1.1 Affymetrix Gene Chip Microarray
In oligonucleotide microarrays (or single-channel microarrays), the probes are designed to match parts of the sequence of known or predicted mRNAs (see in Figure 1.1). There are commercially available designs that cover complete genomes from companies such as GE Healthcare1, Affymetrix2, Ocimum Biosolutions3, or Agilent4. These microarrays give estimations of the absolute value of gene expression and therefore the comparison of two conditions requires the use of two separate microarrays. Oligonucleotide Arrays can be either produced by piezoelectric deposition with full length oligonucleotides or in-situ synthesis.
Oligonucleotide Arrays are composed of 25-mer or 30-mer and are produced by photolithographic synthesis (Affymetrix) on a silica substrate or piezoelectric deposition (GE Healthcare) on an acrylamide matrix. Oligonucleotide microarrays often contain control probes designed to hybridize with RNA spike-ins. The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes.
1 GE Healthcare: http://www.gehealthcare.com/worldwide.html
2 Affymetrix: http://www.affymetrix.com/index.affx
3 Ocimum Biosolutions: http://www.ocimumbio.com/web/default.asp
4 Agilent: http://www.home.agilent.com/agilent/home.jspx?cc=US&lc=eng&cmpid=4533
There are a lot of researches that use the microarray technology to the study of mammalian organogenesis. It can provide great insights into the steps necessary to elicit a functionally competent tissue. Previous researches often focused on maybe one species embryo differentiation [1-3], sex determination of the mammalian gonad [4], gene expression patterns in one organ’s development [5, 6], or analyzing expression profiles during the period from fertilization to implantation [7]. These studies that just mentioned never compare one organ between two species in embryonic development time. Our approach is to synchronize heart development stage between human and mouse and provide an opportunity to identify those functional genes that might be important for controlling embryogenesis and organogenesis.
Figure 1.1
Affymetrix GeneChip Array.1.2 Heart Development
The heart is the first organ to form during embryogenesis and its function is imperative and intricate from early on for the viability of the mammalian embryos. And it is the one of the few organs that has to function almost it is formed [8]. The developmental mechanisms that control the formation and morphogenesis of this organ have received much attention among classical and molecular embryologists. Due to the evolutionary conservation of many of these
processes, major insights have been gained from the studies of vertebrate model. Heart development in all vertebrates follows the same general pattern: fusion of myocardium and endocardium in the ventral midline to form a simple tubular heart, onset of function, looping to the right side, chamber specification and formation, and at last, development of specialized conduction tissue, coronary circulation, innervation, and mature valves [9] (see in Figure
1.2).
Although, many genes important for heart development or organogenesis have been studied for a long time, global analysis of gene expression will provide more information about how the genes work and their interaction networks. In recent years, microarray technology has widely used for researchers to learn how genes’ expression levels in different developmental stages, and to identify the cellular processes in which they participate.
Figure 1.2 Formation of the heart.
1.3 Experimental Objectives
It is not practical to use multiple fetuses at the same gestational age to obtain statistical significances in gene expression level, because of the scarcity of useable fetal specimens at same gestational age. On the other hand, the change in gene expression along various fetal gestational weeks using the expression profiles derived from one fetus at a gestational age may be misleading, considering the existing variations among individual fetus even at the same age. Therefore, mouse has been adopted as a model system for studies of vertebrate
development because of its similar features with human and favorable for genetic studies compared with other vertebrate systems. Using the mouse model will allow us to evaluate the changes in gene expression along various developmental stages, because we can use as many mice as necessary for each time point of a gestational age to eliminate the potential variations, which the result only from individual biological variations.
After mapping the gene expression profiles with the two species, we choose the best 250 match orthologous genes and cluster these genes into groups. As a preliminary analysis, each group of genes has its unique biological meaning after doing time warping. Moreover, specific characteristics were found to be associated with some features of the gene expression patterns. We employed an integrated analytical approach that encompasses Gene Ontology, biological pathway, and some previous research validations to provide more information for identifying the development-specific genes and get more understanding of their function in cardiogenesis. Our works presents a good example in which the combination of microarray technology with human and mouse model will not only consolidate our existing knowledge, but will also help us to identify novel factors that might be important for organogenesis. It also provides us with a global view on how genes are coordinated to form a genetic network to control heart embryogenesis.
The aims of this research are shown as below:
1. Constructing the mapping system between human and mouse 2. Aligning two different time series profiles by using microarray data 3. Identification of heart development-related genes
4. Understanding developmental related genes’ function, pathway, regulation, and how they are coordinated to form a genetic network to control heart embryogenesis 5. Achieve new insights into the heart developmental biology