scholarly journals Characterisation of a dual-colour retinoic acid reporter system for analysing cells and embryos

2021 ◽  
Author(s):  
◽  
Bianca Black
Keyword(s):  
Retinoic Acid ◽  
Embryonic Development ◽  
Hox Genes ◽  
Fluorescent Proteins ◽  
Expression Patterns ◽  
Tissue Type ◽  
Cell Populations ◽  
Growth Media ◽  
Reporter System ◽  
Transgenic Mouse Line

<p>Vitamin A is an important component in the diet as its metabolites, the retinoids, play key roles in a vast range of cellular functions, from production of germ cells, to embryonic development and right through until adulthood. The function of retinoids, in particular retinoic acid (RA), is especially important during early embryonic development, where it is responsible for many different key developmental events. Some of the processes controlled by RA include brain region patterning, Hox gene expression, axis establishment and somite formation. Here, we aimed to characterise the expression pattern of retinoic acid in the early murine embryo and isolate cell populations from a range of RA concentrations to analyse the mRNA expression.  To do this, we used a transgenic mouse line which expressed a reporter plasmid that was able to show, through the expression of two fluorescent proteins, areas of high RA concentration and area of low RA concentration. We tested the function of this reporter system in vitro, using cell lines which were transfected with the plasmid and exposed to RA in their growth media. This worked showed a somewhat does-dependent response from the reporter system expressing the fluorescent proteins. We then imaged transgenic embryos at various stages of early development, to ascertain the areas of RA expression and repression. Here, we saw fluorescent protein expression patterns that indicated both high and low concetrations of RA. Using this information, we dissociated transgenic E8.5 embryos and sorted the cells based on their levels of expression of the two fluorescent proteins, as well as by tissue type, which had been marked with antibodies. mRNA was extracted from these populations and PCR was performed to identify the presence of Hox genes and to see any difference in expression patterns across the various cell populations.</p>

scholarly journals Characterisation of a dual-colour retinoic acid reporter system for analysing cells and embryos

2021 ◽  
Author(s):  
◽  
Bianca Black
Keyword(s):  
Retinoic Acid ◽  
Embryonic Development ◽  
Hox Genes ◽  
Fluorescent Proteins ◽  
Expression Patterns ◽  
Tissue Type ◽  
Cell Populations ◽  
Growth Media ◽  
Reporter System ◽  
Transgenic Mouse Line

<p>Vitamin A is an important component in the diet as its metabolites, the retinoids, play key roles in a vast range of cellular functions, from production of germ cells, to embryonic development and right through until adulthood. The function of retinoids, in particular retinoic acid (RA), is especially important during early embryonic development, where it is responsible for many different key developmental events. Some of the processes controlled by RA include brain region patterning, Hox gene expression, axis establishment and somite formation. Here, we aimed to characterise the expression pattern of retinoic acid in the early murine embryo and isolate cell populations from a range of RA concentrations to analyse the mRNA expression.  To do this, we used a transgenic mouse line which expressed a reporter plasmid that was able to show, through the expression of two fluorescent proteins, areas of high RA concentration and area of low RA concentration. We tested the function of this reporter system in vitro, using cell lines which were transfected with the plasmid and exposed to RA in their growth media. This worked showed a somewhat does-dependent response from the reporter system expressing the fluorescent proteins. We then imaged transgenic embryos at various stages of early development, to ascertain the areas of RA expression and repression. Here, we saw fluorescent protein expression patterns that indicated both high and low concetrations of RA. Using this information, we dissociated transgenic E8.5 embryos and sorted the cells based on their levels of expression of the two fluorescent proteins, as well as by tissue type, which had been marked with antibodies. mRNA was extracted from these populations and PCR was performed to identify the presence of Hox genes and to see any difference in expression patterns across the various cell populations.</p>

Tissue-specific expression of the fibril-associated collagens XII and XIV

1994 ◽  
Vol 107 (2) ◽  
pp. 669-681 ◽  
Author(s):  
C. Walchli ◽  
M. Koch ◽  
M. Chiquet ◽  
B.F. Odermatt ◽  
B. Trueb
Keyword(s):  
Embryonic Development ◽  
Type I Collagen ◽  
Expression Patterns ◽  
Collagen Fibrils ◽  
Tissue Type ◽  
Type I ◽  
Connective Tissues ◽  
Specific Expression ◽  
Temporal Regulation ◽  
Fibrillar Collagens

