1. Expression Profiling Using DNA MicroArrays - Each cell type within an organism expresses a unique combination of genes – this is, in part, what makes cells different from each other. For example, photoreceptor neurons (but not epidermal skin cells) will express opsin genes. Similarly, neuronal cells (but not muscle cells) will express voltage-gated sodium and potassium channel genes. One way to determine the character of a cell is to determine which genes are expressed within it and at what levels. This type of analysis can be accomplished by using technology called DNA microarrays. - In a DNA microarray tens of thousands of small DNA fragments are spotted onto a glass slide. Usually a fragment of each gene or a cDNA corresponding to each gene is spotted onto the slide. For example, if the human genome contains ~35,000 genes then you would have ~35,000 genomic or cDNAs spotted onto the glass slide. These fragments of DNA will serve as the target for a hybridization reaction with mRNA that is collected from tissue samples. - In order to determine the expression profile of tissues, mRNA is collected and converted into cDNAs. During this process fluorescently labeled nucleotides are added to the reactions such that one sample will emit red or green light when excited with a laser beam. As you can surmise, two samples are usually being compared to each other (ie wild type vs mutant tissue). These labeled cDNAs are allowed to hybridize with the genomic fragments that have been affixed to the glass slide. cDNAs from each sample will be able to simultaneously hybridize with the target sequences. After an incubation period the glass slide is washed and prepared for the detection step.

2. Expression Profiling Using DNA MicroArrays - A laser is focused on each spot on the microarray and a computer records the level of hybridization from the two respective samples. At the most basic level there are four types of recordings (1) a red spot; (2) a green spot; (3) a yellow spot; and (4) a black spot. These colors correspond to the level of hybridization from the two samples. Just for the sake of argument lets assume that the green labeled cDNA sample corresponds to wild type tissue and the red labeled cDNA sample corresponds to the mutant tissue. From the image to the left you will notice that there are shades of red, green and yellow. This corresponds to the level of hybridization from each sample. - If a spot on the microarray is green then it indicates that the corresponding gene is expressed at higher levels in the wild type tissue. - If a spot on the microarray is red then it indicates that the corresponding gene is expressed at higher levels in the mutant tissue. - If a spot on the microarray is yellow then it indicates that the corresponding gene is expressed at equal levels in both normal and mutant samples. - If a spot on the microarray is black then it indicates that the corresponding gene is not expressed in either tissue.

3. Tiling Arrays: Leaving No Stone Unturned - A cDNA based microarray by definition will involve the spotting of fragments that only correspond to the exons of coding genes. All intronic and intergenic regions will be ignored. Therefore an mRNA that was transcribed from intronic or intergenic DNA would not have a target to hybridize to on the array slide. Small genes and non-coding RNAs are often missed when a cDNA microarray library is used. Also, the computer programs that predict the existence of genes are not perfect thus some bona fide genes are not placed on the array slide. - One way to overcome these shortcomings is to generate tiling arrays. The ideal tiling array uses overlapping DNA fragments (top left). A human tiling array will require several hundred thousand genomic fragments to be spotted onto the glass slide. Other tiling arrays will make use of fragments that are separated by gaps. The size of the fragments and gaps can vary from experiment to experiment. In all versions of tiling arrays RNA samples are still hybridized to the slide just like a cDNA array. - The last image (bottom left) shows the transcriptional profile or a genomic region that contains two genes. As you will see, in addition to the two genes there is a lot of transcription being initiated in the intergenic regions. These could correspond to new genes. tiling array with overlapping fragments tiling array with non-overlapping fragments

4. DNA Microarrays and D/V Patterning - During dorsal-ventral pattering of the Drosophila embryo there is a gradient of Dorsal nuclear localization. Cells on the ventral surface contain high levels of nuclear Dorsal. Cells along the lateral surface have moderate to low levels of nuclear Dorsal. Cells on the dorsal embryonic surface have no nuclear Dorsal. This asymmetry results in differential gene expression and the adoption of distinct cell fates across the D/V axis. - Prior to the publication (Stathopoulos et al., 2002) only a handful of Dorsal target genes were known. These included twist (twi), rhomboid (rho), short gastrulation (sog) and snail (sna). The authors had some bioinformatic evidence that additional Dorsal targets were encoded in the genome. In order to identify new targets of Dorsal the author used DNA microarrays to identify genes that are expressed along the ventral surface which gives rise to the mesoderm, the lateral surface which gives rise to the neuroectoderm and the dorsal surface which gives rise to the ectoderm.

