Tissue distribution of melanocytes in Silky Fowl (SF).

<p>Spleen capsule and parenchyma (a). Thymus (b). Bursa of Fabricius (c). Testis (d). Oviduct (e). Ovary (f). Leg muscle (g). Dorsal skin (h). Leg skin (i). Brain (j). Trachea (k). Lung (l). Kidney (m). Stomach (n). Intestine (o). Melanocyte: green arrow. Lymph node: blue arrow. Hematoxylin and eosin (H&E) stain. Scale bar = 100 μm.</p

TUNEL analysis of apoptosis in SF and WL.

<p>(A) Apoptotic cells were observed in the bursa of Fabricius (a) and thymus (b) of SF, and the bursa of Fabricius (c) and thymus (d) of WL. Scale bar = 100 μm. (B) There were significant differences between the numbers of apoptotic cells in the spleen, thymus, and bursa of Fabricius in SF, compared with those in WL. **<i>P</i><0.01.</p

Bu-1⁺ cells in the spleen, thymus, and bursa of Fabricius of SF and WL.

<p>(A) Bu-1⁺ cells were observed in the spleen (a, b), thymus (e, f), and bursa of Fabricius (i, j) of SF and in the spleen (c, d), thymus (g, h), and bursa of Fabricius (k, l) of WL. Scale bar = 100 μm. (B) There were significant differences in the numbers of Bu-1⁺ cells in the spleen, thymus, and bursa of Fabricius of SF, compared with those in WL, during early development. *<i>P</i> < 0.05, **<i>P</i><0.01.</p

Tissue-dependent co-localization of melanocytes with heterophils and mast cells.

<p>Melanocytes co-localized with heterophils in the bursa of Fabricius (a) and ovaries (b). H&E stain. Melanocytes co-localized with mast cells in the lungs (c) and ovaries (d). Toluidine blue stain. Scale bar = 100 μm.</p

Melanocyte morphology.

<p>Numerous melanocytes (indicated by a blue arrow), with the appearance of dendritic cells and with round nuclei and abundant melanin, were observed in the skin (a), testis (b), thymus (c), and ovary (d). Macrophages in the thymus were stained by 3, 4-dihydroxy-l-phenylalanine (DOPA; c, green arrow). Scale bar = 100 μm.</p

Organ indices and immune gene expression in the F2 population.

<p>(a) Lower indices of spleen, thymus, and bursa of Fabricius were detected in Black-boned chickens, with a significance difference (<i>P</i> < 0.05) in the thymus. (b) Lower expression levels of <i>IFN-γ</i> and <i>IL-4</i> were detected in Black-boned chickens, compared with those in non-Black-boned chickens (<i>P</i> < 0.05). Higher expression level of <i>GAL-7</i> was detected in Black-boned chickens, compared with that in non-Black-boned chickens, but the difference was not statistically significant. B, Black-boned chicken. Non B, non-Black-boned chicken.</p

Immunohistochemical analysis of Bax expression.

<p>(A) Bax expression (brown) was detected in the spleen (a), thymus (b), and bursa of Fabricius (c) and of SF and the spleen (d), thymus (e), and bursa of Fabricius (f) of WL. Scale bar = 100 μm. (B) There was a significant difference in the numbers of Bax⁺ cells in the spleen, thymus, and bursa of Fabricius of SF, compared with those of WL. **<i>P</i><0.01.</p

Tissue-specific ultrastructural characteristics of melanocytes and neighboring cells.

<p>Transmission electron microscopy was used to observe the melanocytes. Melanosomes were observed in the melanocytes (blue arrow) and the immune cells (green arrow) of the thymus (a). Melanosomes were present in other cells (green arrow) in the skin (b). Melanosomes were secreted by exocytosis from melanocytes in the skin (c). Melanosomes were observed in the interstitial cells (green arrow) in the ovaries (d). Melanocytes and neighboring cells were connected by dendrites in the ovaries (e). Long dendrites were detected in melanocytes and neighboring cells in the ovaries (f).</p

CD3⁺ cells in the spleen and thymus of SF and WL.

<p>(A) CD3⁺ cells were observed in the spleen (a, b) and thymus (e, f) of SF and in the spleen (c, d) and thymus (g, h) of WL. Scale bar = 100 μm. (B) There were significant differences during early development between the numbers of CD3⁺ cells in the spleen and thymus of SF and WL. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Additional file 2 of Epigenetic mechanism of Gtl2-miRNAs causes the primitive sheep characteristics found in purebred Merino sheep

Additional file 2: Table S1. The pedigree relationship between ALC and MF sheep in this study. Table S2. miRNAs in the Dlk-Gtl2 locus and their predicted target genes in the candidate pathway. Table S3. The predicted target genes of sITS-miRNA in the candidate pathway. Table S4. The separate enrichment analysis of the downregulated proteins in metabolic pathways of the ALC group. Table S5. Primers used in this study. Table S6. Guide RNA sequence information in Meg3 (IG-DMR)-KO mice