The molecular mechanisms for cardiac morphogenesis are not completely understood. In chicken development, it has been shown that presumptive cardiac forming cells migrate anteriorly and medially, and then the right and left regions of these cells (lateral plate mesoderm) fuse at the midline to generate the primitive heart tube at stage 10 which starts to beat almost immediately (1). The cell lineage studies of the development of zebrafish embryos have led to the identification of cardiac forming regions in the early blastula (2). However, little is known about specific genes involved in the early cardiac morphogenesis except for several structural genes, such as ventricular myosin heavy chain (3 ), atrial myosin heavy chain (4), cardiac troponin I (5) and vascular smooth muscle alpha-actin ( 6), as well as few regulatory genes, such as MEF2 (7) and Drosophila homeobox gene tinman (8) and its mouse homologue Csx/Nkx-2.5 (9,10). In an effort to identify genes that are differentially expressed in embryonic hearts at the time of the formation of endocardial cushions, the progenitors of the valves and membranous septa, we have employed the differential mRNA display method (11) in this study. Since the differential display is highly sensitive, and likely to detect a large number differentially expressed genes, we have focused on comparing differences of mRNA expression between stage 15 and stage 21 of the atrioventricular (AV) canal regions of developing chicken hearts. It has been demonstrated that the epithelial-mesenchymal cell transformation occurring between stage 15 and 21 may be a key process during the AV cushion morphogenesis (12). Furthermore, this cell transformation appears to be induced by local stimuli derived from the adjacent myocardium (13-15). Thus, genes which are differentially expressed in the AV canal regions of stages 15 and 21 embryonic hearts may be important for the formation of the cardiac valves and septum. The cloned gene fragments we isolated using differential display were sequenced and compared against sequences in the GenBank database. In addition, cloned cDNA fragments were used as probes in whole-mount in situ hybridization. In total 14 differentially expressed fragments were cloned. Here, we report three examples of such clones: 15H16 for cardiac phospholamban, E13 for skeleton alpha-tropomyosin and 21C for a novel gene with a message size of 9.5 Kb.

Tissue Collection and Total RNA Isolation
Fertilized White Leghorn chicken eggs (Hy-Vac, Adel, Iowa) were incubated at 38°C in a humidified incubator with automatic rotation. All embryos were staged according to Hamburger and Hamilton (16). Hearts of stage 15 and stage 21 embryos were quickly dissected and tissues from atrioventricular (AV) canal region were collected and frozen in liquid nitrogen, then stored in -70°C freezer, or immediately used for isolating total RNA. Total cellular RNAs were isolated either by using Tri Reagent RNA isolation kit ( Molecular Research Center, Inc., Cincinnati, OH ) according to manufacturer's manual, or by using guanidinium thiocyanate method (17,18). Total RNAs were also prepared from both stage embryos without hearts and used for the comparison of differentially expressed genes.

Differential mRNA Display
Before use, total RNA samples were treated with RNase-free DNase I ( Promega, Madison, WI ) to remove potential chromosomal DNA contamination. About 2µg each of total RNAs from stage 15 and stage 21 AV canal tissues or embryos without hearts were used for reverse transcription. Reverse transcription and subsequent PCRs were performed as previously described (11,19) with some modifications. The PCR cycling parameters were as follows: 94°C for 45 sec, 42°C for 1 min and 30 sec, 72°C for 45 sec for 30 cycles followed by 72°C for 5 min. The PCR products together with size markers of known sequence were then separated on a 6% denaturing polyacrylamide gel. Differentially expressed gene fragments detected in either stage 15 or stage 21 AV canal tissues were identified and further compared to the displayed patterns obtained from stage 15 and stage 21 embryo tissues without hearts. Only those fragments with clear differential expression and not detected in embryos lacking hearts were selected for further cloning and characterization. The sequencing gel was run to resolve cDNA fragments sized from 200bp to 500bp. Bands of interest were cut out from the gel and cDNA fragments eluted by boiling the gel pieces in 100µl H₂O for 15 min were used as templates for PCR re-amplification using the same condition as described above. A portion of the re-amplified PCR product was analyzed on a 2% agarose gel to check the efficiency of re-amplification and to confirm the size of cDNA fragments. All primers used for reverse transcription and PCR were synthesized by Oligo Etc. Inc. (Guilford, CT). Primers used included: AP1 (T₁₁CA), AP2 (T₁₁GC), AP3 (T₁₁AG), AP6 (T₁₁AA), AP8 (T₁₁AT), AR1 (CTGATCCATG), AR2 (CTTGATTGCC), AR3 (CTGCTCTCAA). AmpliTaq DNA polymerase was purchased from Perkin-Elmer Corp. (Norwalk, CT.). alpha-[³⁵S]dATP (1200Ci/mmole) was obtained from New England Nuclear (Boston, MA.).