5. Identification of New D/V Genes Using DNA Microarrays - DNA microarrays requires the isolation of RNA from tissue samples. In the ideal experiment one would isolate RNA from the cells along the ventral surface, the lateral surface and the dorsal surface. The expression profile of each population of cells could then be determined. However this experiment is not feasible due to technical limitations. - Instead the authors manipulated the Toll pathway using mutants in order to generate whole embryos that mimicked high, low and absent levels of nuclear localized Dorsal. This was accomplished by using a gain-of-function Toll allele (high nuclear Dorsal), a combination of Toll loss-of-function alleles (low nuclear Dorsal) and a pipe loss-of-function allele (no nuclear Dorsal). RNA from populations of these embryos were isolated and hybridized to a DNA microarray. - This comparison led to the identification of dozens of new genes. For example 89 genes were expressed at higher levels in the Toll 10B mutant than in the other two mutants. The Toll 10B mutant has high nuclear Dorsal levels and thus mimics the ventral cells of wild type embryos. These 89 genes could be new genes that are required for the formation of the mesoderm. These genes would be predicted to be expressed along the ventral surface just like twist and snail.

6. Expression Patterns of New D/V Genes - The authors determined the expression patterns of the newly discovered 353 genes and compared them to genes that were already known to be expressed in the mesoderm, neuroectoderm and the ectoderm. - As we have discussed in the past the snail (sna) gene is expressed within cells of the ventral surface (panel a). It is known that sna is a target of Dorsal, in part, because the embryonic enhancer contains a cluster of Dorsal binding sites. In the Toll 10B mutant, sna is expressed in nearly all cells of the embryo (panel b). This is because the Toll 10B mutant is a gain-of-function allele and thus the Dorsal is localized to the nucleus in all embryonic cells. One of the 89 new genes that was expressed at higher levels in the Toll 10B mutant is called Mes3. In wild type embryos it is expressed within cells just along the ventral surface – just like snail. And in the Toll 10B mutant Mes3 is also expanded throughout the entire embryo. - Similarly, the dpp gene is normally expressed with cells that are found along the dorsal surface. This is the region where Dorsal is absent from the nucleus. In pipe mutants Dorsal protein is prevented from entering the nucleus of all cells thus the entire embryo mimics the dorsal surface. In these embryos dpp is expressed throughout the entire embryo as expected. In the microarray screen a gene called Ect1 was found to be expressed at higher levels in pipe mutants than in the two other genotypes. In wild type embryos the Ect1 gene is transcribed only in cells of the dorsal embryo – just like dpp. In pipe mutants Ect1 expression, like that of dpp, is expanded throughout the entire embryo. - The authors used expression profiling to confirm each of the 353 genes that were first detected in the DNA microarray.

7. Molecular Biology Study Questions - What kind of information is provided by a DNA microarray? - What is the difference between a cDNA microaray and a tiling array? - Which method (cDNA array or tiling array) provides the most coverage of the the genome? - What does it mean if a spot on the microarray is black? - What does it mean if a spot on the microarray is yellow? - What does it mean if a spot on the microarray is either red or green? - What is the purpose of using embryos that are mutant for the Toll pathway?

8. Preview of Upcoming Lecture Topics to be Covered Next Time Protein-Protein Interactions Yeast 2-Hyrbid (Y2H) Assays Co-Immunoprecipitation (co-IP) Assays Split YFP Assays Protein Detection using Western Blots Textbook Chapter Chapter 7 pg. 173-177, 182 Weekly Article(s) “Death Breath” “Getting Close and Personal” “Regulating Evolution”