Sumio Takahashi

A possible involvement of melanocortin 1-receptor in regulating feather color pigmentation in the chicken

The Extended black (E) locus on chromosome 1 acts within the melanocyte to regulate feather color pigmentation in the chicken. Several alleles exhibiting different pigmentation have been described and their phenotypes are similar to those of the murine extension locus which encodes melanocortin 1-receptor (MC1-R), the receptor for alpha-melanocyte-stimulating hormone (alpha-MSH). To investigate whether the MC1-R gene is responsible for E-locus function, we examined the structure of MC1-R in E-locus mutants by RFLP analysis and genomic DNA sequencing. In the most recessive allele (ey), which exhibits a uniformly red-yellow pigmentation, MC1-R was found to contain amino acid substitutions possibly causing functional deficiency. On the other hand, in the most dominant allele (E), which confers a uniformly black pigmentation, MC1-R possessed mutation responsible for a constitutively active MC1-R and resultant black coat color in mice. Our finding that the structure of MC1-R was affected by individual E-locus alleles strongly suggests that MC1-R is associated with the E-locus. Furthermore, since the same mutation of MC1-R was found in mice and chickens that exhibit the same pigmentation, it is possible that the regulatory mechanism of MC1-R function is shared in chickens and mammals.

MOLECULAR CLONING AND CHARACTERIZATION OF THE CHICKEN PRO-OPIOMELANOCORTIN(POMC) GENE

The gene for pro-opiomelanocortin (POMC), a common precursor of melanocortins, lipotropins and beta-endorphin, was isolated in the chicken first among avian species. The chicken POMC gene was found to be a single copy gene and appeared to show the same structural organization as that of other species of different classes. The predicted POMC displayed the highest identity to Xenopus POMC(A) (60. 1%), and consisted of 251 amino acid residues with nine proteolytic cleavage sites, suggesting that it could be processed to give rise to all members of the melanocortin family, including adrenocorticotropic hormone and alpha-, beta- and gamma-melanocyte-stimulating hormones, as well as the other POMC-derived peptides. RT-PCR analysis detected the POMC mRNA in the brain, adrenal gland, gonads, kidney, uropygial gland and adipose tissues, each of which has been demonstrated to express melanocortin receptors. These results suggest that melanocortins act in a paracrine and/or autocrine manner to control a variety of functions both in the brain and in the peripheral tissues in the chicken.

Widespread expression of Agouti-related protein (AGRP) in the chicken: a possible involvement of AGRP in regulating peripheral melanocortin systems in the chicken.

Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action. It is expressed mainly in the arcuate nucleus where it plays an important role in the hypothalamic control of feeding and energy homeostasis by antagonism of central melanocortin 4 receptors in mammals. Besides in the brain, the melanocortin 4 receptor is expressed in numerous peripheral tissues in the chicken. To examine whether or not the peripheral melanocortin 4 receptor signaling could be regulated by AGRP, we cloned and localized the expression of the AGRP gene in the chicken. The chicken AGRP gene was found to encode a 154 or 165 amino acid protein, depending on the usage of two alternative translation initiation sites. The coding sequence consisted of three exons, like that of mammalian species. The C-terminal cysteine-rich region of the predicted AGRP displayed high levels of identity to mammalian counterparts (78-84%) and all 10 cysteine residues conferring functional conformation of AGRP were conserved; however, other regions showed apparently no homology, suggesting that biological activities of AGRP are located in its C-terminal region. RT-PCR analysis detected the AGRP mRNA in all tissues examined: the brain, adrenal gland, heart, liver, spleen, gonads, kidney, uropygial gland, skeletal muscle and adipose tissues. Interestingly, the skin also expressed the AGRP mRNA, where Agouti, another melanocortin receptor antagonist regulating hair pigmentation, is expressed in rodents. Most of those AGRP-expressing tissues have been demonstrated to express melanocortin 4 receptors and/or other subtypes of melanocortin receptor whose mammalian counterparts can bind AGRP. These results imply the possibility that some peripheral melanocortin systems could be regulated by the functional interaction between melanocortins and AGRP at melanocortin receptors in the chicken.