cDNA Cloning and Sequencing
Re-amplified cDNA fragments were cloned into either pBluescript II SK (pBK) vector (Stratagene, San Diego, CA) or the pCRII vector using the TA cloning system from Invitrogen (San Diego, CA). For cloning into pBluescript II SK vector, the vector DNAs were linearized at the EcoRV site and a T residue was tailed to the blunt ends according to Marchuk et al.(20) to facilitate the direct cloning of PCR fragments. DNA sequencing was performed using Sequenase Kit Version 2.0 (United States Biochemical, Cleveland, OH). The nucleotide sequences obtained were compared with GenBank databases using the GCG (Genetics Computer Group) FASTA program software (Madison, WI).

Northern Blot Analysis
For Northern blot analysis, total RNAs were isolated from stage 15 and stage 21 chicken hearts. 10µg of total RNA was loaded on a 0.8% formaldehyde/agarose gel. After electrophoresis, the RNA was eletrophoretically transferred to Zetaprobe nylon membrane (Bio-Rad, Richmond, CA). DNA probes were labeled either by nick translation (Promega, Madison, WI) for longer cDNA fragments or by radioactive PCR-labeling protocol (21) for shorter fragments. Hybridization and washes were performed as described previously (21).

PhosphorImager Analysis After hybridization and washing, the Northern blot membranes were exposed to a PhosphorImager plate (Molecular Dynamics, Sunnyvale, CA.). The radioactivity on the plate was scanned and analyzed by a PhosphorImager 450 (Molecular Dynamics, Sunnyvale, CA.).

Whole-Mount In Situ Hybridization
For the in situ hybridization, plasmids containing cDNA fragments in either pBK or pCRII vector were used as templates for the preparation of digoxigenin(DIG)-labeled riboprobes. DIG-labeling kit was purchased from Boehringer Mannheim (Indianapolis, IN) and riboprobes were synthesized according to the manufacturer's protocols. Both sense and antisense DIG-labeled RNA probes were synthesized by the in vitro transcription of linearized DNA templates, in the presence of either SP6, T3, or T7 RNA polymerase using DIG-labeled uridine-triphosphate (DIG-UTP) as one of nucleotide substrates.

