1. The Cone Cells of the Drosophila Retina
- Each unit eye within the Drosophila retina contains four lens secreting cells called cones (remember that these are different than the cone photoreceptor cells of the vertebrate retina). These cells secrete the overlying lens. - The lens is made up of roughly 30 protein layers – the main protein constituent of each layer is called Drosophila crystallin (dcy). This protein is homologous to the crystallin proteins that make up the human lens. In both systems the lens has a different refractive index than the surrounding air. The lens channels photons of light onto the underlying photoreceptor lens. - During the development of the insect retina the photoreceptor cells (which are specified earlier) instruct four undifferentiated cells to adopt a cone cell fate. The process of cone cell recruitment is thought to occur through the activation of two signal transduction cascades: the EGF Receptor Pathway and the Notch Pathway. - As we will see later, a key target gene of these two pathways is the D-Pax2 gene. This gene is absolutely required for the formation of the cone cells. The job of the EGF Receptor and Notch pathways is to activate the D-Pax2 gene in cells that are destined to become cone cells. - The photoreceptor cells produce ligands that bind to the membrane bound receptors on the presumptive cone cell surface.

2. The Paired Box (Pax) Family of Transcription Factors
paired domain OP
homeodomain
- The founding member of the Paired box (Pax) family of transcription factors is the Drosophila Paired protein. It contains a special DNA binding domain called the PAIRED domain. Over the years a number of similar proteins have been identified in all organisms including humans. The different Pax proteins are grouped into distinct classes based on their protein structure.
- Pax1 and Pax9 contain the paired DNA binding domain and an eight amino acid octapeptide that is used in transcriptional repression.
- Pax2, Pax5 and Pax8 contain the paired DNA binding domain, the octapeptide and the first helix of the homeo DNA binding domain. Since it lacks the second and third helices these proteins do not interact with DNA through this motif.
- Pax4 and Pax6 have intact paired and homeodomains therefore these factors can bind to DNA using both motifs.
- Pax3 and Pax7 have all three motifs: the paired and homeodomains as well as the octapeptide.
Drosophila D-Pax2 belongs to the Pax2/5/8 class of Pax proteins. Mutations in human Pax 2 lead to renal-coloboma syndrome.
Pax1/9
Pax2/5/8 1 2 3
Pax4/6
Pax3/7

3. DPax2 is Required in Cone Cells
- In the wild type retina the DPax2 gene is expressed in just the four cone cells (far left panels).
- The loss of DPax2 expression leads (near left bottom panel) leads to the complete loss of all cone cells and a roughening of the external surface of the compound eyes.
- The isolation of flies that have a “rough eye” is a convenient way to screen for genes that affect eye development. To date, hundreds of genes that control various aspects of eye development have been identified by this method.
Different laboratories have used different types of mutagens. Some groups have used X-rays which will cause deletions within the genome. Others have used chemicals such as ethyl methanesulfonate (EMS) to induce single base changes. And finally some groups used transposable elements to jump around the genome thereby inserting themselves into genes.
- The mutations that eliminate expression of DPax2 within the cone cells are located within an eye-specific enhancer. Note that DPax2 is also expressed in other tissues – however in these flies only the eye enhancer has been disrupted.
wild type
D-Pax2 mutant

4. The D-Pax2 Eye Specific Enhancer
- The DPax2 eye specific enhancer is approximately 500bp in length. This enhancer is sufficient to drive expression of a reporter construct just within the cone cells (top right panel). - An analysis of this enhancer revealed that it contains binding sites for at least four proteins. The Pointed and Yan transcription factors are downstream members of the EGF Receptor signaling cascade. The Su(H) DNA binding proteins is the most downstream member of the Notch pathway. And finally Lozenge is a transcription factor that is expressed in all cells of the developing retina – it does not appear to be developmentally regulated. - These binding sites make up only a small fraction of the DNA that is found within this enhancer. Some of the other sequences are conserved within other Drosophila species. These sequences may represent binding sites for additional proteins. - Some sequences within the DPax2 enhancer are found solely within Drosophila melanogaster. Such sequences may simply be necessary for proper spacing between binding sites. Binding sites are nearly always separated from each other since the proteins that bind to them are large and bulky. If the sites are too close to each other then it is possible that the DNA binding proteins will not have enough room to bind onto the enhancer.

5. Structure of Enhancers Combinatorial Code of Binding Sites
- Eukaryotic enhancers are bound by different DNA binding proteins. In the examples to the left each color represents a different sequence of bases that is recognized by a unique DNA binding protein. Some enhancers contain single recognition sites for multiple unique transcription factors (example A). Mutations in any one of these sites can inactivate the entire enhancer and gene expression can be inhibited. - Other enhancers can contain multiple recognition sites for several different DNA binding proteins (example B). The loss of a single site within these classes of enhancers usually lowers the transcriptional levels of the gene of interest. The decrease in transcriptional output is often severe enough to result in phenotypic defects. - The transcriptional output of a gene is often dependent upon the number of transcription factor binding sites that are found within an enhancer. The B enhancer is likely to direct a higher level of expression than enhancer C because it contains 4 (vs 1) of the type blue binding sites. - The quality of binding sites can also affect the transcriptional output of an enhancer. Quality refers to the whether or not the DNA sequence is optimal for the DNA binding protein. In example D there are three shades of blue class sites. These are meant to represent the quality of the blue site. In this example the sequence of the dark blue site is optimal for the transcription factor and thus the residence time on this site will be high. The medium blue site represents a sequence that is recognized by the transcription factor but is not optimal. The residence time on the medium blue site is less than that of the dark blue site. And finally the light blue site is one in which the residence time is the shortest. The transcriptional output from enhancers B, D and E can be ordered in the following way B > E > D. A B C D E
CGATTAGC
AGATTAGC
AGATCCGC

