Print version ISSN 0716-9760
Biol. Res. vol.34 n.1 Santiago 2001
Ventricular Myosin Light Chain-2 Gene Expression in
Developing Heart of Chicken Embryos
Recent gene knock-out studies in mice have suggested that ventricular myosin light chain-2 (vMLC2) has a role in the regulation of cardiogenic development and that perturbation in expression of vMLC2 is linked to the onset of dilated cardiomyopathy. In an attempt to develop an avian model for such studies, we examined the expression pattern of vMLC2 in chicken embryos at various stages and analyzed the effect of antisense oligonucleotide-mediated interference of vMLC2 function in cultures of whole embryos. Our results showed vMLC2 to be a specific marker for ventricular chamber throughout chicken embryonic development and antisense vMLC2 treatment of primitive streak stage (stage 4) embryos to produce pronounced dilation of heart tube with severe deficiency in formation of striated myofibrils. Further studies with antisense mRNA techniques of whole embryo cultures should, therefore, be useful to evaluate the role of vMLC2 and other putative regulatory factors in cardiac myofibrillogenesis.
Key terms: Cardiogenesis, cardiomyopathy, myosin light chains, tissue culture
In avian embryo, heart development begins with the onset of gastrulation at stage 4 when the committed cells of the splanchnic mesoderm migrate through the primitive streak and come to lie on either side of the Hensen's node. The mesodermal cells in the two bilateral heart
The cellular and molecular basis of chamber specific cell lineages in the developing heart is not well understood. It appears that the position of mesodermal precursor cells along the antero-posterior axis, which is established as early as gastrulation, is important for chamber specification; the ventricular myocytes arise from the anterior and the atrial myocytes from the posterior cardiac mesoderm . Cardiac specific gene expression ensues with the fusion of cardiac primordia at early neurula stages in chicken  as well as in mouse embryos . Of the various sarcomeric protein genes, the expression of ventricular myosin light chain 2 (vMLC2) gene is considered to be the earliest marker of both mouse  and chicken  ventricular muscle lineages, with its transcripts becoming detectable at 8dpc in mouse  and stage 5 in chicken  development.
The precise physiological role of MLC2 in vertebrate striated muscle is not clear. Earlier studies by Margossian et al.  indicated that vMLC2 in hearts of patients with dilated cardiomyopathy is selectively degraded and is accompanied by a lower actin-activated ATPase activity and an impaired ability to assemble the thick filaments. A potential role of vMLC2 in cardiac hypertrophy was also underscored by a selective increase of this light chain in the hypertrophic state in both rat and humans [10,11,16] and by the recent findings  that at least one signaling pathway for the assembly of new sarcomeres during cardiac hypertrophy is through phosphorylation of vMLC2. In a vMLC2 gene knock-out study , the atrial form of MLC2 (aMLC2) was upregulated in the ventricles of vMLC2 -/- embryos and replaced the vMLC2 in the thick filaments. Despite the substitution, ultrastructural analysis revealed defects in sarcomeric assembly and an embryonic form of dilated cardiomyopathy developed. It was concluded vMLC2 had a unique requirement for functional maturation of the ventricular chamber. Furthermore, a study of mice harboring null mutation in the homeobox gene Nkx2.5 revealed a selective downregulation of vMLC2 expression, suggesting that vMLC2 gene is somehow associated with the developmental regulation of cardiogenic specification . The precise manner in which vMLC2 is involved during early cardiogenesis and in cardiac atrophy/hypertrophy remains unknown. Our study was undertaken to examine this problem in chicken embryos which offer significant advantages for an experimental approach.
MATERIALS AND METHODS
Embryo preparation and culture
Fresh fertile white leghorn eggs (Spafas, CT) were incubated at 37ºC long enough to obtain stage 4 embryos . Blastoderms were freed from the yolk and explanted along with vitelline membrane as described by New . Each embryo was examined for the developmental characteristics, and only definitive primitive streak stage embryos were selected. For RNA isolation embryos of defined stages (st 1 through 10) were collected under sterile conditions (see below). Stage 4 cultured embryos were treated with 2.5 nmoles of antisense vMLC2 oligonucleotide (catctgatcgatctcctc) (n=20) with an equal number of sense oligonucleotide (gaggagatcgatcagatg) treated controls. Oligonucleotides were dissolved in chick saline and added directly over the embryos. Embryos treated with oligonucleotides (ODN) were observed after 24 hr for gross morphology and photographed without staining. Selected embryos were processed for electron microscopy.
Whole mount in situ hybridization
Whole mount in situ hybridization was performed according to Harland . Digoxigenin-labeled sense and antisense riboprobes were prepared from linearized plasmid containing 186 bp vMLC2 specific PCR amplified DNA  with T3 and T7 RNA polymerase and used in hybridization.
Isolation of mRNA and RT-PCR
Poly (A+) RNA was isolated from embryos using the Micro Fast Track mRNA isolation kit (Invitrogen) and the manufacturer's protocol. The first strand cDNA was synthesized from poly (A+) RNA by oligo (dT) primer and Moloney murine leukemia virus reverse transcriptase (RT) with the first strand synthesis kit from Stratagene. Specific DNA primers for cytoplasmic beta actin, ventricular myosin light chain-2 (vMLC2), and cardiac alpha-actin were used as described earlier . Thirty cycles of polymerase chain reactions were performed with ampliTaq DNA polymerase (Perkin Elmer). The authenticity of PCR-amplified DNA was verified by electrophoresis on 1% agarose gels with appropriate DNA size markers and by sequence determination when necessary using an automated DNA sequencer (Applied Biosystems).
