scholarly journals Enhancer analysis of the Drosophila zinc finger transcription factor Earmuff by gene targeting

Hereditas ◽  
2021 ◽  
Vol 158 (1) ◽  
Author(s):  
Kirsten Hildebrandt ◽  
Sabrina Kübel ◽  
Marie Minet ◽  
Nora Fürst ◽  
Christine Klöppel ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Gene Targeting ◽  
Zinc Finger ◽  
Neural Progenitor Cells ◽  
Expression Patterns ◽  
Brain Structures ◽  
Systematic Analysis ◽  
Major Function ◽  
Stem Cell Maintenance ◽  
The Brain

Abstract Background Many transcription factors are involved in the formation of the brain during the development of Drosophila melanogaster. The transcription factor Earmuff (Erm), a member of the forebrain embryonic zinc finger family (Fezf), is one of these important factors for brain development. One major function of Earmuff is the regulation of proliferation within type II neuroblast lineages in the brain; here, Earmuff is expressed in intermediate neural progenitor cells (INPs) and balances neuronal differentiation versus stem cell maintenance. Erm expression during development is regulated by several enhancers. Results In this work we show a functional analysis of erm and some of its enhancers. We generated a new erm mutant allele by gene targeting and reintegrated Gal4 to make an erm enhancer trap strain that could also be used on an erm mutant background. The deletion of three of the previously analysed enhancers showing the most prominent expression patterns of erm by gene targeting resulted in specific temporal and spatial defects in defined brain structures. These defects were already known but here could be assigned to specific enhancer regions. Conclusion This analysis is to our knowledge the first systematic analysis of several large enhancer deletions of a Drosophila gene by gene targeting and will enable deeper analysis of erm enhancer functions in the future.

scholarly journals Functional analysis of enhancer elements regulating the expression of the Drosophila homeodomain transcription factor DRx by gene targeting

Hereditas ◽  
2021 ◽  
Vol 158 (1) ◽  
Author(s):  
Christine Klöppel ◽  
Kirsten Hildebrandt ◽  
Dieter Kolb ◽  
Nora Fürst ◽  
Isabelle Bley ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Brain Development ◽  
Gene Targeting ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Adult Brain ◽  
Central Complex ◽  
Larval Brain ◽  
Endogenous Expression ◽  
The Brain

Abstract Background The Drosophila brain is an ideal model system to study stem cells, here called neuroblasts, and the generation of neural lineages. Many transcriptional activators are involved in formation of the brain during the development of Drosophila melanogaster. The transcription factor Drosophila Retinal homeobox (DRx), a member of the 57B homeobox gene cluster, is also one of these factors for brain development. Results In this study a detailed expression analysis of DRx in different developmental stages was conducted. We show that DRx is expressed in the embryonic brain in the protocerebrum, in the larval brain in the DM and DL lineages, the medulla and the lobula complex and in the central complex of the adult brain. We generated a DRx enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the endogenous expression of DRx. With the help of eight existing enhancer-Gal4 strains and one made by our group, we mapped various enhancers necessary for the expression of DRx during all stages of brain development from the embryo to the adult. We made an analysis of some larger enhancer regions by gene targeting. Deletion of three of these enhancers showing the most prominent expression patterns in the brain resulted in specific temporal and spatial loss of DRx expression in defined brain structures. Conclusion Our data show that DRx is expressed in specific neuroblasts and defined neural lineages and suggest that DRx is another important factor for Drosophila brain development.

scholarly journals Molecular Cloning ofphd1and Comparative Analysis ofphd1, 2, and3Expression inXenopus laevis

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Dandan Han ◽  
Luan Wen ◽  
Yonglong Chen
Keyword(s):  
Comparative Analysis ◽  
Embryonic Development ◽  
Gall Bladder ◽  
Molecular Cloning ◽  
Gene Targeting ◽  
Expression Patterns ◽  
Systematic Analysis ◽  
Adult Tissues ◽  
Inxenopus Laevis ◽  
And Function

Intensive gene targeting studies in mice have revealed that prolyl hydroxylase domain proteins (PHDs) play important roles in murine embryonic development; however, the expression patterns and function of these genes during embryogenesis of other vertebrates remain largely unknown. Here we report the molecular cloning ofphd1and systematic analysis ofphd1,phd2, andphd3expression in embryos as well as adult tissues ofXenopus laevis. All threephdsare maternally provided duringXenopusearly development. The spatial expression patterns ofphdsgenes inXenopusembryos appear to define a distinct synexpression group. Frogphd2andphd3showed complementary expression in adult tissues withphd2transcription levels being high in the eye, brain, and intestine, but low in the liver, pancreas, and kidney. On the contrary, expression levels ofphd3are high in the liver, pancreas, and kidney, but low in the eye, brain, and intestine. All threephdsare highly expressed in testes, ovary, gall bladder, and spleen. Among threephds,phd3showed strongest expression in heart.

