SAGE) in ovoid (Ovd; 7 mm diameter) and filamentous (Fil; 150 mm length) conceptuses has identified unique transcriptomes associated with these transitional states. The initial utility of SAGE prompted an investigation of the intermediate tubular conceptus (Tub; 15–20 mm length) as well as the development of micro-SAGE methodologies, small-amplified RNA-SAGE (SAR-SAGE) and PCR-amplified SAGE (m-SAGE), for future use with early pre-implantation conceptus stages. Total RNA from in vivo-derived Tub conceptuses was used to construct an unamplified SAGE library (Tub SAGE) and two micro-SAGE libraries (160X less template RNA) by SAR-SAGE and m-SAGE. Comparative analyses of the amplification proportionality between the Tub SAGE and amplified libraries, consisting of 12,000 tags each, demonstrated that SAR-SAGE was a more reliable amplification method. Seventy-five percent of the SAR-SAGE tags, in contrast to 43% of m-SAGE tags, had less than a 2-fold frequency difference when compared to Tub SAGE tags occurring at least 10 times in a library. The Tub SAGE library size was extended to a total of 42,415 tags representing 12,779 unique putative transcripts. Comparing the Tub SAGE tags with previously generated Ovd and Fil SAGE libraries (NCBI GEO Acc. Nos. GSM24470 and GSM24471, respectively) yielded a total of 33,191 unique tags, of which 3,575 tags (10.8%) were common to all three libraries. Chi-square-based analyses (SAGE2000 software; 1997 Gen. Res. 7, 986–995) of the tag frequencies revealed the differential expression of 479 tags between Ovd:Tub libraries and 362 tags between Tub:Fil libraries at a P < 0.05 significance. The Tub SAGE tags were annotated following batch BLASTS against the TIGR porcine index database and then classified according to function. Real-time RT-PCR was used to confirm the differential expression patterns identified between Ovd:Tub, Ovd:Fil, and Fil:Tub. For example, the relative quantity of mRNA for steroidogenic acute regulatory protein, 1.0:6.4:11.1 (Ovd:Tub:Fil), and cytokeratin 8, 1.1:0.6:0.4 (Ovd:Tub:Fil), demonstrated an increase and decrease (ANOVA; P < 0.05), respectively, throughout these three developmental stages. Based on their putative functional annotations, the differentially expressed genes revealed by SAGE are potentially involved in regulating embryo growth, cellular differentiation, apoptosis, steroidogenesis, and energy metabolism during embryo elongation. The present SAGE analyses have elucidated the tubular conceptus transcriptome and enabled the identification of genes that are differentially regulated during the 24-h period that encompasses pig embryo elongation. Furthermore, the decreased template requirement of SAR-SAGE makes this technique a suitable option for SAGE analyses of early stage embryos where the sample is small and limited.

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Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽

10.3390/plants10040776 ◽

2021 ◽

Vol 10 (4) ◽

pp. 776

Author(s):

Shipra Kumari ◽

Bashistha Kumar Kanth ◽

Ju young Ahn ◽

Jong Hwa Kim ◽

Geung-Joo Lee

Keyword(s):

Abiotic Stress ◽

Biotic Stress ◽

Developmental Stages ◽

Expression Patterns ◽

Lilium Longiflorum ◽

Biotic And Abiotic Stress ◽

Biotic Stresses ◽

Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

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p-FAK-Tyr397 regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00254.2013 ◽

2013 ◽

Vol 305 (6) ◽

pp. E687-E699 ◽

Cited By ~ 37

Author(s):

Hin-Ting Wan ◽

Dolores D. Mruk ◽

Stephen Y. T. Li ◽

Ka-Wai Mok ◽

Will M. Lee ◽

...

Keyword(s):

Actin Polymerization ◽

Early Stage ◽

Regulatory Protein ◽

Growth Factor Receptor ◽

Actin Organization ◽

Rat Testis ◽

Mammalian Expression ◽

Ectoplasmic Specialization ◽

Stage Viii

During spermatogenesis, the molecular mechanism that confers spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES), a testis-specific F-actin-rich adherens junction, in the rat testis remains elusive. Herein, the activated form of focal adhesion kinase (FAK), p-FAK-Tyr397, a component of the apical ES that was expressed predominantly and stage specifically in stage VII-early stage VIII tubules, was found to be a crucial apical ES regulator. Using an FAK-Y397E phosphomimetic mutant cloned in a mammalian expression vector for its transfection vs. FAK and vector alone in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation were manifested by entrapment of spermatids in the seminiferous epithelium in late stage VIII–X tubules and were mediated by a disruption on the spatiotemporal expression and/or mislocalization of actin regulatory protein actin-related protein 3, which induces branched actin polymerization, epidermal growth factor receptor pathway substrate 8 (an actin barbed end capping and bundling protein), and palladin (an actin cross-linking and bundling protein). This thus perturbed changes of F-actin organization at the apical ES to facilitate spermiation, which also led to a concomitant alteration in the distribution and upregulation of adhesion proteins nectin-2 and nectin-3 at the apical ES. As such, nectin-2 and -3 remained at the apical ES to anchor step 19 spermatids on to the epithelium, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr397 that regulates spermatid adhesion at the apical ES in vivo.

