LMO4 is a transcription cofactor expressed during retinal development and in amacrine neurons at birth. A previous study in zebrafish reported that morpholino RNA ablation of one of two related genes, LMO4b, increases the size of the eye in embryos. However, the significance of LMO4 in mammalian eye development and function remained unknown since LMO4 null mice die prior to birth. We observed the presence of a smaller eye and/or coloboma in ~40% of LMO4 null mouse embryos. To investigate the postnatal role of LMO4 in retinal development and function, LMO4 was conditionally ablated in retinal progenitor cells using the Pax6 alpha-enhancer Cre/LMO4flox mice. We found that these mice have fewer Bhlhb5-positive GABAergic amacrine and OFF-cone bipolar cells. The deficit appears to affect the postnatal wave of Bhlhb5+ neurons, suggesting a temporal requirement for LMO4 in retinal neuron development. In contrast, cholinergic and dopaminergic amacrine, rod bipolar and photoreceptor cell numbers were not affected. The selective reduction in these interneurons was accompanied by a functional deficit revealed by electroretinography, with reduced amplitude of b-waves, indicating deficits in the inner nuclear layer of the retina. Thus, LMO4 is necessary for normal GABAergic amacrine and OFF-cone bipolar cell development during retina development.

Vertebrate photoreceptors are polarized cells that consist of a specialized sensory structure termed the outer segment required for phototransduction and an inner segment that contains the cellular organelles. Proteins synthesized in the inner segment are transported to the outer segment via a connecting cilium by a process called Intraflagellar Transport (IFT). The IFT mechanism refers to the movement of a multisubunit complex along the flagellar axoneme, and mutations in some IFT components cause retinal degeneration. To better understand the role of IFT in early photoreceptor development, we studied zebrafish with mutations in genes encoding three specific subunits of the IFT particle: IFT57, IFT88 and IFT172. These mutants exhibit photoreceptor defects by five days post fertilization (dpf); however, it is not known whether outer segment formation initiates at earlier time points and then degeneration occurs or if outer segments never form at all. To understand this, we performed transmission electron microscopy to study the ultrastructure of photoreceptors at 60, 72 and 96 hours post fertilization (hpf). At 60 hpf, developing outer segments were seen in IFT57 mutant and wild type embryos, however, disorganized membranous structures were observed in IFT88 and IFT172 mutants. At 72 hpf, the number of outer segments in the IFT57 mutants was reduced by 88% when compared to wild type, indicating a defect in initiation of outer segment formation. By 96 hpf we see a reduction in both outer segment length and number in IFT57 mutants. In comparison, the IFT88 and IFT172 mutants do not grow outer segments at any time point. To complement our ultrastructural analysis, we performed immunohistochemistry to understand cell morphology and protein trafficking in these mutants. Zpr1, a marker for cone morphology, showed the presence of normal cones initially that began to degenerate at later time points. Immunohistochemistry with rhodopsin, a phototransduction protein that localizes to the outer segment, revealed that rhodopsin was mislocalized in all the three mutants by 96 hpf. Connecting cilia labeled with acetylated tubulin were highly reduced in IFT57 mutants whereas none were observed in IFT88 and IFT172 mutants. Together these data indicate that IFT57 is required for maintenance and growth of outer segments whereas IFT88 and IFT172 are required for initiating outer segment formation.

Thesis (M.Med.Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 92-102).

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 180-186). Available also in a digital version from Dissertation Abstracts.