MOLECULAR CLONING OF THE CHICKEN MELANOCORTIN 2 (ACTH) -RECEPTOR GENE

The chicken melanocortin 2-receptor (MC2-R) gene was isolated. It is found to be a single copy gene encoding a 357 amino acid protein, sharing 65.8-68.7% identity with mammalian counterparts. The chicken MC2-R mRNA is expressed in the adrenal and spleen, suggesting that the receptor mediates both endocrine and immunoregulatory functions of ACTH in the chicken. The amino acid sequence of the chicken MC2-R is collinear with those of other subtypes of MC-R, whereas all cloned mammalian MC2-Rs contain a gap in the third intracellular loop, suggesting that mammalian MC2-R molecules have evolved by lacking a part of the domain which determines the specificity of signal transduction in G-protein coupled receptors. Interestingly, the codon usage differs dramatically between MC1-R and MC2-R in the chicken; the GC-contents at the third codon position in MC1-R and MC2-R are 94.6 and 50.6%, respectively. It may reflect selective constraints on the usage of synonymous codons.

Development and Heterogeneity of Prolactin Cells

Prolactin (PRL) is synthesized in pituitary cells called mammotrophs (PRL cells). Ample evidence demonstrates that the PRL cell population consists of structurally and functionally heterogeneous PRL cells. Multiple variants of PRL molecules are found in various species. Prolactin cells may be divided into various subtypes in the rat and mouse. Secretory activities differ among the PRL cell population. These heterogeneities may reflect various phases of the maturation process of PRL cells, or the integrated outcome of various functional differences in PRL cells. To clarify the significance of heterogeneities among PRL cells, we present updated reports on the differentiation, proliferation, and development of PRL cells, and discuss factors responsible for the functional differences in PRL cell population. The age-related alteration in PRL secretion in the rat is summarized, because it is one of the most important aspects of the developmental changes in PRL cells. A mammosomatotroph, which secretes growth hormone and PRL, is found in various species. Prolactin cells and somatotrophs are derived from the same lineage. The possible relationship among PRL cells, somatotrophs, and mammosomatotrophs is discussed.

Avian Melanocortin System: α-MSH May Act as an Autocrine/Paracrine Hormone

Abstract: The interest in the physiological role of α‐MSH in birds has been limited because they lack the intermediate lobe of the pituitary, the main source of circulating α‐MSH in most vertebrates. Recent studies have improved our understanding of the avian melanocortin system. We have cloned and characterized all five MC‐R subtypes, POMC, and AGRP in chicken. Analyses of the tissue distribution of expression of these genes revealed widespread expression throughout the body, corresponding to the situation in mammals in which α‐MSH exerts a multiplicity of effects in different tissues by acting as a local mediator. We showed that the extended black locus controlling feather pigmentation in the chicken encodes MC1‐R. Moreover, black chickens carrying the dominant allele, the extended black, express the MC1‐R with ligand‐independent activity as the somber‐3J black mice. α‐MSH and AGRP were expressed in the infundibular nucleus of POMC and NPY neurons, respectively, in the brain of Japanese quail. Furthermore, fasting stimulated AGRP expression and lowered POMC expression. These data indicate that at least two of the major melanocortin systems reported in mammals, that is, regulation of pigmentation and energy homeostasis, was developed in a common ancestor to chicken and mammals at least 300 million years ago. Furthermore, α‐MSH peptide was identified in developing chicken eye, suggesting a possible involvement of α‐MSH in regulation of ocular development. Collectively, the data reviewed here indicate that α‐MSH is produced locally and acts as an autocrine/paracrine hormone in birds.

Epidermal Growth Factor and Transforming Growth Factor-α Stimulate the Proliferation of Mouse Uterine Stromal Cells

Abstract Growth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17β (E2) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-α increased the percentage of BrdU-labeled cells in a dose-dependent manner. Administration of the combination of E2 (10−9 M) and P (10−7 M) for 2 days increased the percentage of BrdU-labeled cells 2.3-fold. The stimulatory effect of EGF, TGF-α and the combination of E2 and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10−5 M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-α-, and EGF-mRNA was carried out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-α, both produced in the stromal cells, acting for DNA synthesis through EGF receptors.

