Pbx and Prdm1a transcription factors differentially regulate subsets of the fast skeletal muscle program in zebrafish

Abstract

The basic helix-loop-helix factor Myod initiates skeletal muscle differentiation by directly and sequentially activating sets of muscle differentiation genes, including those encoding muscle contractile proteins. We hypothesize that Pbx homeodomain proteins direct Myod to a subset of its transcriptional targets, in particular fast-twitch muscle differentiation genes, thereby regulating the competence of muscle precursor cells to differentiate. We have previously shown that Pbx proteins bind with Myod on the promoter of the zebrafish fast muscle gene mylpfa and that Pbx proteins are required for Myod to activate mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Here we have investigated the interactions of Pbx with another muscle fiber-type regulator, Prdm1a, a SET-domain DNA-binding factor that directly represses mylpfa expression and fast muscle differentiation. The prdm1a mutant phenotype, early and increased fast muscle differentiation, is the opposite of the Pbx-null phenotype, delayed and reduced fast muscle differentiation. To determine whether Pbx and Prdm1a have opposing activities on a common set of genes, we used RNA-seq analysis to globally assess gene expression in zebrafish embryos with single- and double-losses-of-function for Pbx and Prdm1a. We find that the levels of expression of certain fast muscle genes are increased or approximately wild type in pbx2/4-MO;prdm1a-/- embryos, suggesting that Pbx activity normally counters the repressive action of Prdm1a for a subset of the fast muscle program. However, other fast muscle genes require Pbx but are not regulated by Prdm1a. Thus, our findings reveal that subsets of the fast muscle program are differentially regulated by Pbx and Prdm1a. Our findings provide an example of how Pbx homeodomain proteins act in a balance with other transcription factors to regulate subsets of a cellular differentiation program.

Keywords: Fiber-type differentiation; Pbx; Prdm1; Skeletal muscle; Zebrafish.

Fig. 1.. pbx2/4 and prdm1a are not required for each other's expression.

(A,B) RNA in situ expression of prdm1a and krox-20 in (A) control and (B) pbx2/4-MO embryos. prdm1a expression in the adaxial cells along the axis of the embryo is maintained, while krox-20 expression is lost, in pbx2/4-MO embryos. Arrowheads point to adaxial cells, and r3 and r5 indicate krox-20 expression in hindbrain rhombomeres. (C,D) Anti-Pbx (red) and DAPI (blue) staining, along with merged images, in (C) control and (D) prdm1a−/− embryos. Pbx expression is maintained in prdm1a−/− embryos. All embryos are shown in dorsal view, anterior towards the left. Scale bars: (A) 50 µm, (C) 10 µm.

Fig. 2.. Expression of Pbx and Prdm1a target genes in pbx2/4-MO;prdm1a−/− embryos.

(A,C,E) RNA in situ expression of (A) mylpfa and krox-20, (C) myhz1.3 and krox-20, and (E) myog and krox-20 in green, smyhc1 in red, and merged images in control, pbx2/4-MO, prdm1a−/−, and pbx2/4-MO;prdm1a−/− embryos. krox-20 expression is included as a control for the pbx2/4-MO knockdown (Maves et al., 2007). r3 and r5 indicate krox-20 expression in hindbrain rhombomeres. smyhc1 is downregulated in prdm1a−/− embryos (Elworthy et al., 2008; von Hofsten et al., 2008). (A,C) embryos are at 14 somites, (E) embryos are at 10 somites. All embryos are shown in dorsal view, anterior towards the left. Somites were counted in all embryos shown to confirm consistent staging. (B,D,F) Graphs of real-time RT-PCR (qRT-PCR) expression levels for (B) mylpfa, (D) myhz1.3, and (F) myog. Expression levels were normalized to odc1 expression. Error bars represent standard deviation. Somite stages (14 s, 10 s) are shown. Scale bar: 50 µm.

Fig. 3.. Adaxial cell expression of mylpfa is absent in pbx2/4-MO embryos but present in pbx2/4-MO;prdm1a−/− embryos.

RNA in situ expression of mylpfa (green) and smyhc1 (red) in (A) control, (B) pbx2/4-MO, (C) prdm1a−/−, and (D) pbx2/4-MO;prdm1a−/− embryos. Embryos are 14 somites stage, shown in dorsal view, anterior towards the left. Somite numbers 2–7 are shown for each embryo. Yellow lines indicate where optical sections are taken through the x and y planes (lower and right panels, respectively, each image). Dorsal–ventral (D–V) axes (z axis in xy view) are indicated. Arrowheads point to lateral domain of mylpfa expression, absent or reduced in pbx2/4-MO and pbx2/4-MO;prdm1a−/− (asterisk) embryos. smyhc1-overlapping mylpfa expression in adaxial cells is present in pbx2/4-MO;prdm1a−/− embryos. Scale bar: 50 µm.