Whole-mount in situ hybridization was performed generally according to the methods of Hemmati-Brivanlou et al.(22) and Riddle et al. (23) with some modifications. Briefly, staged chicken embryos were dissected in PBS buffer and fixed in M buffer (100mM MOPS pH 7.4, 2mM EGTA, 1mM MgSO₄, and 3.7% formaldehyde) at 4°C overnight. After bleaching in M buffer with 6% hydrogen peroxide for 48 hr at room temperature, embryos were dehydrated through an ascending methanol series in PBS ( 25, 50, 75 and 100% methanol) and stored in 100% methanol at -20°C until needed. Embryos were rehydrated through a descending methanol series in PBT (PBS plus 1% Tween 20) and PBT, permeabilized in PBT containing proteinase K (1µg/ml in PBT) for 30 minutes at room temperature. After post-fixing in 4% paraformaldehyde in PBT, embryos were washed three times in PBT, rinsed once each in 0.1M triethanolamine (pH 8) and in 0.1M triethanolamine plus acetic anhydride (2.5µl/ml). After incubation with prehybridization solution (0.3M NaCl, 20mM Tris pH 8.0, 1mM EDTA, 0.1M DTT, 1X Denharts, 10% dextran sulfate, 50% formamide, 250g/ml yeast tRNA, and 100µg/ml salmon sperm DNA) for 2 hr at 50°C or 60°C, embryos were hybridized with DIG-labeled riboprobes in hybridization solution ( prehybridization solution plus 0.5-1µg/ml riboprobe ) at 50°C or 60°C overnight. After hybridization, embryos were washed two times (30 min each) with prehybridization solution at the hybridization temperature, followed by washing two times (30 min each) with 50% formamide, 2X SSC at the same temperature. Embryos were then treated with RNase A (1µg/ml) and RNase T1(1 unit/ml) for 30 min at 37°C. After a final wash with 0.2X SSC, 1% Tween 20 at 50°C or 60°C, embryos were incubated with PBT plus 10% heat inactivated sheep serum (Sigma, St.Louis, MO) for 2 hr at room temperature and then with alkaline phosphatase conjugated Fab anti-DIG solution (1 to 2000 dilution in PBT plus 10% sheep serum) overnight at 4°C. Embryos were washed at least 8 times with PBT (1 hour each) and left in PBT overnight at 4°C to completely wash out excess amounts of alkaline phosphatase conjugated Fab. Color reaction was performed by washing embryos three times for 10 min each with buffer A containing 100 mM Tris pH 9.5, 50mM MgCl₂, 100mM NaCl, 1% Tween 20, and 2mM levamisol, followed by incubating with detection solution (buffer A plus 4.5µl/ml nitroblue tetrazolium and 3.5µl/ml 5-bromo-4-chloro-3-indolyl-phosphate toluidinium ) for various times at room temperature. When the color reaction was complete, embryos were washed twice with buffer A, once with PBT, and then post-fixed in 4% paraformaldehyde in PBT. After several washes with PBT, embryos were observed and photographed with a Wild M420(Swiss) stereo microscope using either bright-field or dark-field optics with either Kodak Ektar 64 or Royal Golden 100 film. Selected embryos were embedded in Paraffin Plus (Oxford, St. Louis, MO) and sectioned at 10-15µm. After deparaffinizing, rehydrating and mounting on Super frost/Plus slides (Fisher), sections were photographed with bright-field or phase-contrast optics using a Leitz Laborlux 12 microscope ( Leitz Wetzlar, Germany ) and Kodak Royal Golden 100 or 25 films.

Differential display of mRNAs from AV canal regions of stage 15 and stage 21 hearts
Different combinations of five (T₁₁)MN anchoring primers and three arbitrary 10-mers were used in the differential display. Of all 15 possible primer combinations, four (AP1/AR1, AP1/AR3, AP2/AR1, AP2/AR3 ) displayed reproducible and differential patterns of expression (Fig 1). The differentially expressed cDNA fragments were readily identified from either stage 15 sample ( bands a-e of Fig 1) or stage 21 sample ( bands 1-6 of Fig 1). In addition, there existed many cDNA fragments showing slightly differential expression.

To identity cardiac-specific differential expression between stage 15 and stage 21, we also displayed cDNAs prepared from RNA samples isolated from the same staged embryo tissues without hearts. Examples of such display and comparison are shown in Fig 2.

One of cDNA fragments indicated by arrow in Fig 2A could be thought as a cardiac-specific and differentially expressed gene if just compared to the differential expression patterns from heart tissue. By the addition of differential expression patterns generated from the same stage embryos without hearts( Fig 2B and 2C), such false positive clone could then be ruled out, because of the presence of an equally expressed band ( indicated by arrows in 15E and 21E of Fig 2B and 2C). Conversely, cardiac-specific but not differentially expressed genes could be readily identified by comparisons of displaying patterns from heart and embryo without heart (indicated by arrowheads in Fig 2).

From the displaying sequencing gels, we cut out over 20 different bands, ranging in size from 200bp to 500bp. These bands were subcloned and their sequences were determined. Table 1 summarizes 14 cDNA clones. From the sequence comparison to the GenBank database, 4 clones represent known genes, including protein synthesis initiation factor eIF-4AII ( clone 15A ), phospholamban (clone 15H16), skeletal alpha-tropomyosin (E13) and alpha-actin (clone H2). These cloned cDNA fragments were further used as probes in the whole-mount in situ hybridization screening. Of 12 clones tested, 10 probes show specific hybridization to the heart and only 2 probes did not detect significant signal. This is in contrast to the evaluation of cloned cDNA fragments by Northern blot analysis. The rate of success in identifying message sizes by Northern blot analysis with these cDNA probes was very low (29%). We chose 3 clones for further evaluation of their expression patterns in the AV canal regions of stage 15 and stage 21 hearts.