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Endothelial CD47 interaction with SIRPγ is required for human T-cell transendothelial migration under shear flow conditions in vitro

Blood ◽

10.1182/blood-2008-01-134429 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1280-1289 ◽

Cited By ~ 43

Author(s):

Michael Stefanidakis ◽

Gail Newton ◽

Winston Y. Lee ◽

Charles A. Parkos ◽

Francis W. Luscinskas

Keyword(s):

T Cells ◽

T Cell ◽

Expression Patterns ◽

Regulatory Protein ◽

Transendothelial Migration ◽

Critical Event ◽

Flow Conditions ◽

Human T Cell

Abstract Leukocyte transendothelial migration (TEM) is a critical event during inflammation. CD47 has been implicated in myeloid cell migration across endothelium and epithelium. CD47 binds to signal regulatory protein (SIRP), SIRPα and SIRPγ. So far, little is known about the role of endothelial CD47 in T-cell TEM in vivo or under flow conditions in vitro. Fluorescence-activated cell sorting and biochemical analysis show that CD3+ T cells express SIRPγ but not SIRPα, and fluorescence microscopy showed that CD47 was enriched at endothelial junctions. These expression patterns suggested that CD47 plays a role in T-cell TEM through binding interactions with SIRPγ. We tested, therefore, whether CD47-SIRPγ interactions affect T-cell transmigration using blocking mAb against CD47 or SIRPγ in an in vitro flow model. These antibodies inhibited T-cell TEM by 70% plus or minus 6% and 82% plus or minus 1%, respectively, but had no effect on adhesion. In agreement with human mAb studies, transmigration of murine wild-type T helper type 1 cells across TNF-α–activated murine CD47−/− endothelium was reduced by 75% plus or minus 2% even though murine T cells appear to lack SIRPγ. Nonetheless, these findings suggest endothelial cell CD47 interacting with T-cell ligands, such as SIRPγ, play an important role in T-cell transendothelial migration.

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Functional in vivo and in vitro effects of 20q11.21 genetic aberrations on hPSC differentiation

Scientific Reports ◽

10.1038/s41598-020-75657-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hye-Yeong Jo ◽

Youngsun Lee ◽

Hongryul Ahn ◽

Hyeong-Jun Han ◽

Ara Kwon ◽

...

Keyword(s):

Cell Fate ◽

Early Stage ◽

Expression Patterns ◽

Transcriptome Profiling ◽

Embryoid Bodies ◽

Differentiation Potential ◽

Negative Effect ◽

In Vitro Effects

Abstract Human pluripotent stem cells (hPSCs) have promising therapeutic applications due to their infinite capacity for self-renewal and pluripotency. Genomic stability is imperative for the clinical use of hPSCs; however, copy number variation (CNV), especially recurrent CNV at 20q11.21, may contribute genomic instability of hPSCs. Furthermore, the effects of CNVs in hPSCs at the whole-transcriptome scale are poorly understood. This study aimed to examine the functional in vivo and in vitro effects of frequently detected CNVs at 20q11.21 during early-stage differentiation of hPSCs. Comprehensive transcriptome profiling of abnormal hPSCs revealed that the differential gene expression patterns had a negative effect on differentiation potential. Transcriptional heterogeneity identified by single-cell RNA sequencing (scRNA-seq) of embryoid bodies from two different isogenic lines of hPSCs revealed alterations in differentiated cell distributions compared with that of normal cells. RNA-seq analysis of 22 teratomas identified several differentially expressed lineage-specific markers in hPSCs with CNVs, consistent with the histological results of the altered ecto/meso/endodermal ratio due to CNVs. Our results suggest that CNV amplification contributes to cell proliferation, apoptosis, and cell fate specification. This work shows the functional consequences of recurrent genetic abnormalities and thereby provides evidence to support the development of cell-based applications.

