Dr. Justin P. Kumar, Principle Investigator

The Development of the Drosophila Eye

Eye formation in Drosophila begins during mid-embryogenesis when two groups of cells within the developing head delaminate from the surface ectoderm, proliferate rapidly and organize themselves into monolayer epithelial sheets called eye-antennal imaginal discs. During the earliest stages of retinal development, cells within the eye imaginal disc, to the naked eye, are completely unpatterned and undifferentiated. However, several key developmental processes such as the determination of the retina, the establishment of compartment boundaries, the initiation of pattern formation, the specification of cell fates and the rotation of ommatidia are initiated at these early stages.

Retinal Determination

All cells within the retina must collectively make the decision to adopt a retinal fate as opposed to that of another tissue such as the wing, leg, antenna, genital etc. This process begins in the embryonic eye imaginal disc when several members of the retinal determination network are expressed in the developing primordium. These factors include the Pax6 genes eyeless (ey) and twin of eyeless (toy) as well as the Pax6(5a) homologs eyegone (eyg) and twin of eyegone (toe). As development proceeds through the first two larval instar stages the remaining network members are sequentially activated. These genes include two SIX family members sine oculis (so) and optix, the Tsh class genes teashirt (tsh) and tiptop (tio), the Meis homolog homothorax (hth), the transcriptional co-activator and tyrosine phosphatase eyes absent (eya), the winged helix-turn-helix transcription factor dachshund (dac), the Nlk protein kinase nemo (nmo) and the pipsqueak-like genes distal antenna (dan) and distal antenna related (danr). As currently understood, the expression of all fourteen genes is a critical step in forcing cells to taken on a retinal fate. Mutations that disrupt the workings of the network lead to severe disruptions in eye development. In contrast, the forced expression of these factors is sufficient to induce ectopic eye formation in a variety of non-retinal cell populations. Taken together these results suggest that the network sits atop the overall eye development hierarchy.

Compartment Boundary Establishment

While the imaginal disc is being channeled towards the adoption of an eye fate, the tissue is simultaneously subdividing itself into dorsal and ventral compartments. The eye primordium itself is initially specified as being of a ventral identity. At the transition between the first and second instar larval stages a dorsal fate is imposed on approximately half of the eye primordium. The establishment of a midline and the two flanking compartments is critical for later steps in visual development such as cell fate selection, ommatidial rotation and axon pathfinding. These all contribute to the proper vision in the adult retina. The founding of these compartments can be traced to back to embryogenesis with fringe (fng) being expressed throughout the entire eye primordium. At the first/second instar transition expression of the GAGA factor pannier (pnr) is initiated exclusively within the dorsal half of the retina. It subsequently activates the Wingless signaling pathway which itself then activates genes of the Iroquois Complex. As a consequence fng is down regulated just within the dorsal retina. The juxtaposition of the fng+ and fng- cells leads to Notch pathway activation and the creation of the midline or equator.

Pattern Formation

Pattern formation initiates during the third and final larval instar stage when a wave of morphogenesis initiates at the posterior edge of the retinal primordium and proceeds across the epithelium. The anterior edge of this wave is visualized as a dorso-ventral indentation within the tissue and is called the morphogenetic furrow. As the furrow traverses the eye, the field of undifferentiated cells is transformed into an array of periodically spaced ommatidial clusters. The Hedgehog (Hh) and Decapentaplegic (Dpp) pathways regulate the initiation of the furrow. Initially hh is expressed at the intersection of the posterior margin and the midline while dpp transcription is activated along the entire posterior margin. Mutations that disrupt either pathway at the margins of the eye field can result in a complete block in furrow initiation. As the furrow progresses across the eye field, the patterns of hh and dpp expression are altered dramatically. The former is now expressed in all developing photoreceptors while the latter is transcribed within the furrow itself. Reductions in the levels of either signal lead to a slowing or a stoppage of pattern formation. In opposition to the activities of the Hh and Dpp pathways are mechanisms that are put in place to prevent pattern formation from initiating ectopically from the dorsal and ventral margins. Namely, the Wg signaling cascade is active along the lateral margins just ahead of the advancing furrow and functions to block inappropriate patterning. Removal of this pathway results in the formation of ectopic furrows at both margins. And finally, the helix-loop-helix protein Extramacrochaetae (Emc) is expressed ahead of the furrow and functions to prevent the rate of pattern formation from outstripping that of cell proliferation. Loss of emc leads to an acceleration of the morphogenetic furrow. Taken together, these pathways function to initiate the process of patterning formation and ensures that it proceeds across the eye field in an orderly and correctly paced manner.