Elaborate color patterns of individual chicken feathers may be formed by the agouti signaling protein

Hair and feather pigmentation is mainly determined by the distribution of two kinds of melanin, eumelanin and pheomelanin, which produce brown to black and yellow to red colorations, respectively. The agouti signaling protein (ASIP) acts as an antagonist or an inverse agonist of the melanocortin 1 receptor (MC1R), a G protein-coupled receptor for α-melanocyte-stimulating hormone (α-MSH). This antagonism of the MC1R by ASIP on melanocytes initiates a switch of melanin synthesis from eumelanogenesis to pheomelanogenesis in mammals. In the present study, we isolated multiple ASIP mRNA variants generated by alternative splicing and promoters in chicken feather follicles. The mRNA variants showed a discrete tissue distribution. However, mRNAs were expressed predominantly in the feather pulp of follicles. Paralleling mRNA distribution, ASIP immunoreactivity was observed in feather pulp. Interestingly, ASIP was stained with pheomelanin but not eumelanin in pulp areas that face developing barbs. We suggest that the elaborate color pattern of individual feathers is formed in part by the antagonistic action of ASIP that is produced by multiple mRNA variants in chicken feather follicles.

Conserved distal promoter of the agouti signaling protein (ASIP) gene controls sexual dichromatism in chickens.

Brilliant plumage is typical of male birds, thus sexual plumage dichromatism is seen in many avian species; however, the molecular mechanism underlying this remains unclear. The agouti signaling protein (ASIP) is a paracrine factor that stimulates yellow/red pigment (pheomelanin) synthesis and inhibits black/brown pigment (eumelanin) synthesis in follicular melanocytes. In mammals, the distal promoter of the ASIP gene acts exclusively on the ventral side of the body to create a countershading pigmentation pattern by stimulating pheomelanin synthesis in the ventrum. Here, we examined the role of the distal ASIP promoter in controlling estrogen-dependent sexual dichromatism in chickens. Reverse-transcription polymerase chain reaction analyses revealed that ASIP class 1 mRNAs transcribed by the distal promoter were expressed exclusively on the ventral side of chicks and adult females displaying countershading. In showy adult males, the ASIP class 1 mRNAs were expressed in gold-colored ornamental feathers grown on the back. In the presence of estrogen, males molted into female-like plumage and ASIP class 1 mRNAs expression was altered to female patterns. These results suggest that the distal ASIP promoter produces countershading in chicks and adult females, similar to the ventral-specific ASIP promoter in mammals. In addition, the class 1 promoter plays an important role for creating sexual plumage dichromatism controlled by estrogen. This is the first evidence for a pigmentation gene having been modified in its expression during evolution to develop phenotypic diversity between individuals of different sexes.

Identification of a feather β-keratin gene exclusively expressed in pennaceous barbule cells of contour feathers in chicken

Feathers are elaborate skin appendages shared by birds and theropod dinosaurs that have hierarchical branching of the rachis, barbs, and barbules. Feather filaments consist of β-keratins encoded by multiple genes, most of which are located in tandem arrays on chromosomes 2, 25, and 27 in chicken. The expansion of the genes is thought to have contributed to feather evolution; however, it is unclear how the individual genes are involved in feather formation. The aim of the present study was to identify feather keratin genes involved in the formation of barbules. Using a combination of microarray analysis, reverse-transcription polymerase chain reaction, and in situ hybridization, we found an uncharacterized keratin gene on chromosome 7 that was expressed specifically in barbule cells in regenerating chicken feathers. We have named the gene barbule specific keratin 1 (BlSK1). The BlSK1 gene structure was similar to the gene structure of previously characterized feather keratin genes, and consisted of a non-coding leader exon, an intron, and an exon with an open reading frame (ORF). The ORF was predicted to encode a 98 aa long protein, which shared 59% identity with feather keratin B. Orthologs of BlSK1 were found in the genomes of other avian species, including turkey, duck, zebra finch, and flycatcher, in regions that shared synteny with chromosome 7 of chicken. Interestingly, BlSK1 was expressed in feather follicles that generated pennaceous barbules but not in follicles that generated plumulaceous barbules. These results suggested that the composition of feather keratins probably varies depending on the structure of the feather filaments and, that individual feather keratin genes may be involved in building different portions and/or types of feathers in chicken.