Phospholamban is differentially expressed in AV canal regions of developing hearts between stage 15 and stage 21
From the DNA sequence, we found that the clone 15H16 contained 320bp upstream from the poly(A) tail of the chicken phospholamban transcript (24). Phospholamban is a protein regulating the sarcoplasmic reticulum Ca⁺⁺ pump activity and thus, plays an important role in the contraction and relaxation of cardiac and slow skeletal muscles (24-27). Although the expression pattern of phospholamban in developing hearts has been recently reported in paraffin-sections by in situ hybridization (26,27), its expression in developing AV canal region remained unclear. The cloned cDNA fragment 15H16 was obtained from RNAs isolated from stage 15 AV canal region by differential display method, suggesting a decreasing expression of phospholamban in the AV region from the stag 15 heart to the stage 21 heart. However, the whole-mount in situ hybridization showed that phospholamban mRNA signals were stronger in the stage 21 heart (Fig 3B) than in the stage 15 heart (Fig 3A).

The overall increase in phospholamban messages between these two developing stage hearts was consistent with the previous reports by Toyofuku et al. (26) and Toyofuku and Zak (24). When spatial expression patterns of phospholamban were examined in cross-sections from the same embryos after whole-mount in situ hybridization, we detected a drastic decrease in the phospholamban expression at the AV canal myocardium and the outflow tract myocardium from stage 15 heart (Fig 3C) to stage 21 heart (Fig 3D). Northern blot analysis of total RNAs isolated from stage 15 and stage 21 hearts revealed that two bands with sizes of 1.1Kb and 0.6Kb hybridized to the 15H16 probe (Fig 4A). The steady-state levels of these two messages increased from stage 15 to stage 21. Therefore, the overall expression of phospholamban in the heart increased from stage 15 to stage 21, although its expression at the AV canal region appeared to be decreased.

Skeletal alpha-tropomyosin expression is temporally and spatially decreased in the developing hearts
The clone E13 was obtained from stage 15 RNAs using the differential display method. E13 contained 268bp upstream from the poly(A) tail of chicken skeletal alpha-tropomyosin transcript (32). In avians, cardiac alpha-tropomyosin has been shown to differ from the skeletal alpha-tropomyosin in respect to its mobility in 2D gels (28,29) and at the nucleotide sequence (30-32). As can be seen in Fig 5, skeletal alpha-tropomyosin messages were detected in developing somites (open arrows) and hearts (arrowheads) at both stage 15 and stage 21 embryos. The hybridization signal was strong in the stage 15 heart and became weaker in the stage 21 heart. On the other hand, the signals became much stronger in the stage 21 somites ( Fig 5A and 5B ).

Examination of cross sections of the heart of the same embryos revealed that E13 was evenly expressed in the myocardium of stage 15 heart (Fig 5C). As the development proceeded, the expression of E13 in the myocardium was gradually diminished and at the stage 21 embryo, only the atrial myocardium expressed the E13 gene (Fig 5D). By Northern blot analysis, the E13 probe detected a 1.3Kb message in the total RNAs derived from both staged hearts (Fig 4B). Consistently, the steady-state level of E13 message was decreased from stage 15 heart to stage 21 heart. However, no change in the level of message of glyceraldehyed-3-phosphate dehydrogenase (GAPDH) was detected by Northern blot analysis of RNA derived from stage 15 and stage 21 hearts (Fig 4C). Therefore, skeletal alpha-tropomyosin recognized by the E13 probe was decreased in the developing heart temporally and spatially.

Expression of a novel 21C gene is increased at AV canal region of developing hearts
From the differential display and whole-mount in situ hybridization screening, one (21C) of the several novel genes isolated showed a cardiac-specific expression pattern during early chicken embryonic development. Whole-mount in situ hybridization using the 21C riboprobe revealed hybridization signals in the hearts of both stage 15 and stage 21 embryos(Fig 6A and 6B).

Sectioning through these embryos after whole-mount in situ hybridization revealed that the 21C gene expression was confined to myocardium (Fig 6C and 6D). At stage 15, a strong signal was found in myocardia of atrium and outflow tract ( Fig 6C ). As the development advanced to stage 21, a strong hybridization signal was detected by the 21C riboprobe in the ventricular myocardium (Fig 6B and section data not shown). The AV canal myocardium from stage 21 heart showed an increase in the expression of this 21C gene as compared to that from stage 15 heart. This is consistent with the isolation of the 21C clone from the stage 21 RNA sample using the differential display method.

