The primary cilium is essential for skin morphogenesis through regulating the Notch, Wnt, and hedgehog signaling pathways. Prior studies on the functions of primary cilia in the skin were based on the investigations of genes that are essential for cilium formation. However, none of these ciliogenic genes has been linked to ciliopathy, a group of disorders caused by abnormal formation or function of cilia. To determine whether there is a genetic and molecular link between ciliopathies and skin morphogenesis, we investigated the role of RPGRIP1L, a gene mutated in Joubert (JBTS) and Meckel (MKS) syndromes, two severe forms of ciliopathy, in the context of skin development. We found that RPGRIP1L is essential for hair follicle morphogenesis. Specifically, disrupting the Rpgrip1l gene in mice resulted in reduced proliferation and differentiation of follicular keratinocytes, leading to hair follicle developmental defects. These defects were associated with significantly decreased primary cilium formation and attenuated hedgehog signaling. In contrast, we found that hair follicle induction and polarization and the development of interfollicular epidermis were unaffected. This study indicates that RPGRIP1L, a ciliopathy gene, is essential for hair follicle morphogenesis likely through regulating primary cilia formation and the hedgehog signaling pathway.

Recent breakthroughs in the generation of induced pluripotent stem cells (iPSCs) have provided a novel renewable source of cells with embryonic stem cell-like properties, which may potentially be used for gene therapy and tissue engineering. Although iPSCs have been differentiated into various cell types, iPSC-derived keratinocytes have not yet been obtained. In this study, we report the in vitro differentiation of mouse iPSCs into a keratinocyte lineage through sequential applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen IV-coated plates. We show that iPSCs can be differentiated into functional keratinocytes capable of regenerating a fully differentiated epidermis, hair follicles, and sebaceous glands in an in vivo environment. Keratinocytes derived from iPSCs displayed characteristics similar to those of primary keratinocytes with respect to gene and protein expression, as well as their ability to differentiate in vitro and to reconstitute normal skin and its appendages in an in vivo assay. At present, no effective therapeutic treatments are available for many genetic skin diseases. The development of methods for the efficient differentiation of iPSCs into a keratinocyte lineage will enable us to determine whether genetically corrected autologous iPSCs can be used to generate a permanent corrective therapy for these diseases.

GORAB is a golgin that localizes predominantly at the Golgi apparatus and physically interacts with small guanosine triphosphatases. GORAB is ubiquitously expressed in mammalian tissues, including the skin. However, the biological function of this golgin in skin is unknown. Here, we report that disrupting the expression of the Gorab gene in mice results in hair follicle morphogenesis defects that were characterized by impaired follicular keratinocyte differentiation. This hair follicle phenotype was associated with markedly suppressed hedgehog (Hh) signaling pathway in dermal condensates in vivo. Gorab-deficient dermal mesenchymal cells also displayed a significantly reduced capability to respond to Hh pathway activation in vitro. Furthermore, we found that the formation of the primary cilium, a cellular organelle that is essential for the Hh pathway, was impaired in mutant dermal condensate cells, suggesting that Gorab may be required for the Hh pathway through facilitating the formation of primary cilia. Thus, data obtained from this study provided insight into the biological functions of Gorab during embryonic morphogenesis of the skin in which Hh signaling and primary cilia exert important functions.

Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes.