Fig. 3: A human-specific LTR5Hs insertion is essential for blastoid formation.
From: A human-specific regulatory mechanism revealed in a pre-implantation model
a, Strategy for testing LTR5Hs sequence dependency. WT, wild-type. b, RT–qPCR results of the indicated genes in wild-type or ΔLTR5Hs hnPSCs. RNA values were normalized to RPL13A. Above each plot, schematics of the locus are included. The grey dots represent expression in each clone, and the diamonds represent median values. n (independent clones) per gene: SUSD2/CABIN, WT = 5, heterozygous = 2 and homozygous = 5; ZNF729, WT = 5, heterozygous = 4 and homozygous = 2; SERPINB9/6, WT = 5 and heterozygous = 4; CEBPZ/NDUFAF7, WT = 5 and homozygous = 4; BARD1, WT = 5 and homozygous = 2; and PCAT14, WT = 5, heterozygous = 6 and homozygous = 6. Significance was determined by an unpaired, two-tailed Student’s t-test. The asterisk colour indicates homozygous (red) or heterozygous (yellow); specific P values are included as source data. NS, not significant. c, Expression and conservation of the LTR5Hs-regulated genes between humans and mice using data in ref. 34. The silhouettes of the human and mouse were created in BioRender. Fueyo, R. (2025) https://BioRender.com/tmgd0pn. d, Cell proliferation curves in wild-type (green) and ΔLTR5Hs ZNF729−/− hnPSCs (red). n = 3 biological replicates. Data represent mean ± s.d. Significance was determined by one-way analysis of variance (ANOVA). e, Cartoon displaying the conservation of the gene ZNF729 and its regulatory landscape across species. The LTR5Hs element is only present in humans. Data adapted from Cactus UCSC tracks53,54. Chr., chromosome. f, ZNF729 expression in human29,55,56 and chimpanzee39 naive PSCs; rhesus monkey naive PSCs lack the transcript. Data are presented as mean ± s.e.m. Each dot represents a bulk RNA-seq biological replicate: hnPSC = 4, naive_H9 = 4, HNES1 = 3, cRH9 = 3, Ume4 = 2, CP127 = 3, CPR1 = 2 and Pen23 = 3. TPM, transcripts per million. g, Representative bright-field images of blastoids or dark spheres generated from wild-type hnPSCs, ΔLTR5Hs ZNF729−/+, ΔLTR5Hs ZNF729−/− hnPSCs or ΔLTR5Hs ZNF729−/− hnPSCs expressing a ZNF729 transgene (rescue, ΔLTR5Hs ZNF729 + overexpression (OE) ZNF729). Scale bar, 400 µm. h, Quantification of blastoid formation efficiency from panel g. Data are presented as mean ± s.e.m. Each dot represents a biological replicate from one clone; we used three clones per condition in biological triplicates. Significance was determined by an unpaired, two-tailed Student’s t-test: P = 7.6 × 10−10 for ZNF729−/+, P = 4.47 × 10−14 for ΔLTR5Hs ZNF729−/− and P = 7.63 × 10−9 for ΔLTR5Hs ZNF729  + OE ZNF729.
