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. 2021 Apr 13;118(16):e2021722118. doi: 10.1073/pnas.2021722118

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Copyright © 2021 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

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Fig. 3. — hECE18 positively regulates Engrailed 1 to promote eccrine gland formation. (A) Fold change normalized EN1 mRNA by qRT-PCR in human GMF24F1A cultured keratinocytes upon dCAS9-KRAB–mediated hECE18 repression. The schematic of strategy and relative positions of EN1, SP1^A and SP1^B, and Cr1 and Cr2 gRNA targets is shown. The fold change calculated relative to dCAS9-KRAB transduction alone is shown. (B) Fold luciferase induction relative to empty reporter vector (Control) by ECE18 orthologs in primary mouse keratinocytes. (C) Strategy to generate hECE18 knock-in (hECE18^KI) mouse model. (D) Fold change in En1 mRNA by qRT-PCR in P2.5 volar forelimb skin of wild-type (^+/+), hECE18^KI heterozygote (hECE18^KI/+), and hECE18^KI homozygote (hECE18^KI/hECE18^KI) mice relative to wild type. (E) Normalized ratio of C57BL/6J:FVB/N En1 allelic expression in volar forelimb skin of wild-type (C57BL/6J/FVB/N) and hECE18^KI (C57BL/6J^(hECE18KI)/FVB/N) hybrid mice. The ratios were normalized to genomic DNA allelic ratio. (F) Representative stained epidermal preparations of volar forelimb skin from En1^KO/+;^+/+ and En1^KO/+;+/hECE18^KI adult mice. The number of eccrine glands in the IFP area (outlined) and excluding the footpads (FP, circled) were quantified in analyses in G. Hair follicle (HF, *), eccrine gland (EG). (G) Quantification of IFP eccrine glands in En1 ^KO/+;^+/+ and En1 ^KO/+;+/hECE18^KI mice. Each point represents the average number of eccrine glands in the IFP across both forelimbs of a mouse. (A, B, G) Median (line), 25 to 75% percentiles (box bounds) and min and max (whiskers) are plotted. (D and E) Genotype mean is shown as a line and each point represents a single biological sample of pooled volar skin from both forelimbs of at least three mice. (A, B, D) significance assessed by one-way ANOVA and Tukey-adjusted P values are reported. (E and G) Significance assessed by a two-tailed t test. ****P < 0.0001, **P < 0.01, *P < 0.05. (+) wild- type allele. (KO) knockout. (KI) knock-in. The dots represent an individual biological replicate.