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123 AGGREGATION OF CLONED EQUINE EMBRYOS: IMPROVEMENT OF IN VITRO AND IN VIVO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab123 ◽

2011 ◽

Vol 23 (1) ◽

pp. 166

Author(s):

A. Gambini ◽

J. Jarazo ◽

R. Olivera ◽

F. Karlanian ◽

D. F. Salamone

Keyword(s):

Expression Patterns ◽

Chi Square ◽

Serum Progesterone ◽

Str Analysis ◽

In Vitro Development ◽

Group Iii ◽

Group Ii ◽

Vitro Development

Development of cloned equine embryo is still inefficient. The aim of our study was to assess the aggregation of zona-free genetically identical cloned embryos as a strategy to improve in vitro and in vivo development. Oocyte collection, maturation, cloning, and activation procedures were performed as described by (Lagutina et al. 2007 Theriogenology 67, 90–98). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 with 5% of FBS in the well of well system in 3 different groups: I, only one RE per well; II, two RE per well; and III, three RE per well. Cleavage and blastocyst formation (7 to 8 days) of all experimental groups was assessed. At day 8, some embryos of each group were either fixed to determine Oct-4 expression by immunocytochemistry or transferred transcervically to a synchronized mare. Pregnancies were assessed by ultrasound from 7 days after embryo transfer until day 45 to 50 of pregnancy every 7 to 10 days, and sizes of vesicles and embryos were measured. In advanced pregnant mares, combined thickness of the uterus and the placenta (CTUP) and serum progesterone levels were also determined. The remaining embryos obtained from each group were maintained in culture from day 7 until day 15. Blastocysts growth was determined every 24 h. In vitro development, on a per-well and RE basis, was compared using the chi-square test. Statistical differences were observed in cleavage among groups I and II (P = 0.0088) and groups I and III (P = 0.0004): (I: 91/111, 82%; II: 74/78, 95%; III: 62/62, 100%). Blastocyst rates differed between groups I and III (I: 10/111, 9%; III: 23/62, 37%); no difference was observed with group II (11/78, 14%). There was no difference on blastocyst rates based on the number of aggregated RE (I: 10/111, 9%; II: 11/156, 7%; III: 23/184, 12.5%). The highest pregnancy rate was obtained in group III (I: 1/3, 33%; II: 2/5, 40%; III: 3/4, 75%). Sizes of vesicles and embryos did not differ statistically in such groups. The CTUP and serum progesterone levels were considered normal (<1.2 cm; >8 ng mL–1, respectively) in ongoing pregnancies. We did not observe any differences in Oct-4 expression patterns among groups. Even though statistical differences were found, surprisingly all embryos grew in vitro until day 15 with good rates and the biggest embryo reached 4.25 mm. Embryo aggregation improved in vitro development of equine cloned embryos until day 7, and pregnancies rates were higher. The in vivo sizes of vesicles and embryos were normal for all groups, and in vitro development beyond day 7 showed the high viability of embryos. To conclude, aggregation of cloned equine embryo does not imply extra oocytes because there is no statistical difference in the number of blastocysts obtained per oocytes used to achieve RE. It is also a good strategy to improve in vitro embryo development without alterations on in vivo progress. This is the first report of pregnancies from aggregated equine cloned embryos, and the first healthy cloned foal from South America, confirmed by STR analysis, was born recently derived from group II. Stumpo, Ignacio, Paola Barboza, and Don Antonio staff.

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A human iPSC-astroglia neurodevelopmental model reveals divergent transcriptomic patterns in schizophrenia

Translational Psychiatry ◽

10.1038/s41398-021-01681-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Attila Szabo ◽

Ibrahim A. Akkouh ◽

Matthieu Vandenberghe ◽

Jordi Requena Osete ◽

Timothy Hughes ◽

...

Keyword(s):

Developmental Stages ◽

Glutamate Uptake ◽

Expression Patterns ◽

Induced Pluripotent Stem Cell ◽

Specific Gene ◽

Specific Expression ◽

Functional Changes ◽

Neurodevelopmental Abnormalities ◽

Induced Pluripotent

AbstractWhile neurodevelopmental abnormalities have been associated with schizophrenia (SCZ), the role of astroglia in disease pathophysiology remains poorly understood. In the present study, we used a human induced pluripotent stem cell (iPSC)-derived astrocyte model to investigate the temporal patterns of astroglia differentiation during developmental stages critical for SCZ using RNA sequencing. The model generated astrocyte-specific gene expression patterns during differentiation that corresponded well to astroglia-specific expression signatures of in vivo cortical fetal development. Using this model we identified SCZ-specific expression dynamics, and found that SCZ-associated differentially expressed genes were significantly enriched in the medial prefrontal cortex, striatum, and temporal lobe, targeting VWA5A and ADAMTS19. In addition, SCZ astrocytes displayed alterations in calcium signaling, and significantly decreased glutamate uptake and metalloproteinase activity relative to controls. These results implicate novel transcriptional dynamics in astrocyte differentiation in SCZ together with functional changes that are potentially important biological components of SCZ pathology.

