Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice

Abstract

BackgroundHairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs.MethodsGeneration of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493–2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation.ResultsPLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice.ConclusionsIn conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don’t display completely hairless phenotype as expected.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1512-9) contains supplementary material, which is available to authorized users.

<p>Loss of Cirbp expression is correlated with the malignant progression and poor prognosis in nasopharyngeal carcinoma</p>

“…RNA isolation and qRT-PCR were previously well described 11–16. Briefly, total RNA was extracted from cells or fresh tissue specimens using Trizol Reagent (TaKaRa, Dalian, China) according to the manufacturer’s instruction.…”

“…Histological analysis and immunohistochemical staining were performed as described previously 11–14,17,18. Briefly, for immunohistochemical staining, 4-µm sections of the TMA blocks were deparaffinized, rehydrated.…”

Phospholipase C families: Common themes and versatility in physiology and pathology

“…Studies from mouse models indicate that PLCδ1 plays a role in keratinocytes. Mice knockouts of PLCδ1 show progressive hair loss, epidermal hyperplasia and epidermoid cyst formation and inflammation of the skin [321][322][323][324]. In these knockout mice, calcium mobilization and activation of calcineurin and NFAT is disrupted in the keratinocytes.…”

Spontaneous Quick Resolution of Uncombable Hair Syndrome-Like Disease

“…In humans, variations in PLCD1 have been connected to hereditary leukonychia [11]; however, no changes in the nail phenotype were clinically observed in the mother or the affected child. Phosphoinositide-specific phospholipase C delta 1 is located downstream of Foxn1, which is of critical importance in normal hair development [12], and indeed, mice knocked-out for PLCD1 showed a clear hair phenotype [13]. Nevertheless, the importance of this variation to the hair phenotype observed in the patient described here is unclear.…”