Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.

Insertional polymorphisms of ETn retrotransposons include a disruption of the wiz gene in C57BL/6 mice.

ETn (early transposon) elements are moderate repetitive sequences present in hundreds of copies in the mouse genome. Their length ranges from 4.4 to 7.1 kb, and, like transposons, they contain long terminal repeats (LTRs) on both sides and are flanked by target site duplications (Kaghad et al. 1985). ETn-related elements can be grouped into three distinct families. Members of the ETn I and ETn II families mainly contain sequences of unknown origin in their core region. Only very short stretches of retrovirus-like sequences are present, and there are no ORFs. ETn I and ETn II elements differ primarily in the 3- half of both the 5- and 3- LTR, and in the 5- end of the core region (see Fig. 1). As a consequence, only ETn II elements contain a primer binding site for tRNALys. In contrast to ETn I and ETn II, members of the recently described MusD family (Mager and Freeman 2000) contain ORFs for (at least parts of) D-type virus Gag, Pro, and Pol proteins. However, in other regions they are structurally similar to ETn II elements and contain an intact primer binding site. It has been shown that MusD sequences are evolutionarily older than ETn II elements, suggesting that the latter might have arisen by recombinatory replacement of the MusD gene-coding sequences with sequences of unknown origin (Mager and Freeman 2000). ETn elements are still active as retrotransposons. In the past years, several germ line and somatic mutations caused by fresh ETn integrations have been found (Table 1). From 19 mutations, sufficient sequence is available in seven cases to show that the insertion was an ETn II element. In eight cases, the sequence data available indicate either an ETn II or a MusD element. ETn I has not been found to be the cause of any mutations, prompting the suggestion that ETn II is the "mobile" family, whereas ETn I elements have lost the capacity to retrotranspose.

Structure and expression of mobile ETnII retroelements and their coding-competent MusD relatives in the mouse.

ETnII elements are mobile members of the repetitive early transposon family of mouse long terminal repeat (LTR) retroelements and have caused a number of mutations by inserting into genes. ETnII sequences lack retroviral genes, but the recent discovery of related MusD retroviral elements with regions similar to gag, pro, and pol suggests that MusD provides the proteins necessary for ETnII transposition in trans. For this study, we analyzed all ETnII elements in the draft sequence of the C57BL/6J genome and classified them into three subtypes (alpha, beta, and gamma) based on structural differences. We then used database searches and quantitative real-time PCR to determine the copy number and expression of ETnII and MusD elements in various mouse strains. In 7.5-day-old embryos of a mouse strain in which two mutations due to ETnII-beta insertions have been identified (SELH/Bc), we detected a three- to sixfold higher level of ETnII-beta and MusD transcripts than in control strains (C57BL/6J and LM/Bc). The increased ETnII transcription level can in part be attributed to a higher number of ETnII-beta elements, but 70% of the MusD transcripts appear to have been derived from one or a few MusD elements that are not detectable in C57BL/6J mice. This element belongs to a young MusD subgroup with intact open reading frames and identical LTRs, suggesting that the overexpressed element(s) in SELH/Bc mice might provide the proteins for the retrotransposition of ETnII and MusD elements. We also show that ETnII is expressed up to 30-fold more than MusD, which could explain why only ETnII, but not MusD, elements have been positively identified as new insertions.