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Differential expression patterns of MafB and c-Maf in macrophages in vivo and in vitro

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2016.03.063 ◽

2016 ◽

Vol 473 (1) ◽

pp. 118-124 ◽

Cited By ~ 10

Author(s):

Dhouha Daassi ◽

Michito Hamada ◽

Hyojung Jeon ◽

Yuki Imamura ◽

Mai Thi Nhu Tran ◽

...

Keyword(s):

Differential Expression ◽

Expression Patterns

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NR4A2 Expression Patterns in Mouse Models of Rheumatoid Arthritis

Journal of Student Research ◽

10.47611/jsr.v4i1.213 ◽

2015 ◽

Vol 4 (1) ◽

pp. 136-143

Author(s):

Jordan Everett ◽

Ellen Gravallese ◽

Kimberlee S. Mix

Keyword(s):

Mouse Models ◽

Cross Sections ◽

Early Stage ◽

Expression Patterns ◽

New Drugs ◽

Late Time ◽

Orphan Nuclear Receptor ◽

Protective Function ◽

Protein Levels

Arthritis is a group of over 100 musculoskeletal disorders affecting approximately 50 million adults in the US. In an effort to develop new drugs to treat arthritis, we are exploring the function of the orphan nuclear receptor 4A2 (NR4A2), a transcription factor over-expressed in inflamed joints. The transcriptional targets of NR4A2 include angiogenesis factors and matrix metalloproteinases (MMPs). NR4A2 appears to have a deleterious effect in synoviocytes by promoting tissue degradation, while in chondrocytes it seems to have a protective function. Previous work on human synoviocytes has shown NR4A2 to rise early in response to inflammation, leading us to hypothesize that NR4A2 may be a preliminary mediator of arthritis. To test this hypothesis in vivo, we studied NR4A2 expression patterns in two mouse models of RA: Antigen Induced Arthritis (AIA) and Serum Transfer Arthritis (STA). Tissue sections were obtained from healthy and arthritic mice at early, mid, and late time-points following induction. Joint cross-sections were examined via immunohistochemical staining, and NR4A2 positive cells were quantified in synovial and cartilage tissues. In the AIA model, NR4A2 protein levels peaked in synovium at day 10 of disease (mid stage, 50% positive) and declined later in disease. In cartilage, protein levels reached a maximum at day 8 (early stage, 70%) and subsequently declined as well. In contrast, NR4A2 was not expressed in the STA model, despite apparent joint degradation. NR4A2 has been shown to be expressed in STA arthritis by other researchers, so it is unknown why STA samples did not show NR4A2 expression in our experiment.

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Roles of cell-autonomous mechanisms for differential expression of region-specific transcription factors in neuroepithelial cells

Development ◽

10.1242/dev.122.8.2449 ◽

1996 ◽

Vol 122 (8) ◽

pp. 2449-2464 ◽

Cited By ~ 7

Author(s):

Y. Nakagawa ◽

T. Kaneko ◽

T. Ogura ◽

T. Suzuki ◽

M. Torii ◽

...

Keyword(s):

Transcription Factors ◽

Differential Expression ◽

Sonic Hedgehog ◽

Expression Patterns ◽

Cellular Level ◽

Neuroepithelial Cells ◽

Islet 1 ◽

Inductive Signal

Although a number of genes have been found to have restricted expression domains in the embryonic forebrain and midbrain, it remains largely unknown how the expression of these genes is regulated at the cellular level. In this study, we explored the mechanisms for the differential expression of region-specific transcription factors in neuroepithelial cells by using both primary and immortalized neuroepithelial cells from the rat brain at embryonic day 11.5. We found that differential expression patterns of Pax-3, Pax-5, Pax-6, Dlx-1, Dlx-2, Emx2, Otx1 and Dbx observed in vivo were maintained even when the cells were isolated and cultured in vitro, free from environmental influences. Furthermore, in response to Sonic hedgehog, which is a major inductive signal from the environment for regional specification, neuroepithelial cells that maintain distinct regional identities expressed different sets of ventral-specific genes including Islet-1, Nkx-2.1 and Nkx-2.2. These results suggest that certain cell-autonomous mechanisms play important roles in regulating both environmental signal-dependent and -independent expression of region-specific genes. Thus, we propose that use of the in vitro culture systems we describe in this study facilitates the understanding of regulatory mechanisms of region-specific genes in neuroepithelial cells.

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