scholarly journals Molecular regulation of fetal brain development in pigs

2021 ◽  
Author(s):  
◽  
Monica P. Strawn
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcription Factor ◽  
Brain Development ◽  
Early Development ◽  
Expression Patterns ◽  
Fetal Brain ◽  
Molecular Regulation ◽  
Fetal Brain Development ◽  
The Brain

Two experiments were conducted to investigate molecular regulation that impacts fetal brain development in pigs. In the first experiment (Chapter 2), gene expression was profiled by RNA sequencing (RNA-seq) to examine the whole transcriptome of the male (M) and female (F) fetal brain at gestation day (d) 45, 60 and 90. The analysis showed fewer differentially expressed genes (DEGs) in the brain of male and female fetuses in earlier gestation (d45-d60) when compared to late gestation (d60-d90). The homeobox (HOX) A5 gene that regulates pattern formation in early development was in the top upregulated DEGs between d45 to d60 in fetuses of both sexes. This study also found HOX B5 and D3 genes were in the top upregulated genes between d45 and d60 of the fetal brain of females, but not males. The second experiment (Chapter 3) investigated DNA methylation in pigs. DNA methylation in the fetal brain of both sexes at the same three gestation days was performed by enzymatic methyl sequencing (EM-seq). Hotspots of methylation in specific chromosomal regions were observed in the analysis. The analysis identified 1,475 sites in the pig genome that were methylated in the fetal brain, irrespective of sex, during development. The same sites were methylated in a canonically correlated manner in the blood of the adult stage, both in sows and boars. This is consistent with the Dilman theory of developmental aging (DevAge), which suggests that aging and early development of the brain are regulated by common molecular processes. A comparative analysis (Chapter 4) compared the gene expression patterns in the fetal brain and placenta between pigs and mice. The analysis identified 112 genes that were expressed (mean FPKM > 10) in the fetal brain of both species but not expressed (mean FPKM < 1) in the placenta of either species, and 10 genes that were expressed in the placenta of both species but not expressed in the fetal brain. In-silico analysis of the transcription factor binding sites in the 500 bp of the upstream DNA of these common genes revealed that they were commonly regulated by the RE1 silencing transcription factor (REST), which is a multifaceted transcription factor that acts as a master regulator of neurogenesis as well as controls neural excitation and the aging processes.

scholarly journals Genome-Wide Analysis of the YABBY Transcription Factor Family in Pineapple and Functional Identification of AcYABBY4 Involvement in Salt Stress

2019 ◽  
Vol 20 (23) ◽  
pp. 5863 ◽  
Author(s):  
Zeyun Li ◽  
Gang Li ◽  
Mingxing Cai ◽  
Samaranayaka V.G.N. Priyadarshani ◽  
Mohammad Aslam ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Salt Stress ◽  
Protein Structure ◽  
Zinc Finger ◽  
Structure Prediction ◽  
Zinc Finger Protein ◽  
Expression Patterns ◽  
Regulatory Role ◽  
Short Root ◽  
Finger Protein

The plant-specific transcription factor gene family, YABBY, belongs to the subfamily of zinc finger protein superfamily and plays an essential regulatory role in lateral organ development. In this study, nine YABBY genes were identified in the pineapple genome. Seven of them were located on seven different chromosomes and the remaining two were located on scaffold 1235. Through protein structure prediction and protein multiple sequence alignment, we found that AcYABBY3, AcYABBY5 and AcYABBY7 lack a C2 structure in their N-terminal C2C2 zinc finger protein structure. Analysis of the cis-acting element indicated that all the seven pineapple YABBY genes contain multiple MYB and MYC elements. Further, the expression patterns analysis using the RNA-seq data of different pineapple tissues indicated that different AcYABBYs are preferentially expressed in various tissues. RT-qPCR showed that the expression of AcYABBY2, AcYABBY3, AcYABBY6 and AcYABBY7 were highly sensitive to abiotic stresses. Subcellular localization in pineapple protoplasts, tobacco leaves and Arabidopsis roots showed that all the seven pineapple YABBY proteins were nucleus localized. Overexpression of AcYABBY4 in Arabidopsis resulted in short root under NaCl treatment, indicating a negative regulatory role of AcYABBY4 in plant resistance to salt stress. This study provides valuable information for the classification of pineapple AcYABBY genes and established a basis for further research on the functions of AcYABBY proteins in plant development and environmental stress response.

scholarly journals Matrix Metalloproteinase (MMP) 9 Transcription in Mouse Brain Induced by Fear Learning

