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Transcriptome analyses of reprogrammed feather / scale chimeric explants revealed co-expressed epithelial gene networks during organ specification.

ABSTRACT: BACKGROUND:The molecular mechanism controlling regional specific skin appendage phenotypes is a fundamental question that remains unresolved. We recently identified feather and scale primordium associated genes and with functional studies, proposed five major modules are involved in scale-to-feather conversion and their integration is essential to form today's feathers. Yet, how the molecular networks are wired and integrated at the genomic level is still unknown. RESULTS:Here, we combine classical recombination experiments and systems biology technology to explore the molecular mechanism controlling cell fate specification. In the chimeric explant, dermal fate is more stable, while epidermal fate is reprogrammed to be similar to the original appendage type of the mesenchyme. We analyze transcriptome changes in both scale-to-feather and feather-to-scale transition in the epidermis. We found a highly interconnected regulatory gene network controlling skin appendage types. These gene networks are organized around two molecular hubs, ?-catenin and retinoic acid (RA), which can bind to regulatory elements controlling downstream gene expression, leading to scale or feather fates. ATAC sequencing analyses revealed about 1000 altered widely distributed chromatin open sites. We find that perturbation of a key gene alters the expression of many other co-expressed genes in the same module. CONCLUSIONS:Our findings suggest that these feather / scale fate specification genes form an interconnected network and rewiring of the gene network can lead to changes of appendage phenotypes, acting similarly to endogenous reprogramming at the tissue level. This work shows that key hub molecules, ?-catenin and retinoic acid, regulate scale / feather fate specification gene networks, opening up new possibilities to understand the switches controlling organ phenotypes in a two component (epithelial and mesenchyme) system.

SUBMITTER: Lai YC

PROVIDER: S-EPMC6206740 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Transcriptome analyses of reprogrammed feather / scale chimeric explants revealed co-expressed epithelial gene networks during organ specification.

Lai Yung-Chih YC Liang Ya-Chen YC Jiang Ting-Xin TX Widelitz Randall B RB Wu Ping P Chuong Cheng-Ming CM

BMC genomics 20181029 1

<h4>Background</h4>The molecular mechanism controlling regional specific skin appendage phenotypes is a fundamental question that remains unresolved. We recently identified feather and scale primordium associated genes and with functional studies, proposed five major modules are involved in scale-to-feather conversion and their integration is essential to form today's feathers. Yet, how the molecular networks are wired and integrated at the genomic level is still unknown.<h4>Results</h4>Here, we ...[more]

PMID: 30373532

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Project description:1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.

Project description:1. Explants of mammary glands of mid-pregnant rabbits that had been cultured for 18h in the presence of insulin, prolactin and cortisol were incubated at 37 degrees C for 2h in Medium 199 containing l-[4,5-(3)H]leucine. After a wash procedure at 4 degrees C, explants were re-incubated at 37 degrees C in fresh medium and the radioactivity of casein polypeptides isolated by isoelectric focusing (at pH 4.6) was followed with time. Casein radioactivity rose during the first hour of re-incubation, but fell markedly during the subsequent hour. 2. Loss of radioactivity represented casein degradation, since less than 10% of newly synthesized casein was found in the incubation medium. 3. Such a loss of radioactivity was not due solely to hydrolysis of signal peptides, since similar results were obtained when l-[5-(3)H]proline, which is not part of casein signal peptides, was the radiolabelled precursor. 4. A dual-isotope experiment using l-[U-(14)C]proline and N-[(3)H]acetyl-d-mannosamine gave similar profiles of radioactivity loss from isoelectrically focused casein, indicating that degradation of mature casein was occurring. 5. Analysis of total pellet and particle-free-supernatant fractions prepared by centrifugation of explant homogenates at 115000g(av.) for 1h did not show loss of radioactivity on re-incubation. Total pellet-protein radioactivity remained constant, whereas total soluble-protein radioactivity increased during the 2h re-incubation period. 6. Radioactivity in a specific particle-free-supernatant polypeptide, the subunit of fatty acid synthetase, mimicked that of the total soluble protein. 7. Addition of cycloheximide (20mug/ml) during the re-incubation period completely blocked the incorporation of radioactivity from l-[5-(3)H]proline into casein and the subsequent fall, indicating that observations were being made on newly synthesized casein. 8. Addition of chloroquine (50mum) did not prevent the increase in radioactivity from l-[5-(3)H]proline into casein during the first hour of re-incubation, but did prevent the loss of radioactivity in the second hour. 9. The intracellular degradation of a newly synthesized milk protein is discussed in relation to the known intracellular degradation of other secretory polypeptides.

Dataset Information

Transcriptome analyses of reprogrammed feather / scale chimeric explants revealed co-expressed epithelial gene networks during organ specification.

Publications

Transcriptome analyses of reprogrammed feather / scale chimeric explants revealed co-expressed epithelial gene networks during organ specification.

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