scholarly journals The Dispensable Roles of X-Linked Ubl4a and Its Autosomal Counterpart Ubl4b in Spermatogenesis Represent a New Evolutionary Type of X-Derived Retrogenes

2021 ◽  
Vol 12 ◽  
Author(s):  
Changping Yu ◽  
Runjie Diao ◽  
Ranjha Khan ◽  
Cheng Deng ◽  
Hui Ma ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Purifying Selection ◽  
Housekeeping Gene ◽  
Reproductive Parameters ◽  
Double Knockout ◽  
Specific Expression ◽  
Normal Spermatogenesis ◽  
First Time ◽  
Testis Specific Expression

X-derived retrogenes contribute to genetic diversity in evolution and are usually specifically expressed in testis and perform important functions during spermatogenesis. Ubl4b is an autosomal retrogene with testis-specific expression derived from Ubl4a, an X-linked housekeeping gene. In the current study, we performed phylogenetic analysis and revealed that Ubl4a and Ubl4b are subject to purifying selection and may have conserved functions in evolution. Ubl4b was knocked out in mice using CRISPR/Cas9 genome editing technology and interestingly, we found no alterations in reproductive parameters of Ubl4b–/– male mice. To get insights into whether Ubl4a could compensate the absence of Ubl4b in vivo, we further obtained Ubl4a–/Y; Ubl4b–/– mice that lack both Ubl4a and Ubl4b, and the double knockout (dKO) mice also displayed normal spermatogenesis, showing that Ubl4a and Ubl4b are both dispensable for spermatogenesis. Thus, through the in vivo study of UBL4A and UBL4B, we provided a direct evidence for the first time that some X chromosome-derived autosomal retrogenes can be unfunctional in spermatogenesis, which represents an additional evolutionary type of X-derived retrogenes.

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)

2009 ◽  
Vol 296 (5) ◽  
pp. R1650-R1660 ◽  
Author(s):  
Yi-Fang Wang ◽  
Yung-Che Tseng ◽  
Jia-Jiun Yan ◽  
Junya Hiroi ◽  
Pung-Pung Hwang
Keyword(s):  
Specific Inhibitor ◽  
In Vivo Model ◽  
Zebrafish Embryos ◽  
Uptake Mechanism ◽  
Ion Regulation ◽  
Specific Expression ◽  
Net Uptake ◽  
First Time

The thiazide-sensitive Na+-Cl− cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na+ and Cl− from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na+ and/or Cl− uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H+-ATPase-rich (HR) cells and Na+-K+-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl− influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na+ and Cl− influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl− influx and Cl− content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl− in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na+ influx and content that resulted from upregulation of mRNA expressions of Na+-H+ exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl− uptake mechanism in zebrafish skin/gills.

scholarly journals Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway

Development ◽  
2020 ◽  
Vol 147 (21) ◽  
pp. dev187187
Author(s):  
Hannah K. Vanyai ◽  
Fabrice Prin ◽  
Oriane Guillermin ◽  
Bishara Marzook ◽  
Stefan Boeing ◽  
...  
Keyword(s):  
Cell Fate ◽  
Target Genes ◽  
Control Cell ◽  
Tissue Growth ◽  
Double Knockout ◽  
Cartilage Growth ◽  
Chondrocyte Proliferation ◽  
Specific Expression

ABSTRACTThe Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Previously, YAP was proposed to inhibit skeletal size by repressing chondrocyte proliferation and differentiation. We find that, in vitro, Yap/Taz double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear nls-YAP5SA accelerates proliferation, in line with the canonical role of this pathway in most tissues. However, in vivo, cartilage-specific knockout of Yap/Taz does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. Cartilage-specific expression of nls-YAP5SA or knockout of Lats1/2 do not increase cartilage growth, but instead lead to catastrophic malformations resembling chondrodysplasia or achondrogenesis. Physiological YAP target genes in cartilage include Ctgf, Cyr61 and several matrix remodelling enzymes. Thus, YAP/TAZ activity controls chondrocyte proliferation in vitro, possibly reflecting a regenerative response, but is dispensable for chondrocyte proliferation in vivo, and instead functions to control cartilage morphogenesis via regulation of the extracellular matrix.

scholarly journals Redundant and additive functions of the four Lef/Tcf transcription factors in lung epithelial progenitors

2020 ◽  
Vol 117 (22) ◽  
pp. 12182-12191 ◽  
Author(s):  
Kamryn N. Gerner-Mauro ◽  
Haruhiko Akiyama ◽  
Jichao Chen
Keyword(s):  
Transcription Factors ◽  
Purifying Selection ◽  
Mouse Lung ◽  
Cell Type ◽  
Genetic Redundancy ◽  
Specific Expression ◽  
Complex Genetics ◽  
Lung Epithelial ◽  
And Function

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling theCtnnb1mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the correspondingCtnnb1mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.

scholarly journals Genetic evidence for partial redundancy between the arginine methyltransferases CARM1 and PRMT6

2020 ◽  
Vol 295 (50) ◽  
pp. 17060-17070
Author(s):  
Donghang Cheng ◽  
Guozhen Gao ◽  
Alessandra Di Lorenzo ◽  
Sandrine Jayne ◽  
Michael O. Hottiger ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Type I ◽  
Gene Promoters ◽  
Double Knockout ◽  
Embryonic Fibroblasts ◽  
Arginine Methyltransferase ◽  
Fetal Size ◽  
Partial Redundancy

