In vitro quantification of pigment production and transfer in 2D co-cultures and 3D skin organotypic.

Here, we present a protocol to set up and study 2D keratinocyte-melanocyte co-cultures and 3D full-thickness human skin equivalents. We describe steps for culturing of keratinocyte and melanocyte lines and the establishment of both 2D and 3D co-cultures. The cultures are utilized to measure melanin content and investigate mechanisms driving melanin production and transfer, through flow cytometry and immunohistochemistry. Culture conditions are highly amendable to different conditions, and analysis is simple and objective-thus allowing for medium to high throughput. For complete details on the use and execution of this protocol, please refer to Ng et al. (2022).1.

Sox2 in the dermal papilla regulates hair follicle pigmentation.

Within the hair follicle (HF) niche, dermal papilla (DP) cells are well known for the hair induction capacity; however, DP cell signaling also regulates HF pigmentation. Here we describe how Sox2 in the DP is a key regulator of melanocyte signaling. To study the largely unknown regulatory role the DP has on hair pigmentation, we characterize leptin receptor (Lepr) expression in the skin and as a genetic tool to target the DP. Sox2 ablation in the DP results in a phenotypic switch from eumelanin to pheomelanin. Mechanistically, we describe a temporal upregulation of Agouti and downregulation of Corin, directly by Sox2 in the DP. We also show that bone morphogenic protein (BMP) signaling regulation by Sox2 is responsible for downregulating MC1R, Dct, and Tyr in melanocytes of Sox2 cKO mice. Thus, we demonstrate that Sox2 in the DP regulates not only the choice of hair pigment but also the overall HF pigment production.

Complex housing causes a robust increase in dendritic complexity and spine density of medial prefrontal cortical neurons.

Prelimbic cortex and infralimbic cortex, parts of the ventromedial prefrontal cortex, are critical brain regions for generating a flexible behavioral response to changing environmental contingencies. This includes the role of these brain structures in the extinction of learned fear, decision making and retrieval of remote memories. Dendritic structure of medial prefrontal cortex neurons retains significant structural plasticity in adulthood. This has been mainly demonstrated as dendritic atrophy and loss of dendritic spines due to chronic stress. It remains unknown if housing condition of the animals itself can cause opposing changes in the dendritic organization. In that backdrop, here we report that short-term increase in complexity of the housing causes a robust increase in complexity of dendritic architecture of prelimbic and infralimbic neurons. This is reflected in the dendritic expansion of prelimbic neurons and increase in spine density of prelimbic and infralimbic neurons. These results suggest that non-invasive changes in the housing environment can be harnessed to study brain reserves for the flexible and species-typical behaviors.