Ultrastructural analysis by electron microscopy
Embryos were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.4 for one hour, washed twice in 0.1M phosphate buffer for 1-2 hours, and placed in cold 1% osmium tetraoxide in 0.1M phosphate buffer for 1 hour. After washing, the embryos were dehydrated in graded series of alcohol and embedded in epon. Sections 30-40 nm thick prepared from the tissues were stained with lead citrate and examined using a Jeol 100C microscope.
Expression of vMLC2 in early Chicken Embryonic Development
In order to study the role of vMLC2 in cardiac development, it was necessary to first check the precise distribution of its transcripts in chicken embryos. We therefore applied the whole mount in situ hybridization technique at different stages of embryonic development and found that vMLC2 transcripts were confined in the anterior part of the cardiac primordia (Fig. 1, A, B) where prospective ventricle cells are located at stage 8/9. The vMLC2 positive signal remained restricted to the ventricle of the heart at stages 11 and 13 of development (Fig. 1C, D). Others have reported a similar expression pattern in murine heart development, where the 8 dpc (equivalent to stage 9 of chicken development) prospective ventricular cells contain vMLC2, while no detectable signal was present in prospective atrial region [2,15]. We could not detect vMLC2 positive signal by whole mount in situ hybridization in embryos prior to stage 9, but as we have reported previously  the more sensitive RT-PCR technique consistently showed vMLC2 as well as cardiac a-actin transcripts from Stage 5 onward (Fig. 2).
Figure 1. Whole-mount in situ expression of vMLC2. Chicken embryos were hybridized with vMLC2 specific antisense riboprobe. The first developmental stage showing a positive signal is stage 9 (A). At stage 10 (B) fused cardiac primordia forming the heart tube exclusively express vMLC2. At stages 11-13 (C and D) the primitive ventricular part of the heart tube expresses vMLC2 gene. Thus vMLC2 expression in the early stages is in the ventricle only.
Figure 2. RT-PCR analysis of cardiac a -Actin (A), vMLC2 (B) and ß-actin (C) transcripts during stages 5-10 of chick embryos. M indicates 123 bp DNA size markers. Cardiac a-actin (225 bp), vMLC (186 bp) and ß - actin (434 bp).
Cardiac anomalies develop after antisense vMLC2 oligonucleotide treatment
We next considered that perturbation of vMLC2 at early stages of development prior to assembly of contractile proteins into myofibrils should be useful. Whole stage 4 embryos were therefore cultured and antisense vMLC2 ODN were directly added over the embryos as described in Materials and Methods. Seventeen out of 20 embryos (85%) developed various degrees of cardiac abnormalities. All the embryonic hearts beat at a much lower rate than those treated with sense vMLC2 ODN (controls). The heart did not show normal looping, but rather appeared as a centrally-located tube that was much larger than in controls (Fig. 3). Representative chicken embryos were subjected to ultrastructural analysis. We found that the sense ODN treated embryo showed normal heart development with typical myofibrillar assembly (Fig. 4, top panel), while in the antisense ODN treated embryonic heart the sarcomere pattern was poorly developed and Z bands often appeared as dense irregular material (Fig. 4, bottom panel). Similar defects in myofibrillogenesis have been reported in vMLC2 knock-out mice .
Figure 3. Whole-mount photographs of embryos growing in New cultures treated with sense (left panel) and antisense vMLC2 (right panel) oligonucleotides. Antisense oligo treated embryo shows short and dilated ventricle as compared to control.
Figure 4. Electron micrograph of cardiocytes form sense vMLC2 (CNT) and anti-sense vMLC2 (AS) ODN-treated embryos. The myofibrils (MF) are well-developed with regular striations in CNT but poorly formed in AS. White arrow heads indicate irregular Z band densities in AS.
It is well known that MLC2 plays a central role in smooth muscle contraction and myosin structure. MLC2 phosphorylation on a specific serine (Ser 19) serves as the switch for turning on the actin-activated myosin ATPase  and its phosphorylation also causes striking structural changes; the folded monomeric form of smooth muscle myosin extends out and assembles into filaments . In vertebrate striated muscle, however, acto-myosin ATP-ase activity is regulated through the troponin/tropomyosin system associated with thin filaments, and MLC2 is considered to have only a minor modulatory effect . The possible role of MLC2 in the in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by Margossian et al. , who showed that on removal of MLC2s the myosins exhibited reduced ability to form regular thick filaments. Subsequently Margossian et al.  found selective degradation of vMLC2 in the hearts of patients with dilated cardiomyopathy and observed that myosin extracted from myopathic hearts formed shorter, stunted filaments when compared to control myosin.
A very marked effect of MLC2 on muscle development was reported in Drosophila, where mutation of the MLC2 gene resulted in a flightless phenotype . Recently Chen et al.  found a selective requirement for vMLC2 in embryonic heart function as the vMLC2 knock-out mice developed an embryonic form of dilated cardiomyopathy. Several other knock-out studies in mice indicate that vMLC2 is a unique cardiac gene since its expression is only affected by homeobox gene Nkx2.5 , but not any other genes significant in cardiac development.
The chick embryo is particularly useful for experimental studies of early events in cardiogenesis. Using the antisense technique we present evidence in this report which strongly supports the concept of special requirement for vMLC2 in cardiac development as proposed from the mouse knock-out studies .
Corresponding Author: M.A.Q. Siddiqui, Chairman Dept. of Anatomy & Cell Biology State University of New York Health Science Center at Brooklyn 450 Clarkson Avenue, Brooklyn, NY 11203. Phone: 18-270-1014, Fax: 718-270-3732, E-Mail: MSiddiqui@netmail.hscbklyn.edu
Received: December 19, 2000. Accepted: January 3, 2001.
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