6. The EGF Receptor Pathway
- Binding of the ligand to a receptor tyrosine kinase leads to the dimerization and trans phosphorylation of the receptor. This leads to the immediate activation of a cytoplasmic signaling cascade. - The first molecule to be activated is Ras, a member of a family of small GTPases. Ras will in turn activate the cytoplasmic Raf kinase, which will then phosphorylate and activate MEK which itself will phosphorylate and activate Mitogen Activated Protein Kinase (MAPK). - Unphosphorylated MAPK is found within the cytoplasm while the dually phosphorylated version is translocated into the nucleus. Upon entering the nucleus, MAPK will phosphorylate several transcription factors. Some are transcriptional activators and will therefore promote gene expression. Others are transcriptional repressors and will thus inhibit gene expression. - All RTKs will transmit signals to the nucleus through the Ras/MAPK signaling cassette. However, the activation of different RTKs will lead to the modification of very different transcription factors. Different RTKs can function in different tissues while multiple RTKs can also function within the same cell. It is an ongoing area of research to determine how a single cell can interpret messages that are simultaneously being sent by different RTKs.
RAS
RAF
MEK
MAPK
MAPK
MAPK TF TF

7. The Notch Pathway Duel Identities: Receptor and Transcription Factor
Delta
- Upon binding of the Delta ligand to the Notch receptor, the receptor is bound by a cytoplasmic scissor protein called Kuzbanian (Kuz). It cleaves intracellular domain of the receptor (Nicd). Note that the Notch receptor remains as a monomer (even in the ligand bound state). - Nicd is then translocated to the nucleus where is interacts with the Suppressor of Hairless (Su[H]) DNA binding protein. In the absence of Notch signaling Su(H) physically interacts with the C terminal Binding Protein (CtBP) and Groucho (Gro) co-repressors to inhibit transcription. Nicd displaces both of these co-repressors thereby turning Su(H) into a transcriptional activator. - Cells that express the Notch receptor will become non-neuronal cells while those that express Delta will become neurons.
Notch
Kuz
CtBP
Su(H)
Gro

8. Combinatorial Code of Transcription Factors Activates the DPax2 Enhancer
- Mutations that alter the ability of Pointed (Pnt), Suppressor of Hairless (Su[H]) or Lozenge (Lz) to bind to the DPax2 eye enhancer results in the complete loss of DPax-2 expression within the presumptive cone cells (left panels). This results in the failure of cone cells to develop and the structure of the eye is disrupted since the lens is not secreted. - This has led to a model in which a combinatorial code of transcription factors is required to activate expression of target genes. In this case the Lz DNA binding protein as well as the EGF Receptor and Notch pathways are required to activate expression of the DPax2 gene and thus specify cone cell fates. The loss of any of these factors will inactivate DPax2 – the presumptive cone cells will either remain undifferentiated or will be transformed into a photoreceptor cell.
wild type
Pnt mutant
Su(H) mutant
Lz mutant

9. Complexity of the DPax-2 Enhancer More than Lz and EGF and Notch Signaling
Within the DPax-2 eye enhancer lies five sites for Su(H) binding, three sites for Lz binding and four sites for Pnt/Yan binding (panel a). A transcriptional reporter that preserves the native spacing of these sites and the nature of the intervening sequences drives expression in all four cone cells (panels c and d). However, a construct in which the native spacing has been preserved but the sequences has been mutated fails to drive expression of the reporter (panel e). Likewise a construct that eliminates the intervening sequences also fails to drive expression of the reporter (panel f). These results imply that the DNA bases that lie between the Lz, Pnt/Yan and Su(H) sites are important for expression of DPax2 within the eye.
The order of the sites is also important as scrambling the order while preserving the correct spacing is insufficient to maintain proper expression of the D-Pax2 enhancer (below – last panel).
- An important questions is whether all of these intervening sequences are important or is it just a subset. Deleting or changing individual regions can be used to test the requirements for each intervening segment.

10. Dissecting the DPax-2 Enhancer
- In this experiment individual intervening segments are either deleted or altered (preserves spacing). The resulting modified enhancers are assayed for their ability (or lack thereof) to drive expression of a reporter construct in the cone cells. The wild type construct is used as a control for comparison (panel b). As you can see most of the intervening sequences are required for proper expression from the DPax-2 enhancer. Some exceptions include deletions of the third intervening sequence (no effect). Interestingly, deletion of the fifth intervening sequence leads to an up-regulation of DPax2 expression. This suggests that during normal development this regions is bound by a transcriptional repressor – in its absence the enhancer has increased activity.

11. Molecular Biology Study Questions
- Which factor in the Drosophila genome is primarily responsible for specifying cone cell fate? - What differentiates the different members of the Pax family of DNA binding proteins? - Where is D-Pax2 expressed in the fly retina? - Which cells signal to the presumptive cone cells? - What are the three known inputs into the D-Pax2 eye specific enhancer? - Is the spacing between known binding sites important? - Do the exact sequences between the Lz, Pnt and Su(H) sites have to be preserved in order to get normal expression of D-Pax2? - Is every sequence within an enhancer element critical for transcriptional regulation? - What does it mean if the loss of a site leads to an increase in transcriptional output?

12. Preview of Upcoming Lecture
Topics to be Covered Next Time
Transcriptional Enhancers Part I
Identifying an Enhancer Element
Generating a Transcriptional Reporter
The UAS/GAL4 system
GFP, lacZ and Luciferase
Weekly Article(s)
“A New Molecule of Life”
“The Orderly Chaos of Proteins”
“Some Needles in the Haystack”