Interstitial collagen fibrils form the supporting scaffold of all connective tissues. The synthesis of this framework is subject to a precise spatial and temporal regulation in order to meet the mechanical needs of every tissue type. A subgroup of non-fibrillar collagens termed FACIT seems to play a role in this regulation by providing specific molecular bridges between fibrils and other matrix components. Collagens XII and XIV represent such FACIT molecules and occur preferentially in tissues containing banded type I collagen fibrils. We have used the techniques of indirect immunofluorescence and in situ hybridization to investigate the expression patterns of the two molecules during chicken embryonic development. We detected specific differences in these patterns, which may be related to the respective functions of the two proteins within the connective tissues. Collagen XIV was expressed at very few sites in the 6-day-old embryo, but occurred in virtually every collagen I-containing tissue (skeletal muscle, cardiac muscle, gizzard, tendon, periosteum, nerve) by the end of embryonic development. In contrast, collagen XII was fairly abundant in the 6-day-old embryo but was, at later stages, restricted to only a few dense connective tissue structures (bone, tendon, gizzard). Thus, our results suggest that collagen XII and collagen XIV serve different functions during embryonic development although their structures are highly similar.

scholarly journals microRNA-138 modulates cardiac patterning during embryonic development

2008 ◽  
Vol 105 (46) ◽  
pp. 17830-17835 ◽  
Author(s):  
Sarah U. Morton ◽  
Paul J. Scherz ◽  
Kimberly R. Cordes ◽  
Kathryn N. Ivey ◽  
Didier Y. R. Stainier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Embryonic Development ◽  
Expression Patterns ◽  
Acid Synthesis ◽  
Specific Gene ◽  
Protein Activity ◽  
Gene Encoding ◽  
Small Noncoding Rnas ◽  
Discrete Domains

Organ patterning during embryonic development requires precise temporal and spatial regulation of protein activity. microRNAs (miRNAs), small noncoding RNAs that typically inhibit protein expression, are broadly important for proper development, but their individual functions during organogenesis are largely unknown. We report that miR-138 is expressed in specific domains in the zebrafish heart and is required to establish appropriate chamber-specific gene expression patterns. Disruption of miR-138 function led to ventricular expansion of gene expression normally restricted to the atrio-ventricular valve region and, ultimately, to disrupted ventricular cardiomyocyte morphology and cardiac function. Temporal-specific knockdown of miR-138 by antagomiRs showed miR-138 function was required during a discrete developmental window, 24–34 h post-fertilization (hpf). miR-138 functioned partially by repressing the retinoic acid synthesis enzyme, aldehyde dehydrogenase-1a2, in the ventricle. This activity was complemented by miR-138-mediated ventricular repression of the gene encoding versican (cspg2), which was positively regulated by retinoic-acid signaling. Our findings demonstrate that miR-138 helps establish discrete domains of gene expression during cardiac morphogenesis by targeting multiple members of a common pathway, and also establish the use of antagomiRs in fish for temporal knockdown of miRNA function.

Expression of AmphiHox-1 and AmphiPax-1 in amphioxus embryos treated with retinoic acid: insights into evolution and patterning of the chordate nerve cord and pharynx

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1829-1838 ◽  
Author(s):  
L.Z. Holland ◽  
N.D. Holland
Keyword(s):  
Retinoic Acid ◽  
Neural Crest ◽  
Nerve Cord ◽  
Hox Genes ◽  
Expression Patterns ◽  
Expression Domain ◽  
Craniofacial Malformations ◽  
Pharyngeal Arches ◽  
Pharyngeal Endoderm ◽  
Cerebral Vesicle

