Gene expression at the pink-eyed dilution (p) locus in the mouse is confirmed to be pigment cell autonomous using recombinant embryonic skin grafts

Development ◽  
1985 ◽  
Vol 87 (1) ◽  
pp. 65-73
Author(s):  
Dennis A. Stephenson ◽  
Janet E. Hornby

Sash (Wsh), a viable and fully fertile allele of the dominant spotting (W) locus (Lyon & Glenister, 1982) has been expression at the pink-eyed dilution (p) locus. Reciprocal recombinant epidermal//dermal skin grafts were constructed from 13-day embryonic skin of p and Wsh homozygotes. Thus in the reciprocal experiments pink-eyed dilution melanocytes were exposed to any environmental influence from the wild-type allele of thep locus in either the epidermis (when WshWsh) or the dermis (when WshWsh). The hair pigmentation of the grafts recovered after three weeks beneath the testicular tunica of adult male mice was always typical of the p phenotype showing thatp is melanocyte autonomous. This result was supported by experiments using a modification of Mayer's (1965) neural crest grafting technique and the construction of 14-day recombinant skin grafts. Sash (WshWsh) epidermis can support melanocyte differentiation and pigment production but lacks functional melanocytes. The advantages of Wsh in experimental systems for testing the site of pigment gene expression have been demonstrated. Control experiments confirmed the dermal influence of agouti (A) over non-agouti (a) epidermis but non-agouti dermis did not overrule agouti pink-eyed dilution (AA pp) epidermis suggesting an epistatic effect ofp in the melanocyte.

1985 ◽  
Vol 46 (2) ◽  
pp. 193-205 ◽  
Author(s):  
Dennis A. Stephenson ◽  
Peter H. Glenister ◽  
Janet E. Hornby

SUMMARYAggregation chimaeras were constructed by fusing embryos homozygous for sash (Wsh) with fuzzy, leaden beige (fz, ln, bg) homozygotes to investigate the site of action of the beige and leaden loci. The genotype of the hair follicle was identified by fuzzy alleles (+ +fz or fzfz). All melanocytes were derived from the fuzzy leaden beige population, as sash homozygotes do not produce functional melanocytes. Reciprocal recombinant epidermal/ /dermal skin grafts were constructed from 14-day embryonic skin of homozygous fzln bg and either albino (aa cc) or pink-eyed dilution (pp) embryos to test for any dermal expression of leaden or beige, since the epidermal and dermal genotype of the chimaeric hair follicles could differ.Patches of fuzzy and non-fuzzy hairs were distributed throughout the coats of two of the three chimaeras obtained. The pigmented regions were blue grey, typical of the leaden beige interactive phenotype. Large abnormal beige granules were found in fuzzy and non-fuzzy hairs. Melanocytes in both classes of growing follicles were nucleopetal, typical of leaden. Similarly, the results of the 14-day skin grafts showed that the beige and leaden loci are melanocyte-autonomous.The chimaeras showed a pigment distribution resembling the heterozygous sash phenotype. Thus the 1:1 gene dosage of sash: wild type in heterozygotes and chimaeras has an overall effect on pigment pattern that overrides the predicted random distribution of the melanocyte precursors.


2019 ◽  
Vol 116 (20) ◽  
pp. 9893-9902 ◽  
Author(s):  
Christopher M. Uyehara ◽  
Daniel J. McKay

The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 646-646
Author(s):  
Alyssa Cull ◽  
Brooke Snetsinger ◽  
Michael J. Rauh