CARM1 is a protein arginine methyltransferase (PRMT) that acts as a coactivator in a number of transcriptional programs. CARM1 orchestrates this coactivator activity in part by depositing the H3R17me2a histone mark in the vicinity of gene promoters that it regulates. However, the gross levels of H3R17me2a in CARM1 KO mice did not significantly decrease, indicating that other PRMT(s) may compensate for this loss. We thus performed a screen of type I PRMTs, which revealed that PRMT6 can also deposit the H3R17me2a mark in vitro. CARM1 knockout mice are perinatally lethal and display a reduced fetal size, whereas PRMT6 null mice are viable, which permits the generation of double knockouts. Embryos that are null for both CARM1 and PRMT6 are noticeably smaller than CARM1 null embryos, providing in vivo evidence of redundancy. Mouse embryonic fibroblasts (MEFs) from the double knockout embryos display an absence of the H3R17me2a mark during mitosis and increased signs of DNA damage. Moreover, using the combination of CARM1 and PRMT6 inhibitors suppresses the cell proliferation of WT MEFs, suggesting a synergistic effect between CARM1 and PRMT6 inhibitions. These studies provide direct evidence that PRMT6 also deposits the H3R17me2a mark and acts redundantly with CARM1.

Mouse testis Pdha-2 promoter upstream sequences confer tissue-and temporal-specific activity in transgenic mice

1994 ◽  
Vol 6 (5) ◽  
pp. 599 ◽  
Author(s):  
RC Iannello ◽  
JC Young ◽  
S Sumarsono ◽  
MJ Tymms ◽  
I Kola
Keyword(s):  
Transgenic Mice ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Somatic Tissue ◽  
Specific Expression ◽  
Complex Process ◽  
Specific Manner ◽  
Cat Gene ◽  
Testis Specific Expression

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine testis-specific isoform of the E1 alpha subunit of the pyruvate dehydrogenase complex. To elucidate the mechanisms regulating its expression in vivo, we have begun to investigate the Pdha-2 promoter in transgenic mice. In this paper, a construct containing 3.0 kb of promoter and upstream sequences is reported to be sufficient for directing the testis-specific expression of a CAT reporter gene in mice harbouring the transgene. Similarly to the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. However, the 3.0-kb Pdha-2 promoter is not active in somatic tissue suggesting that repressor elements may be present within these sequences.

scholarly journals Direct visualization of a native Wnt in vivo reveals that a long-range Wnt gradient forms by extracellular dispersal

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ariel M Pani ◽  
Bob Goldstein
Keyword(s):  
Long Range ◽  
Direct Evidence ◽  
Signaling Proteins ◽  
Long Distance ◽  
Biologically Relevant ◽  
C Elegans ◽  
Neuroblast Migration ◽  
Evolutionarily Conserved ◽  
First Time

Wnts are evolutionarily conserved signaling proteins with essential roles in development and disease that have often been thought to spread between cells and signal at a distance. However, recent studies have challenged this model, and whether long-distance extracellular Wnt dispersal occurs and is biologically relevant is debated. Understanding fundamental aspects of Wnt dispersal has been limited by challenges with observing endogenous ligands in vivo, which has prevented directly testing hypotheses. Here, we have generated functional, fluorescently tagged alleles for a C. elegans Wnt homolog and for the first time visualized a native, long-range Wnt gradient in a living animal. Live imaging of Wnt along with source and responding cell membranes provided support for free, extracellular dispersal. By limiting Wnt transfer between cells, we confirmed that extracellular spreading shapes a long-range gradient and is critical for neuroblast migration. These results provide direct evidence that Wnts spread extracellularly to regulate aspects of long-range signaling.

scholarly journals Redundant and additive functions of the four Lef/Tcf transcription factors in lung epithelial progenitors

2020 ◽  
Author(s):  
Kamryn N. Gerner-Mauro ◽  
Haruhiko Akiyama ◽  
Jichao Chen
Keyword(s):  
Transcription Factors ◽  
Purifying Selection ◽  
Cell Type ◽  
Genetic Redundancy ◽  
Specific Expression ◽  
Lung Epithelial ◽  
Single Cell Type ◽  
Canonical Wnt ◽  
Multicellular Organisms

ABSTRACTIn multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multi-member paralogs, as exemplified by the 4 obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the dauntingly complex genetics involved. Using the developing mouse lung as a model system, we generate 2 quadruple conditional knockouts and myriad triple and double combinations, and show that the 4 Lef/Tcf genes function redundantly in the presence of at least 2 Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Pre-lung-specification, pan-epithelial double knockouts have no lung phenotype, triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas, and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Post-lung-specification deletion of all 4 Lef/Tcf genes leads to branching defects, downregulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors and represents, to our knowledge, the first in vivo analysis of cell-type-specific genetic redundancy among the 4 Lef/Tcf paralogs.SIGNIFICANCE STATEMENTParalogs represent genetic redundancy and survive purifying selection by evolving overlapping and distinct functions. In multicellular organisms, such functional diversification can manifest as tissue and cell type specific expression, which masks possible selective pressure for genetic redundancy within a single cell type. Using in vivo genetic and genomic analyses, we show that although the 4 mammalian Lef/Tcf transcription factors have evolved organ-specific functions, they function additively and redundantly, depending on gene dosage, to promote lung epithelial progenitors and do so in a monotonic, positive manner with beta-Catenin in the canonical Wnt signaling pathway.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

2019 ◽  
Vol 20 (15) ◽  
pp. 3679 ◽  
Author(s):  
Lin Chen ◽  
Alyne Simões ◽  
Zujian Chen ◽  
Yan Zhao ◽  
Xinming Wu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Oral Mucosa ◽  
Wound Closure ◽  
Expression Patterns ◽  
Skin Wound ◽  
Skin Wound Healing ◽  
Specific Expression ◽  
Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

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