Research

Vertebrate Eye Development

The eye collects, integrates and delivers visual information to processing regions within the brain. In development, eye primordium coalesces into a lens placode in the surface ectoderm, and an underlying optic vesicle that becomes the retina (Panel A). The optic vesicle ceases outgrowth when it contacts the lens placode, and becomes transformed into a cup (Panels B,C). The optic cup also becomes bilayered, but remains contiguous with the diencephalon via the optic stalk, whose lumen will hold the optic nerve (Panels C,D). Lens development starts in the surface ectoderm when this tissue undergoes localized thickening to form the lens placode, which then changes shape to produce a lens pit. The pit fills the space created when the optic vesicle invaginates to form an optic cup. Once the lens pit closes off fully it detaches and forms a hollow lens vesicle.

What controls optic vesicle outgrowth and patterning?

A fundamental component of embryonic development is cell-cell communication, mediated in part by paracrine signaling pathways (ex. Shh), whose secreted ligands bind to surface receptors on nearby cells. The activity of this pathway must be exquisitely controlled, by fine-tuning the timing, spatial localization, duration of the signal - and response. Cells in the off Shh signaling mode have Patched (Ptch)-Smoothened (Smo) protein complexes at the cell membrane. Upon Shh binding to Ptch, Smo is released, allowing its accumulation on primary cilia, which triggers a signaling cascade, culminating in Gli transcriptional regulation of downstream target genes in the nucleus. The Shh morphogen diffuses from the embryonic midline in two waves. Shh is first secreted during gastrulation by the prechordal plate, in a narrow time window, and later by the ventral diencephalon. Disruption of the first wave causes holoprosencephaly (HPE, cyclopia), and suppresses optic stalk genesis. Loss of the second wave causes septo-optic dysplasia, and impairs hypothalamus, pituitary and optic nerve head formation. In the vertebrate embryo, a central eye field is specified in the anterior neural plate at the end of gastrulation, which subsequently splits to form bilateral optic vesicles by evagination from the ventral diencephalon. The growing optic vesicles become regionalized by demarcating the optic stalk (OS), neural retina (NR) and retinal pigment epithelium (RPE). Cells in the optic vesicle and stalk are primed to receive midline Hh signals, via the Patched (Ptch) receptor, but their responses are modulated by multiple mechanisms.

Another commonly used signaling system, the Notch pathway, relies on physical interactions between membrane-bound ligands and receptors on adjacent cells, severely limiting its range of action. During retinal development, this pathway orchestrates the appearance of distinct neuronal and glial cell types. Notch ligand-receptor binding induces sequential proteolytic cleavages to the receptor protein, ultimately releasing the intracellular domain (N-ICD) to form a nuclear protein complex with Rbpj, MAML, p300 and other proteins. Then the complex transcriptionally activates downstream genes, for example Hes1. Notch pathway mutations are linked to human birth defects such as Alagille Syndrome and Tetralogy of Fallot, SIgnal dysregulation can cause multiple types of cancer. We are exploring genetic and molecular intersections between the Shh and Notch signaling pathways during ocular growth, morphogenesis and patterning. We are using two spatiotemporally distinct Cre drivers (Rax-Cre and Chx10-Cre) to examine the phenotypes caused by loss- or gain-of-function of each pathway.

Vertebrate Retinal Neurogenesis

Retinal neurons integrate and transmit essentially all visual information our eyes see to the brain. Defects in retinal development lead to abnormal vision. Intriguingly, developing retinal cells are capable of choosing multiple fates, but ultimately become a single cell type. A better understanding of these principles will aid in the future design of more effective retinal disease therapies. During early growth of the optic vesicle and cup, progenitor cells contact both sides of the tissue, except when dividing ((Top panel). During mitosis, progenitor cells retract cell processes from each surface, divide apically, and then reattach to both edges. Moreover, RPC nuclei undergo interkinetic nuclear movements, between the apical and basal surfaces of the eye. At S-phase, the nuclei are basally located, but migrate to the apical surface during G2 phase. Early RPCs exiting the cell cycle migrate basally (green cell). An architectural landmark that accompanies retinal neurogenesis is tissue lamination, induced by waves of progenitors, differentiating and accumulating in layers, where they extend an axon and form a synaptic connection. The mature retina contains three cell layers: the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL)(bottom panel). The primary sensory neurons of the retina are photoreceptors, which absorb photons of light in their outer segments, and are divided into two major classes: rods (detect dim light) and cones (detect bright light and color). Bipolar neurons relay information from photoreceptors to retinal ganglion cells (RGCs), which in turn convey this sensory information to particular regions of the brain. Amacrine and horizontal neurons modulate and integrate visual signals within the retinal circuitry. In the adult vertebrate retina, Müller glia nuclei reside in the INL, but their cell bodies stretch across the entire width.

bHLH transcription factors in the retina

In the vertebrate retina basic helix-loop-helix (bHLH) factors are instructional molecules for retinal neuron formation. The expression of "proneural" bHLH factors coincides with the onset of specification and differentiation of particular retinal neuron classes (e.g. Atoh7/Math5 and RGCs). RGCs appear first in the vertebrate retina (diagram at left). RGC neurons require Atoh7 bHLH gene in progentior cells for their proper development. At least four neuron-promoting bHLH genes have been characterized: Atoh7, Neurog2, Neurod1 and Ascl1. The expression of each proneural gene coincides with a peak of genesis for distinct retinal cell type(s) and their activation is staggered across several days of development. Mutational and ectopic expression experiments have shown that progenitors are biased to particular fates by their expression of one or more bHLH proteins. Although Atoh7 and Neurog2 are coexpressed, Neurog2 plays a separable role in controlling the rate of neuronal differentiation, which moves across the forming retina. Another class of bHLH factors are Hes genes, which typically suppress neuronal fates. The Hes gene family is often, but not exclusively, activated by Notch signaling, to promote retinal progenitor cell proliferation and block initation of neurogenesis (in part by suppressing proneural gene expression).