Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish

Parts used

Key findings

• Huang et al. fused a 1.6-kb zebrafish cmlc2 fragment containing -870 bp upstream sequence, exon 1, intron 1, and exon 2 with GFP cDNA flanked by AAV inverted terminal repeats. Microinjection into single-cell embryos produced germ-line transmitted zebrafish with GFP exclusively in myocardial cells of the heart chambers and atrioventricular canal, recapitulating endogenous cmlc2 spatial-temporal expression.
• Systematic deletion analysis identified the -210/34 fragment of the zebrafish cmlc2 promoter as sufficient for heart-specific GFP expression. The -210/-73 region served as a basal promoter, while the -210/-174 motif suppressed ectopic non-cardiac expression, defining the minimal cis-regulatory elements for myocardium-specific transcription in zebrafish.
• A single G-to-A point mutation at position -119 of the cmlc2 promoter caused GFP to appear ectopically in all muscle types including heart, skeletal, and smooth muscle. This mutation created a consensus binding sequence for myocyte-specific enhancer factor-2, identifying position -119 as the critical specificity determinant restricting cmlc2 expression to cardiac myocytes.

Abstract

In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (-870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from -210 to 34 (-210/34) was sufficient to drive heart-specific expression, with a -210/-73 motif as a basal promoter and a -210/-174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at -119, evidence that A at -119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2.