Fig. 4.. Pbx2/4 and Prdm1a independently regulate genes.

Graphs of qRT-PCR expression levels for (A,B) krox-20 and (C,D) smyhc1. Expression levels were normalized to odc1 expression. Error bars represent standard deviation. Somite stages (10 s, 14 s) are shown.

Fig. 5.. pbx2/4 and prdm1a regulate overlapping and distinct subsets of fast muscle differentiation genes, identified through RNA-seq.

(A) Venn diagram identifying the intersection (12 genes) of the set of genes downregulated in pbx2/4-MO embryos (pbx2/4-MO<control, supplementary material Table S1) with the set of genes upregulated in prdm1a−/− embryos (prdm1a−/−>control, supplementary material Table S2). (B) List of the 12 genes identified in A. Also shown are fast muscle genes regulated by Pbx2/4, independently of Prdm1a, and those regulated by Prdm1a, independently of Pbx2/4, as identified from supplementary material Tables S1 and S2. The asterisk refers to fast myosin heavy chain genes that were not identified as Pbx2/4-dependent through RNA-seq, but that are known through additional validation to be Pbx2/4-dependent (see Fig. 2 and Discussion).

Fig. 6.. Visualization and validation of RNA-seq analysis.

Mapping of RNA-seq reads as viewed in the Integrative Genomics Viewer for (A) mylpfa, (B) myog, (C) myhz1.3, (D) krox-20, and (E) smyhc1. Mapped reads are represented by grey histograms. Blue bars at the bottom of each panel represent annotated exons and UTRs (blue boxes) and introns (blue lines with arrowheads for orientation of transcript from 5′ to 3′) for each gene. For C, myhz1.3 is not annotated but the genomic position is confirmed relative to neighboring annotated zebrafish myosin heavy chain genes and mammalian orthologues. For each gene, rows for reads from control, pbx2/4-MO, prdm1a−/−, and pbx2/4-MO;prdm1a−/− are shown, as in A.

Fig. 7.. RNA-seq analysis identifies subsets of the fast muscle program that are differentially regulated by Pbx2/4 and Prdm1a.

(A–C) Venn diagrams showing overlap of genes whose expression is downregulated in pbx2/4-MO embryos relative to control (green circles) compared to (A) genes that are upregulated in pbx2/4-MO;prdm1−/− embryos, (B) genes that are expressed at about control levels in pbx2/4-MO;prdm1−/− embryos, and (C) genes that are downregulated in pbx2/4-MO;prdm1−/− embryos. The number of genes in each overlap is shown in parentheses; genes are listed in supplementary material Table S3.

Fig. 8.. Validation of fast muscle genes differentially regulated by Pbx and Prdm1a.

(A,C,E) RNA in situ expression of (A) myl1 and krox-20, (C) atp2a1 and krox-20, and (E) srl and krox-20 in green, smyhc1 in red, and merged images in control, pbx2/4-MO, prdm1a−/−, and pbx2/4-MO;prdm1a−/− embryos. krox-20 expression is included as a control for the pbx2/4-MO knockdown (Maves et al., 2007). r3 and r5 indicate krox-20 expression in hindbrain rhombomeres. smyhc1 is downregulated in prdm1a−/− embryos (Elworthy et al., 2008; von Hofsten et al., 2008). (A,C) embryos are at 14 somites, (E) embryos are at 18 somites. All embryos are shown in dorsal view, anterior towards the left. Somites were counted in all embryos shown to confirm consistent staging. (B,D,F) Graphs of real-time RT-PCR (qRT-PCR) expression levels for (B) myl1, (D) atp2a1, and (F) srl. Expression levels were normalized to odc1 expression. Error bars represent standard deviation. Somite stage for qRT-PCR (14 s) is shown. Scale bar, 50 µm.

Fig. 9.. Multiple combinations of Pbx and Prdm1a inputs regulate fast muscle gene expression.

Cartoons illustrating inputs for the expression of three fast muscle genes (A) myl1, (B) mylpfa, and (C) srl. Evidence for inputs comes from A (Burguière et al., 2011; this work), B (Maves et al., 2007; von Hofsten et al., 2008; Liew et al., 2008; this work), and C (Maves et al., 2007; this work). Inputs reflect genetic requirements, although, in the case of mylpfa, direct regulation by Pbx, Myod and Prdm1a has been demonstrated (Maves et al., 2007; von Hofsten et al., 2008; Liew et al., 2008).