Figure 1. Positive regulators of CMP formation. (A) Schematic of screening strategy to identify new regulators of cardiogenic mesoderm differentiation. (B,C) Immunostaining of Kdr-eGFP (cardiogenic mesoderm) and Alexa fuor568-Foxa2 (endoderm) showing increased mesoderm differentiation in response to siAcvr1b as compared with siControl at day 6 of differentiation. Bar, 50 μm. (D) Microarray data reveal that 33 transcripts are up-regulated (P < 0.05) at day 4 in response to siAcvr1b as compared with siControl 24 h after transfection. (E)Quantitative RT–PCR (qRT–PCR) confirmation of the microarray results in D, showing that 14 genes are robustly up-regulated in response to siAcvr1b as compared with siControl. (F) siRNAscreen of the 14 candidates fromE to evaluate their requirement for cardiogenic mesoderm formation induced by siAcvr1b. Differentiation was quantified by induction of Kdr-eGFP reporter (total integrated intensity) (see the Materials and Methods for details). siGrrp1, siEvx1, and siId1 strongly repressed siAcvr1b-induced cardiogenic mesoderm. (G–J) Representative images of Kdr-eGFP and Alexa fluor 568-Foxa2 illustrating results presented in F. Bar, 50 μm. (K) qRT–PCR results showing that siGrrp1, siEvx1, and siId1 markedly repress cardiogenic mesoderm-specific marker (Kdr, Mesp1, Snai1, and Cdh11) expression. (L–O)Temporal expression profiles of Id1, Grrp1, Evx1, and Mesp1 in response to siAcvr1b or siControl from day 3 to day 6 of differentiation. (P–W′) Endogenous expression of Id1, Grrp1, Evx1, and Mesp1 in embryonic day 6.5 (E6.5) and E7.25 mouse embryos by in situ hybridization. (P– W) Whole-mount view. Transverse histological section of the proximal region of E7 embryos indicating Id1 (T′) expression in the gastrulating epiblast (yellowarrow) and migrating mesoderm(white arrow), Grrp1 (U′) expression in the gastrulating epiblast (yellow arrow), and Evx1 (V′) and Mesp1 (W′) expression in the primitive streak (yellowarrow). (X) Schematic representation of an E7.25 embryo transverse section illustrating the different domains of expression of the three candidates. The gastrulating epiblast (blue) indicates the domain where Id1 and Grrp1 expression overlaps. In the primitive streak region (gray), high levels of Evx1 expression are observed with decreased Grrp1 expression. As cells exit the primitive streak and migrate laterally (purple), they start to express Mesp1 along with Evx1. As mesodermcells migrate more anteriorly (orange), they resume Id1 expression. All qRT–PCR data were normalized to β-actin mRNA levels. Quantitative data are presented as means ± SD. (∗) P < 0.05. All experiments were performed at least in biological quadruplicates.
Image published in: Cunningham TJ et al. (2017)
Copyright © 2017. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article.
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