Cell Fate Specification

As the furrow passes the process of ommatidial assembly begins. Each ommatidium consists of approximately twenty cells: eight photoreceptors and twelve non-neuronal accessory cells. Cell fate specification occurs in a stereotyped fashion. The first cell to be specified is the R8 whose fate is controlled by the Notch signaling pathway and the basic helix-loop-helix transcription factor Atonal (Ato). The next set of photoreceptor cells to be specified is the R2/5 pair that are then followed by the R3/4 and R1/6 pairs. Like the R8, each cell is specified by a unique combination of transcription factors. The last photoreceptor neuron to be added to the developing unit eye is the R7 cell and it is the one whose specification is the best known. Prior to its specification, the presumptive R7 expresses the Sevenless (Sev) receptor tyrosine kinase. When it comes into contact with the Bride of Sevenless (Boss) ligand, which is expressed exclusively in the R8 cell, the downstream Ras/MAPK signaling cascade is activated thereby committing the cell to adopting the R7 neuron fate. Construction of the ommatidium is complete with the specification of the cone and pigment cells.

Ommatidial Rotation

As cells within the ommatidium are being specified they are also rotating as a unit and while each unit eye can rotate independently, all ommatidia in the dorsal half of the retina will rotate in the opposite direction of the ommatidia in the ventral compartment. This rotation is important for the establishment of the diametric opposite orientations of ommatidia across the equator in the adult eye. The direction of rotation is dependent upon which precursor cell within the unit eye adopts the R3 fate and which one is specified to be the R4. The cell that lies the closest to the equator will ultimately become the R3. Notch-Delta signaling and differential Frizzled activity within the two cells are among the factors that regulate this cell fate decision. Once the R3/4 photoreceptors adopt their fate, the ommatidium rotates 90° (in two steps of 45° each) so that its final position is perpendicular to the equator. This means that in the dorsal retina ommatidia will rotate counterclockwise while those in the ventral compartment will rotate clockwise (assuming the anterior is defined as pointing to the right). Over the years two classes of genes that regulate ommatidial rotation have been identified. One class is defined as consisting of "rotation genes" in that they only affect the degree of rotation of each ommatidium. Examples of these include the nemo (nmo) and roulette (rlt) genes. Mutations in either one of these factors result in ommatidia that under rotate. The second class of genes is referred to as “tissue polarity genes” as they affect both cell fate and the chirality of the ommatidium.

Cell Proliferation and Programmed Cell Death

Eye development (at least its determination and patterning) is complete when the eye field has generated and specified the fate of enough cells to produce the approximately 800 unit eyes that comprise the adult retina. The growth of the retina is regulated by a wide-ranging list of genes and signaling pathways. For example, the retinal determination network itself contributes at least four genes (eyegone, teashirt, tiptop and homothorax) to the task of maintaining and proliferating retinal progenitor cells. Additionally, all of the major signaling cascades including the Notch, EGF Receptor, JAK/STAT, Wingless, Hedgehog and Decapentaplegic pathways also promote growth of the developing eye field. The combined activities of all of these genes and signaling pathways produces an excess of cells that are later removed by programmed cell death. Most of this death occurs during the pupal stage when the final form of retina is being sculpted.

Research Overview Collage
Dr. Justin Kumar- PI

Kumar, J.P. (2013) Catching the next wave: patterning of the Drosophila eye by the morphogenetic furrow. In Molecular genetics of axial patterning, growth and disease in the Drosophila eye. Springer-Verlag 75-97

Kumar, J.P. (2012) Building an ommatidium one cell at a time. Developmental Dynamics 241: 136-149

Kumar, J.P. (2011) My what big eyes you have: how the Drosophila retina grows. Developmental Neurobiology 71: 1133-1152

Kumar, J.P. (2010) Retinal Determination: The Beginning of Eye Development. Current Topics in Developmental Biology 93: 1-28