To further examine the expression pattern of the 21C gene in early developing heart, we performed whole-mount in situ hybridization on stage 8⁺ and stage 13 embryos. At stage 8⁺, the 21C gene was exclusively expressed in the initially paired primordia located on either side of the embryonic midline (Fig 7A).Careful sectioning through the embryo demonstrated that 21C expression was restricted to the cardiac mesoderm and not found in any other tissues (Fig 7B). This cardiac-specific expression pattern was still detected in stage 13 embryo (Fig 7C and 7D) in which the heart tube had formed and looped to the right side of the embryo.

Northern blot analysis of total RNAs isolated from stage 15 and stage 25 hearts suggested that the 21C gene encodes a single 9.5Kb message (Fig 8B). It is also clear that the steady-state level of the 21C expression in stage 25 heart is much higher than that in the stage 15 heart (Fig 8B). This result together with the in situ hybridization data suggest an increased expression in the heart as the development of embryos proceeded. However, our in situ data do not allow us to distinguish whether more cells at the AV regions of stage 21 heart express the 21C gene or each cell at this region expresses more 21C gene or both. In a preliminary experiment, we found that the 21C expression diminished in the day 12 embryonic heart (data not shown).

For all cloned cDNA fragments used for making anti-sense riboprobes, sense probes were also made and used as negative controls. One example is shown in Fig 9, where the 21C sense riboprobe was used. This probe did not hybridize to either stage 15 (Fig 9A) or stage 21 (Fig 9B) embryos. Same results were obtained from 15H16 and E13 sense probes (data not shown).

The decrease in the phospholamban at the AV canal region of developing hearts between stage 15 and stage 21 may correlate to the drastic change from heart tubes to heart chambers. The heart tube at the stage 15 performs a peristaltic contraction throughout the whole tube. An evenly distributed phospholamban in the myocardium of tubular heart may be needed to control the sarcoplasmic reticulum Ca⁺⁺ pump activity during myocardial contraction. When the valves and septa are partially formed at the stage 21, a rapid and regulated contraction is mainly carried out by the myocardia of atria and ventricles. Therefore, an increase in phospholamban expression is needed in these areas of the heart. In contrast, the AV myocardium may not require this type of contraction and therefore, a decrease in expression of phospholamban occurrs. What mechanism/signal is responsible for the down-regulation of phospholamban gene in the AV myocardium between stage 15 and stage 21 remains to be determined. It is of interest to note that a down-regulation of N-CAM gene in the AV endocardium also occurs during this period of development and is believed to be required for the epithelial-mesenchymal cell transformation (14). One plausible mechanism is that the down-regulation of both phospholamban and N-CAM genes in the same AV canal region at this period of development may be a developmental response to a similar signal. In this regard, it should be of interest to examine whether a common regulatory element might exist in the promoters/enhancers of both genes.

The decrease in the expression of skeletal alpha-tropomyosin in hearts from stage 15 to stage 21 did not appear to be restricted to the AV myocardium. Thus, the mechanism/signal responsible for this decrease in skeletal alpha-tropomyosin may be different from that inducing the down-regulation of phospholamban in the AV myocardium during the same period of development. It has been previously shown that the decrease in skeletal alpha-tropomyosin synthesis in the developing heart was accompanied with an increase in cardiac-specific alpha-tropomyosin (29). This type of developmentally regulated isoform switching during heart development was also seen for many contractile and regulatory proteins (35-37), although the molecular mechanism(s) for isoform switches is not completely understood.

In this study, a novel, cardiac-specific and differentially expressed gene, 21C, was identified and cloned. In a preliminary screening of cDNA libraries with the 21C fragment probe, we have obtained several overlapping clones. The composite insert sequenced contains about 3Kb of sequence upstream from the poly (A) tail of the 21C transcript. Using larger cDNA insert as a probe for the whole-mount in situ hybridization, an identical temporal and spatial expression pattern as that shown in this study was obtained (data not shown). The expression of the 21C gene appears to start at very early stage and possibly diminishes during the later stages of embryonic heart development. This suggests that the 21C gene may play an important role in cardiac morphogenesis.

We thank Rebecca Reiter and Sandra Kolker for helping collect embryonic heart tissues, Dr. Yi-Ping Chen for teaching us the whole-mount in situ hybridization technique and for his stimulating discussion. This work was supported by National Institutes of Health Grant HL42266 (SCOR in Congenital Heart Diseases).

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