Abstract
To study the molecular mechanisms that control patterning of the heart tube during early cardiogenesis, we have used the ventricular myosin regulatory light chain (MLC-2v), which is expressed in the ventricular segment of the primitive heart tube, as a genetic marker for ventricular specification in rodents. To assess whether the atrial isoform, MLC-2a, could also serve as a chamber-specific marker, we cloned an atrial MLC-2 cDNA (554 base pairs) which displayed homology to the human MLC-2a cDNA at both the nucleotide (87%) and amino acid (95%) levels. Northern blot, reverse transcriptase-linked polymerase chain reaction, RNase protection, and Western blot analysis revealed atrial restricted expression in the adult mouse heart, very low levels in aorta, and no detectable expression in ventricle, skeletal muscle, uterus, or liver. In situ hybridization studies during mouse embryogenesis revealed cardiac specific expression throughout days 8-16 postcoitum, with atrial restricted expression from day 12 and qualitatively greater atrial expression than ventricular from day 9. Thus, preferential pattern of expression in the atria occurs prior to septation. The MLC-2a gene was differentially regulated when compared with MLC-2v expression during embryonic stem cell cardiogenesis in vitro with MLC-2a transcript levels detectable from day 6 in suspension cultures as compared with day 9 for MLC-2v. The region-specific expression of the MLC-2a and MLC-2v genes in their respective chambers during early cardiogenesis provides genetic markers for chamber specification (atrial and ventricular) in both the in vitro and in vivo context.
Highlights
To study the molecular mechanisms that control pat- The formationof distinct atrial and ventricular chambers is terning of the heartube during early cardiogenesis, we a criticalstep during normal hedaretvelopment
A preferential pattern of expression in the atria Utilizing the ventricularisoform of the myosin light chain-2 occurs prior to septation
The remaining 16% of the day 8 postcoitum embryo which included the ventricular segclones were most similar to thesmooth muscle-specific isoform ment of the heart tube (Fig. 4, E and G ) displayed positive of vascular smooth muscle myosin reportedly cloned from hu- signals to both the MLC-2a (Fig. 4 F ) and MLC-2v (Fig. 4 H )
Summary
Val. 269, No 24, Issue of June 17, pp. 16961-16970,1994 Printed in U.S.A. Chamber Specificationof Atrial Myosin Light Chain-2 Expression Precedes Septation during Murine Cardiogenesis”. Studies in an in vitro model of cardiogenesis in embryonic stem (ES) cells document that the expression of the MLC-2v gene canoccur independently of cues that require an intact heart tube, again suggesting that ventricular specification can occur relatively early during mammalian cardiogenesis (Miller-Hance et al, 1993). In situ hybridization studies indicate that atria- RLC-C specific expression is achieved relatively early during cardiac chamber development and clearly priorto septation. During invitro ES cell cardiogenesis, the temporalonset of MLC-2a expression precedes by several days the expression of MLC-Bv, suggesting thaMt LC-2a might representa n excellent marker for the earliest steopfscardiogenesis as well as a marker for atrial chamberspecification, in both in vitroand in vivomurine model systems Based on these results, a model for the sequential stagesof ventricular muscle development is presented
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