Fig 2: Generation of zebrafish rtf1 null mutants with CRISPR/Cas9.(a) Schematic of CRISPR/Cas9 mutagenesis of the rtf1 gene. Two mutant alleles, rtf1LA2678 and rtf1LA2679, were recovered after targeting of rtf1 exon 3. Both alleles are predicted to disrupt translation of the Rtf1 protein and eliminate the HMD, Plus3, Pol II interaction, and PAF1C interaction domains. (b) Agarose gel electrophoresis results of genotyping rtf1 mutants by PCR. Deletions in rtf1LA2678 and rtf1LA2679 alleles can be distinguished from wild type allele using primers rtf1-e3-F and rtf1-e3-R. (c) Western blot detecting Rtf1 and β-actin (loading control) proteins in lysates of wild type and rtf1 mutant embryos. Image is representative of two independent experiments.

Fig 3: Failure of cardiac differentiation from Rtf1 knockdown mouse embryonic stem cells.(a) Diagram of shRNA knockdown of Rtf1 in mouse embryonic stem cells (mESCs) and differentiation of plated EBs. mESCs were transduced with non-target control or Rtf1 shRNA lentivirus and selected with puromycin. Transduced mESCs were grown into embryoid bodies (EBs) using the hanging drop method which were then plated to examine differentiation into cell types including beating cardiomyocyte clusters. (b) Western blot verifying reduction of Rtf1 protein in Rtf1 shRNA mESCs (~70% reduced based on densitometry) compared to unchanged level of the loading control protein β-actin. Image is representative of three independent experiments. (c) qPCR analysis of Myh6, Nkx2–5, and Nppa expression in non-target control (NT) and Rtf1 shRNA plated EBs. Data are normalized to the mean expression level in NT shRNA samples and error bars indicate the standard error of the mean. (d) Mean percentage (± standard error) of plated EBs exhibiting beating cardiomyocyte differentiation (cEBs) in NT and Rtf1 shRNA plated EBs. (e) qPCR analysis of Brachyury expression in non-target control (NT) and Rtf1 shRNA plated EBs. (f) qPCR analysis of Afp expression in non-target control (NT) and Rtf1 shRNA plated EBs. (g) qPCR analysis of Ncam1 expression in non-target control (NT) and Rtf1 shRNA plated EBs. Data in e, f, and g are normalized to the mean expression level in NT shRNA samples at day 0 (D0) and error bars indicate the standard error of the mean. *: p < 0.05, ***: p < 0.001.

Fig 4: Rtf1’s Plus3 domain is necessary for cardiac progenitor formation.(a) Schematics of Rtf1 wildtype (wt) and Rtf1 mutant constructs (ΔHMD and ΔPlus3). Domains are indicated by colored boxes (HMD: red, Plus3: blue, PolII and PAF1C interaction: grey). Deleted regions are represented by lines. Numbers refer to the amino acid positions in wildtype Rtf1. (b) Projections of confocal z-stacks of whole mount immunostaining detecting N-terminal FLAG-tagged Rtf1 constructs (red) expressed in 75% epiboly zebrafish embryos. Nuclei are labeled by DAPI staining (blue). Scale bars represent 20 μm. (c) Representative images of RNA in situ hybridization detecting nkx2.5 expression in 10 somite stage (10S) zebrafish embryos coinjected with rtf1 morpholino and mRNA encoding Rtf1 wildtype or mutant proteins. Scale bars represent 0.1 mm.

Fig 5: Rtf1 is required for transcriptional progression of cardiac precursors to a more mature state.(a-c) UMAP plots of anterior lateral plate mesoderm (ALPM) and derivatives from merged and integrated single cell RNA-seq datasets of 11–12 somite stage uninjected control and rtf1 morphant embryos. Cells in plots are colored by cell type (a), sample (b), and pseudotime (c). (d,f) Expression dynamics plots displaying expression levels (y-axis) for selected genes in uninjected control (d) and rtf1 morphant (f) cells over the pseudotime trajectory (x-axis) from ALPM (root) to cardiac precursor state shown in c. (e,g) UMAP plots of uninjected control (e) and rtf1 morphant (g) cells colored by gene expression levels.