Excess all-trans retinoic acid (RA) causes severe craniofacial malformations in vertebrate embryos: pharyngeal arches are fused or absent, and a rostrad expansion of Hoxb-1 expression in the hindbrain shows that anterior rhombomeres are homeotically respecified to a more posterior identity. As a corollary, neural crest migration into the pharyngeal arches is abnormal. We administered excess RA to developing amphioxus, the closest invertebrate relative of the vertebrates and thus a key organism for understanding evolution of the vertebrate body plan. In normal amphioxus, the nerve cord has only a slight anterior swelling, the cerebral vesicle, and apparently lacks migratory neural crest. Nevertheless, excess RA similarly affects amphioxus and vertebrates. The expression domain of AmphiHox-1 (homologous to mouse Hoxb-1) in the amphioxus nerve cord is also extended anteriorly. For both the amphioxus and mouse genes, excess RA causes either (1) continuous expression throughout the preotic hindbrain (mouse) and from the level of somite 7 to the anterior end of the nerve cord (amphioxus) or (2) discontinuous expression with a gap in rhombomere 3 (mouse) and a gap at the posterior end of the cerebral vesicle (amphioxus). A comparison of these expression patterns suggests that amphioxus has a homolog of the vertebrate hindbrain, both preotic and postotic. Although RA alters the expression of AmphiHox-1 expression in the amphioxus nerve cord, it does not alter the expression of AmphiHox-1 in presomitic mesoderm or of alkali myosin light chain (AmphiMlc-alk) in somites, and the axial musculature and notochord develop normally. The most striking morphogenetic effect of RA on amphioxus larvae is the failure of mouth and gill slits to form. In vertebrates effects of excess RA on pharyngeal development have been attributed solely to the abnormal migratory patterns of Hox-expressing cranial neural crest cells. This cannot be true for amphioxus because of the lack of migratory neural crest. Furthermore, expression of Hox genes in pharyngeal tissues of amphioxus has not yet been detected. However, the absence of gill slits in RA-treated amphioxus embryos correlates with an RA-induced failure of AmphiPax-1 to become down-regulated in regions of pharyngeal endoderm that would normally fuse with the overlying ectoderm. In vertebrates, RA might similarly act via Pax-1/9, also expressed in pharyngeal endoderm, to impair pharyngeal patterning.

3 ANALYSIS OF HOX EXPRESSION IN BOVINE OOCYTES AND EARLY EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 159
Author(s):  
D. Paul ◽  
W. Sonnet ◽  
R. Rezsohazy ◽  
I. Donnay
Keyword(s):  
Embryonic Development ◽  
Rna Polymerase Ii ◽  
Oocyte Maturation ◽  
Hox Genes ◽  
Early Stage ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Main Body ◽  
Bovine Oocytes

HOX genes encode transcription factors known to play a major role in patterning the main body axis of vertebrate embryos from the gastrulation stage onward. A few studies have provided evidence that some HOX genes might be expressed before implantation in mammalian embryos. Translation of maternally inherited transcripts is regulated by modifications of the poly(A) tail length until embryonic genome activation (EGA), occurring during the 4th cell cycle in the bovine. The objective of this work was to establish the expression pattern of various HOX genes and to study the polyadenylation of their transcripts during oocyte maturation and early embryonic development. Pools of 20 bovine oocytes before and after in vitro maturation and 20 in vitro-produced embryos at different stages of development up to the blastocyst stage were collected. Three to 12 pools were used for each stage. RNA was extracted and reverse transcribed (RT) using random hexamers. Quantitative real-time PCR (qPCR) was performed to establish expression profiles of 4 HOX genes: HOXD1, HOXA3, HOXB9, and HOXC9. Two distinct patterns of expression were observed. First, relative amounts of HOXD1, HOXA3, and HOXC9 were lower in morulae and blastocysts than in oocytes. On the other hand, relative expression of HOXB9 increased between the 5 to 8 cell stage and the morula stage (Mann-Whitney, P < 0.05). Those expression patterns were not modified when embryos were cultured in presence of α-amanitin, a RNA polymerase II inhibitor, indicating the maternal origin of the transcripts until EGA. Total amount of mRNAs, estimated by RT-qPCR with random hexamers, was stable for all studied genes during oocyte maturation. The relative amount of polyadenylated GAPDH mRNAs, estimated by RT-qPCR with poly(dT), decreased greatly in mature oocytes compared with immature oocytes indicating massive deadenylation of those transcripts. The relative amount of polyadenylated HOXC9 transcripts decreased slightly but significantly during oocyte maturation (Mann-Whitney, P < 0.05).The relative amount of polyadenylatedm RNAs corresponding to HOXD1, HOXA3, and HOXB9 was stable during oocyte maturation. This indicates that those transcripts escape the default deadenylation pathways followed by housekeeping genes. This experiment has been repeated 3 to 4 times. In conclusion, we confirmed the presence of HOXD1, HOXA3, HOXB9, and HOXC9 transcripts in bovine oocytes and early-stage embryos. Their role during oocyte maturation and the first stages of embryonic development will be investigated through loss of function studies. This work is funded by the Fonds National de la Recherche Scientifique (Belgium).