Abstract Introduction: The epigenetic regulator, TET2, catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. Inactivating TET2 mutations are common in myeloid cancers such as chronic myelomonocytic leukemia (CMML). Although TET2 has been characterized in hematopoietic stem and progenitor cells, little is known about its role in disease-relevant monocytes/macrophages (MΦ). Previously, we found increased expression of M2 MΦ-associated arginase 1 (Arg1) in TET2 -mutant CMML and Tet2 -deficient MΦ. Therefore, our goals were to (1) characterize Tet family expression during normal murine MΦ differentiation and polarization, (2) determine the effect of Tet2 -deficiency on broader M1-M2 MΦ spectrum gene signatures. Methods: Hematopoietic-specific Tet2+/- and Tet2-/- knockout mice were generated by breeding floxed Tet2(f/f) with Vav-Cre mice (JAX), in accordance with Queen's University's Animal Care protocols. MΦs obtained by peritoneal lavage (PMΦ) and bone marrow differentiation (BMMΦ) from 9-13 week old Tet2-/- and 20-40 week old Tet2+/- mice were treated with an M1 stimulus (100ng/mL LPS) or an M2 stimulus (10ng/mL Il-4). Comparative gene expression analysis was conducted using a 591 candidate gene Mouse Immunology Gene Expression CodeSet (NanoString). Blood plasma samples collected from Tet2f/f and Tet2-/- mice were sent for cytokine/chemokine array analysis (Eve Technologies). Results: A survey of Tet mRNA expression in wild-type C57BL/6 mouse whole BM showed that Tet1 was most abundantly expressed, with Tet2 and Tet3 having relative abundances of 0.56±0.05 and 0.09±0.01 respectively. In contrast, Tet2 expression peaked, while Tet1 expression diminished during BMMΦ differentiation. Suggesting a functional role, loss of murine Tet2 is associated with skewed myelomonocytic differentiation (i.e. CMML phenotype). In terminally-differentiated MΦ, Tet2 was the most abundantly expressed Tet gene, suggesting MΦ-specific functions. Consistent with this, following a 3-hour LPS stimulation, Tet2 mRNA levels increased 2- to 4-fold, whereas Il-4 failed to induce a similar increase in expression. Overall, our results suggested that Tet2 plays a role in M1 but not M2 macrophage polarization. Based on these findings, we hypothesized that loss of Tet2 would lead to M1 program dysregulation. PMΦs were obtained from Tet2f/f and Tet2-/- mice (n=2/ genotype) and RNA was harvested from untreated and LPS- or Il-4-treated cells. Pools of these RNA samples were then screened using Nanostring. Overall, M1-associated markers such as Stat1, Socs1, Nfkbiz, Il-6, Il-27, Il-12, Il-1 and Ccl2 were markedly increased by 2- to 50-fold in resting Tet2-/- PMΦs compared to matched Tet2f/f samples. These same M1 genes demonstrated a reduced ability to be induced by LPS treatment. We also found that while the expression of most M2 genes was similar in controls versus knockouts, Il-1rn and Arg1 were overexpressed, and Marco was decreased. This suggested that Tet2 -deficient MΦs possess a complex phenotype with a potential homeostatic response to M1 gene dysregulation. We have previously seen variable upregulation of Arg1 in mouse BMMΦs and PMΦs. Approximately 60% of Tet2-deficient mice (+/- and -/-) (n=20) tested for MΦ Arg1 mRNA expression demonstrated 2- to 90-fold increases in Arg1 compared to pooled Tet2f/f controls (n=5). We were interested in investigating the underlying mechanisms contributing to this dramatic increase in expression. Using Nanostring on pooled Tet2-deficient PMΦs with low (n=7) or high (n=8) Arg1 mRNA expression, we were able to identify genes whose expression significantly correlated with Arg1 overexpression: Cxcl3 (p=0.0329), Ppbp (p=0.0015), Cxcl1 (p=0.0104) and Ccl6 (p=0.0185). Of note, Ppbp was the most divergently expressed gene (46-fold difference) in Arg1 low vs Arg1 high macrophages, followed by Arg1 itself (14-fold difference). Suggesting a further environmental influence, blood plasma levels of TNF-alpha, Il-1b, Il-4, Il-10, Il-12 and Il-13 were significantly elevated in mice with high PMΦ Arg1 mRNA expression (n=5) compared to those with low expression (n=10). Conclusions: Tet2 is a novel regulator of murine MΦ, induced during MΦ differentiation and M1-polarization. Tet2 loss leads to complex disruption of the M1-M2 spectrum. We are currently exploring whether human TET2 mutations contribute to the abnormal immune environment of myeloid cancers. Disclosures No relevant conflicts of interest to declare.


2009 ◽  
Vol 425 (2) ◽  
pp. 295-302 ◽  
Author(s):  
Victoria H. Cowling

The 7-methylguanosine cap added to the 5′ end of mRNA is essential for efficient gene expression and cell viability. Methylation of the guanosine cap is necessary for the translation of most cellular mRNAs in all eukaryotic organisms in which it has been investigated. In some experimental systems, cap methylation has also been demonstrated to promote transcription, splicing, polyadenylation and nuclear export of mRNA. The present review discusses how the 7-methylguanosine cap is synthesized by cellular enzymes, the impact that the 7-methylguanosine cap has on biological processes, and how the mRNA cap methylation reaction is regulated.


1995 ◽  
Vol 44 (4) ◽  
pp. 347-351 ◽  
Author(s):  
Atsushi TSUJI ◽  
Takao WAKAYAMA ◽  
Akira ISHIKAWA

1992 ◽  
Vol 89 (15) ◽  
pp. 6968-6972 ◽  
Author(s):  
M. F. Lyon ◽  
T. R. King ◽  
Y. Gondo ◽  
J. M. Gardner ◽  
Y. Nakatsu ◽  
...  

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