2013 ◽  
Vol 288 (29) ◽  
pp. 20978-20991 ◽  
Author(s):  
Krishnendu Ganguly ◽  
Emilia Rejmak ◽  
Marta Mikosz ◽  
Evgeni Nikolaev ◽  
Ewelina Knapska ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Prefrontal Cortex ◽  
Matrix Metalloproteinase ◽  
Gene Promoter ◽  
Brain Structures ◽  
Contextual Fear Conditioning ◽  
Fear Learning ◽  
Contextual Fear ◽  
The Brain

Memory formation requires learning-based molecular and structural changes in neurons, whereas matrix metalloproteinase (MMP) 9 is involved in the synaptic plasticity by cleaving extracellular matrix proteins and, thus, is associated with learning processes in the mammalian brain. Because the mechanisms of MMP-9 transcription in the brain are poorly understood, this study aimed to elucidate regulation of MMP-9 gene expression in the mouse brain after fear learning. We show here that contextual fear conditioning markedly increases MMP-9 transcription, followed by enhanced enzymatic levels in the three major brain structures implicated in fear learning, i.e. the amygdala, hippocampus, and prefrontal cortex. To reveal the role of AP-1 transcription factor in MMP-9 gene expression, we have used reporter gene constructs with specifically mutated AP-1 gene promoter sites. The constructs were introduced into the medial prefrontal cortex of neonatal mouse pups by electroporation, and the regulation of MMP-9 transcription was studied after contextual fear conditioning in the adult animals. Specifically, −42/-50- and −478/-486-bp AP-1 binding motifs of the mouse MMP-9 promoter sequence have been found to play a major role in MMP-9 gene activation. Furthermore, increases in MMP-9 gene promoter binding by the AP-1 transcription factor proteins c-Fos and c-Jun have been demonstrated in all three brain structures under investigation. Hence, our results suggest that AP-1 acts as a positive regulator of MMP-9 transcription in the brain following fear learning.

scholarly journals Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sayyed M Azimi ◽  
Steven D Sheridan ◽  
Mostafa Ghannad-Rezaie ◽  
Peter M Eimon ◽  
Mehmet Fatih Yanik
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Progenitor Cells ◽  
Dopaminergic Neurons ◽  
Neural Progenitor Cells ◽  
Expression Patterns ◽  
Neural Progenitor ◽  
Dynamically Reconfigurable ◽  
Neuronal Progenitors ◽  
Copy Numbers

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.

scholarly journals A zinc-finger fusion protein refines Gal4-defined neural circuits

2017 ◽  
Author(s):  
Shamprasad Varija Raghu ◽  
Farhan Mohammad ◽  
Chua Jia Yi ◽  
Claudia S. Barros ◽  
Joanne Lam ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Fusion Protein ◽  
Zinc Finger ◽  
Neural Circuits ◽  
Expression Patterns ◽  
Genetic Fidelity ◽  
Dna Binding Specificity ◽  
Expression Control ◽  
Gal4 Expression

AbstractThe analysis of behavior requires that the underlying neuronal circuits are identified and genetically isolated. In several major model species—most notably Drosophila, neurogeneticists identify and isolate neural circuits with a binary heterologous expression-control system: Gal4–UASG. One limitation of Gal4–UASG is that expression patterns are often too broad to map circuits precisely. To help refine the range of Gal4 lines, we developed an intersectional genetic AND operator. Interoperable with Gal4, the new system’s key component is a fusion protein in which the DNA-binding domain of Gal4 has been replaced with a zinc finger domain with a different DNA-binding specificity. In combination with its cognate binding site (UASZ) the zinc-finger-replaced Gal4 (‘Zal1’) was functional as a standalone transcription factor. Zal1 transgenes also refined Gal4 expression ranges when combined with UASGZ, a hybrid upstream activation sequence. In this way, combining Gal4 and Zal1 drivers captured restricted cell sets compared with single drivers and improved genetic fidelity. This intersectional genetic AND operation presumably derives from the action of a heterodimeric transcription factor: Gal4-Zal1. Configurations of Zal1–UASZ and Zal1-Gal4-UASGZ are versatile tools for defining, refining, and manipulating targeted neural expression patterns with precision.

scholarly journals Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development

Hereditas ◽  
2022 ◽  
Vol 159 (1) ◽  
Author(s):  
Kirsten Hildebrandt ◽  
Dieter Kolb ◽  
Christine Klöppel ◽  
Petra Kaspar ◽  
Fabienne Wittling ◽  
...  
Keyword(s):  
Brain Development ◽  
Gene Targeting ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Adult Brain ◽  
Specific Cell ◽  
Larval Brain ◽  
Developmental Defects ◽  
The Brain

Abstract Background The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. Results In this study we conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. We generated a new hbnKOGal4 enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. We characterized several enhancers that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, we generated deletions of four of these enhancer regions through gene targeting and analysed their effects on the expression and function of hbn. Conclusion The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.

Sign in / Sign up

Export Citation Format

Share Document