scholarly journals Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Zain Umer ◽  
Jawad Akhtar ◽  
Muhammad Haider Farooq Khan ◽  
Najma Shaheen ◽  
Muhammad Abdul Haseeb ◽  
...  
Keyword(s):  
Hox Genes ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Gene Activation ◽  
Rnai Screen ◽  
Polycomb Group ◽  
Reporter System ◽  
Trithorax Group ◽  
Genome Wide ◽  
A Genome

Abstract Background Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the specialization of cell types by maintaining differential gene expression patterns. Initially discovered as positive regulators of HOX genes in forward genetic screens, trxG counteracts PcG-mediated repression of cell type-specific genes. Despite decades of extensive analysis, molecular understanding of trxG action and regulation are still punctuated by many unknowns. This study aimed at discovering novel factors that elicit an anti-silencing effect to facilitate trxG-mediated gene activation. Results We have developed a cell-based reporter system and performed a genome-wide RNAi screen to discover novel factors involved in trxG-mediated gene regulation in Drosophila. We identified more than 200 genes affecting the reporter in a manner similar to trxG genes. From the list of top candidates, we have characterized Enoki mushroom (Enok), a known histone acetyltransferase, as an important regulator of trxG in Drosophila. Mutants of enok strongly suppressed extra sex comb phenotype of Pc mutants and enhanced homeotic transformations associated with trx mutations. Enok colocalizes with both TRX and PC at chromatin. Moreover, depletion of Enok specifically resulted in an increased enrichment of PC and consequently silencing of trxG targets. This downregulation of trxG targets was also accompanied by a decreased occupancy of RNA-Pol-II in the gene body, correlating with an increased stalling at the transcription start sites of these genes. We propose that Enok facilitates trxG-mediated maintenance of gene activation by specifically counteracting PcG-mediated repression. Conclusion Our ex vivo approach led to identification of new trxG candidate genes that warrant further investigation. Presence of chromatin modifiers as well as known members of trxG and their interactors in the genome-wide RNAi screen validated our reverse genetics approach. Genetic and molecular characterization of Enok revealed a hitherto unknown interplay between Enok and PcG/trxG system. We conclude that histone acetylation by Enok positively impacts the maintenance of trxG-regulated gene activation by inhibiting PRC1-mediated transcriptional repression.

Exposure to retinoic acid before or after the onset of somitogenesis reveals separate effects on rhombomeric segmentation and 3′ HoxB gene expression domains

Development ◽  
1994 ◽  
Vol 120 (8) ◽  
pp. 2279-2285 ◽  
Author(s):  
H. Wood ◽  
G. Pall ◽  
G. Morriss-Kay
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Hox Genes ◽  
Expression Patterns ◽  
Genetic Identity ◽  
Normal Expression ◽  
Contrast Exposure ◽  
Altered Gene ◽  
The Individual ◽  
Rhombomere 4

We have compared the relationship between the patterns of altered morphogenesis and of altered gene expression in mouse embryos exposed to excess retinoic acid (RA) (a) just before and (b) just after the onset of somitic segmentation (day 7.75 to day 8.25). Exposure to RA prior to the onset of somitic segmentation results in suppression of rhombomeric (but not somitic) segmentation, and conversion of the genetic identity of the whole preotic hindbrain to that of rhombomere 4. In contrast, exposure to RA at early somite stages results in near-normal rhombomeric segmentation; rhombomeric gene expression domains indicate that only rhombomere 2 has changed its genetic identity to that of rhombomere 4, the other preotic segments showing normal expression patterns for HoxB genes and Krox-20. The results indicate that RA has separable effects (1) on the genes mediating the process of rhombomeric segmentation per se, such as Krox-20, and (2) on the genes that influence the nature of the structures that subsequently develop from the individual rhombomeres, such as the Hox genes.

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