Experimental setup and phenotypic classification. (A) Melanin-based plumage pigmentation differs between all-black carrion crows (CC) and grey-coated hooded crows (HC). Growing feather follicles were sampled in the head region (circle) and on the ventral part of the torso (rectangle). Photographs adjacent to the schematic representation of birds show an example of semiplume, pennaceous body feathers (scale bar=1 cm) from the torso or head for each taxon. Squares define the area of the mature feather represented by the ensheathed feather follicle (scale bar=2 mm), which forms the raw material of the experiment. Lighter pigmentation of mature feathers of HC torso is already visible in ensheathed feather follicles. Symbol color imitates the pigmentation of mature feather tips. Bird drawings courtesy of Dan Zetterström. (B) Bright-field images from sections of ensheathed feather follicles. The dashed square on the longitudinal sections defines the corresponding position of the cross-section at 1000 μm above the dermal papilla. The arrowhead in blue indicates the location of the rachis (see Fig. 1). Dark areas represent regions where light-absorbing eumelanin is accumulated. Follicles from black-fathered regions show relatively higher eumelanin content in barb ridges than those sampled from the grey torso of hooded crows (scale bar=0.5 mm).

Melanocyte characterization. The schematic of the proximal end of a feather follicle on the left side depicts the cutting planes for the images to the right. Melanocytes are visualized by immunostaining against the TYRP1 protein (in green); nuclei were stained with Hoechst 33342 (in blue). (A) The longitudinal section represented by the area of the dashed square illustrates the emergence of melanocytes in the collared bulge (CB) and their subsequent maturation in the ramogenic zone (RGZ) with increasing accumulation of the TYRP1 protein; scale bar=50 μm. (B) Cross-section showing barb ridges at an early developmental stage at 500 μm above the dermal papilla. The TYRP1 protein begins accumulating in a tubular cell body located in the ventral growth zone; scale bar=20 μm. (C) Protein immunostaining combined with in situ mRNA padlock probe detection on a cross-section at 1000 μm above the dermal papilla. At this developmental stage, melanocytes appear fully mature; the TYRP1 protein (in green) is predominantly accumulated in a spherical cell body sending dendritic cytoplasm into barbule plates (white arrow). Signals of rolling circle amplification (RCA) of in situ mRNA padlock probing tagging individual TYRP1 mRNA transcripts (shown in red) are confined to the cell body. In contrast to melanocyte-specific gene expression of TYRP1, mRNA of the internal control gene ACTB (in white) is ubiquitously expressed across other cell types; scale bar=20 μm.

Longitudinal gene expression patterns of targeted mRNA transcripts in feathers from torso. Histogram summarizing mRNA transcript abundance along the proximal–distal axis of feather follicles sampled from black torso of carrion crows (CC, left column) and grey torso of hooded crows (HC, right column). Each bin represents the raw counts of targeted RCA signals from mRNA transcripts of each candidate gene. Grey and black arrows indicate the position of cross-sections at 500 and 1000 μm above the dermal papilla, respectively.

Five-way in situ gene expression in an explicit histological context. Illustration of in situ stained mRNA transcripts of five genes on the same histological cross-section of carrion crow (CC black) and hooded crow (HC grey) follicles sampled from torso at 1000 μm above the dermal papilla. For orientation, these cross-sections reflect the schematic shown in Fig. 1B. Bright-field images are superimposed for orientation and direct comparison among genes. The images represent one of five serial sections used for statistical analyses. ACTB and MITF (left column) are ubiquitously expressed in all cell types of a follicle. In contrast, HPGDS, SLC45A2 and TYRP1 (right column) are restricted to melanocyte cell bodies. Scale bar=50 μm.

Specificity of gene expression and differentiation by taxon and pigmentation intensity. (A) Boxplot of the specificity index of targeted genes. TYRP1, HPGDS and SLC45A2 are near-exclusively expressed in melanocytes and do not differ in specificity (post hoc Tukey test, P>0.05 for all comparisons). MITF transcripts are significantly less specific to melanocytes, but include a variety of other cell types. The ACTB control is ubiquitously expressed. Highly significant comparisons (P<0.001, post hoc Tukey test) are indicated among groups of genes with a triple asterisk (n=8). (B) Notched boxplot of raw counts (ACTB gene) or normalized in situ RCA signals of targeted mRNA transcripts (HPGDS, SLC45A2, TYRP1 and MITF) in melanocytes at 1000 μm above the dermal papilla of carrion crow (CC) and hooded crow (HC) torso feathers (n=30). Gene expression of all four genes is significantly higher in melanocytes of carrion crow (CC black) compared with hooded crows (HC grey) (see Table 2). For gene expression of the head, see Fig. S5.