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CAVEAT LECTOR
DIFFERENTIAL DISPLAY OF mRNAs FROM THE ATRIOVENTRICULAR REGION OF DEVELOPING CHICKEN HEARTS AT STAGES 15 AND 21
Da-Zhi Wang, Xiaoyun Hu, Jenny L-C. Lin, Gregory T. Kitten, Michael Solursh, and Jim J-C. Lin.
Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242-1324
Received 11/15/95; Accepted 11/28/95; On-line 1/1/96
In this study, the differential mRNA display method was employed to identify and to clone differentially expressed genes, which may be important for the formation of AV valves and septa. A critical period in the valve and septum formation has been previously identified to be between stage 15 and stage 21 of chicken development (15). During this period, an epithelial-mesenchymal cell transformation appears to occur through the induction of certain endocardial cells in the AV canal region of developing heart to become mesenchymal cells by local signals produced from the AV myocardium (13-15). Therefore, we performed our differential display on the RNAs prepared from AV tissues of these two stages to specifically identity candidate genes involved in the valve and septum formation. As a potentially rapid and efficient method of screening differentially expressed genes, differential display has several technical advantages (11) as well as several problems (19). The amount of total RNAs used in the differential display is 2-3µg and this amount of RNA, in combination with 10 different sets of primers, is enough to display at least 5% of the total mRNA represented. Moreover, there is no need to isolate poly(A)+ RNA in this approach. However, one of the obstacles is the rather high number of 'false positive' cDNA products from the primary screening that cannot be confirmed by Northern blot analysis. In this regard, in order to limit the positive clones to those that are cardiac-specific and are differentially expressed genes during this developmental period, we differentially displayed RNAs isolated from hearts and from whole embryos without hearts at both stages of development. Although the majority of clones obtained (Table 1) were not cardiac-specific, three examples given in this study did show differential expression in the AV canal regions of stage 15 and stage 21 hearts.
| Clone Name | Size(bp) | Sequence Homology | Whole-Mount In Situ Pattern |
| 15Aa | 491 | eIF-4AIIb | ubiquitousf |
| 15H1 | 240 | none | heart, tail |
| 15H16a | 320 | phospholambanc | heart |
| 15H2 | 250 | none | heart, brain |
| 15H3 | 280 | none | not tested |
| 21Ca | 251 | none | heart |
| 21Ga | 365 | none | undetectable |
| 21H1 | 280 | none | undetectable |
| 21H2 | 400 | none | ubiquitousf |
| E10 | 380 | none | heart, neural tube, limb |
| E13 | 268 | alpha-tropomyosind | heart, somite |
| H1 | 200 | none | ubiquitousf |
| H2 | 200 | alpha-actine | not tested |
| H3 | 400 | none | ubiquitousf |
a. These clones were generated by subcloning respective cDNA fragments into the pBK vector, whereas the rest of clones were done in the pCRII vector.
b. Mouse protein synthesis initiation factor eIF-4AII (38 ).
c. Chicken cardiac phospholamban (24).
d. Chicken skeletal alpha-tropomyosin (32).
e. Chicken cardiac alpha-actin (39).
f. Their messages were expressed in more than 5 different organs/tissues including heart. However, among these clones, there were different expression patterns observed in the whole-mount in situ hybridization.
Furthermore, whole-mount in situ hybridization was also employed in this study as a secondary screening method. This approach allowed us to quickly confirm the spatial and temporal expression patterns of isolated genes. In addition, the success rate (83%) of the whole-mount in situ hybridization in this study was much higher than that for Northern blot analysis (29%). Moreover, the Northern blots required a large amount of total RNAs, which, in general, was difficult to obtain from the early developing embryos. In addition, the information regarding the spatial pattern of expression could not be obtained by the Northern blot analysis. Moreover, contradictory results were observed when data from the Northern blot analysis was compared with the differential display results. One such example was seen with the clone 15H16 (phospholamban). The phospholamban messages were significantly higher in the AV canal region of stage 15 heart than that of stage 21 heart (Fig 3). However, the Northern blot analysis of RNAs from stage 15 and stage 21 hearts showed an opposite result (Fig 4). Therefore, the whole-mount in situ hybridization is more useful for validation of the differential display data. In fact, in a recent report, whole-mount in situ hybridization was used to screen randomly sequenced cDNA clones to identify expression patterns of novel genes (33). The redundancy and under-representation of certain mRNA species in the differential display may represent another problem. However, it has been suggested that this problem may be overcome by using one-base anchored oligo-dT primers instead of two-base anchored oligo-dT primers (34).
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.