Skin regional specification and higher-order HoxC regulation [RNA-Seq]
The integument plays a critical role in functional adaptation, with macro-regional specification forming structures like beaks, combs, feathers, and scales, while micro-regional specification modifies skin appendage shapes. However, the molecular mechanisms remain largely unknown. Craniofacial integument displays dramatic diversity, exemplified by the Polish chicken (PC) with a homeotic transformation of comb-to-crest feathers, caused by a 195–base pair (bp) duplication in HoxC10 intron. Micro-C analyses show that HoxC-containing topologically associating domain (TAD) is normally closed in the scalp but open in the dorsal and tail regions, allowing multiple long-distance contacts. In the PC scalp, the TAD is open, resulting in high HoxC expression. CRISPR-Cas9 deletion of the 195-bp duplication reduces crest feather formation, and HoxC misexpression alters feather shapes. The 195-bp sequence is found only in Archelosauria (crocodilians and birds) and not in mammals. These findings suggest that higher-order regulation of the HoxC cluster modulates gene expression, driving the evolution of adaptive integumentary appendages in birds. Overall design: To understand how chromatin architecture relates to region-specific HoxC gene expression, we performed Micro-C experiments using embryonic day 9 (E9) WL scalp, dorsal, and tail dermis as well as  juvenile PC crest mesenchyme (Cm). To examine the Hox gene expression profile in different chicken skin regions, we performed RNA-seq analysis from E9 dermis and adult feather follicles in five regions along the anterior-posterior axis (scalp, neck, anterior back, posterior back and tail) in WL chickens. We then identified differentially expressed genes (DEGs) between the scalp vs back, back vs tail, and scalp vs tail, and showed specific marker genes for each region. To explore how different skin regions modulate their chromatin accessibility to control their HoxC cluster expression profiles, we conducted ATAC-seq on E9 scalp, anterior back, and tail dermis. To test HoxC8 gene function, replication-competent avian sarcoma virus (RCAS) was used to modulate HoxC8 expression. Virus was injected into WL E4 scalp skin, and the embryos were harvested at E13 to characterize skin phenotype changes. To identify downstream mediators and possible signaling pathways associated with the phenotypic change, we performed RNA-seq on HoxC8 over-expressing and empty vector control samples. To examine HoxC cluster regulation differences between PC long crest feathers and neighboring short cranial feathers at both transcriptomic and epigenetic levels, we performed RNA-seq and ATAC-seq comparisons of juvenile PC Cm and short cranial feather mesenchyme (Fm) .