The retina is an internal photosensitive neural tunic which absorbs light and prevents it from reflecting back. The light receptors and neurons of the retina are initial processor of visual information. Various anomalies of the retina such as retinitis pigmentosa, cone-rod dystrophy to retinal degenerative diseases cause severe loss of vision since they affect photoreceptors directly or indirectly. Conventional therapies have never been fully successful in restoring vision in such diseases. However current research in stem cell therapies has shown remarkable potential. In this project, induced pluripotent stem cells from mouse were coxed into photoreceptor fate in presence and absence of Dorsomorphin using specific media in a stepwise differentiation process. Dorsomorphin is an inhibitor of Bone Morphogenic Protein (BMP) whose suppression may influence neural differentiation. Studies were done using conventional inverted microscopy and fluorescent microscopy on mouse induced pluripotent stem cells (miPS cells). Immunolabelling techniques involving Pax6, Crx, RPE65, Rhodopsin and Opsin were used to evaluate the advantage of these as markers for stem cells differentiation. Reverse Transcriptase PCR was done to confirm the gene expression on the differentiated cells. Human iPS derived Mesenchymal stem cells were cultured and the effect of different concentrations of Retinoic Acid such as 0mM, 0.1mM and 0.5mM on cell proliferation was tested in both presence and absence of Dorsomorphin. The results revealed both control and Dorsomorphin treated miPS cells successfully differentiated into photoreceptors-like cells as detected by positive staining of Rhodopsin and Opsin. The cells were however negative for Pax6, and very weak staining for RPE and Crx. The presence or absence of Dorsomorphin did not make any difference on miPS differentiation. The same observation was made on differentiating human iPS-MSC where Dorsomorphin did not reveal much effect. However highest cell count of proliferating cells was observed in the subgroups containing 0.1mM Retinoic Acid on Day7 groups as control had an average of 590 ±±317.23 and treatment 1206 .33 ±±114.99 cells, with statistical significance of P<0.05. It appears that the presence of Retinoic Acid facilitated the proliferation of human iPS-MSC. In conclusion, the study reveals that iPS can be another potential stem cell source for therapies of retinal diseases involving photoreceptors where the question of ethical issue is not a problem unlike embryonic stem cells. Also it reveals the concentration of Retinoic Acid most suited for human iPS-MSC cell proliferation. Dorsomorphin did not seem to have much effect on either type of stem cells in terms of promoting photoreceptor differentiation. / published_or_final_version / Medicine / Master / Master of Medical Sciences

The complex circuitry of the visual system contains around one hundred functionally distinct neuronal types that become specified and connect with the appropriate synaptic partners during development. Previous studies have indicated that immature retinal ganglion cells already express subset-specific molecules that guide them to make precise synaptic choices. In the mammalian retina, members of the cadherin family of adhesion molecules are attractive candidates for this role. To test this idea I began by investigating the expression of cadherins 1-26 in the mouse retina and superior colliculus using in situ hybridization. I then studied the connectivity of cadherin-expressing neurons by analyzing mouse lines in which a marker was inserted after the start codon of each of six cadherin genes of interest. In this way, I identified functional circuits in the visual system that are marked by cadherins. One such circuit is formed of direction-selective retinal ganglion cells (DSGCs), which fire in response to objects moving in one (preferred) direction, and their synaptic partners, the starburst amacrine cells. There are four DSGC subsets, distinguished by their preference for dorsal, ventral, nasal, or temporal motion on the retina. I determined that cadherin 6 is selectively expressed by the two DSGCs subtypes that respond to dorsal or to ventral movement. In collaboration with other lab members I used in situ hybridization and gene expression profiling to identify other molecular markers that distinguish between the four DSGC subsets and that distinguish DSGCs from other retinal ganglion cells. Finally, I used birthdating and lineage tracing methods to ask when DSGCs become molecularly specified. I determined that at least two subsets of DSGCs are specified at or shortly after their birth. For cadherin 6-positive DSGCs, I went on to show that they are specified even before their birth, and that they arise from committed retinal progenitors. Globally, my experiments aimed not only to examine cadherin expression and function in the visual system, but also to demonstrate a method of using molecular signatures to probe the mechanisms of neural circuit assembly in the central nervous system.

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DLX transcriptional targets have been implicated during retinal development. The Crx (Cone-Rod homeobox) gene is required for the differentiation and maintenance of cone and rod photoreceptors. Otx2 (Orthodenticle homeobox 2) is a key regulator of photoreceptor cell fate. The Dlx1/Dlx2 mutant mouse retina has a significant reduction of retinal ganglion cells with aberrant Crx expression in the neuroblastic layer and increased retinal Otx2 expression. We hypothesized that the Dlx homeobox genes directly repress Crx and Otx2 expression during retinal development. Expression of CRX demonstrates increased transcript and protein expression in the Dlx1/Dlx2 double knockout retina at E18.5, suggesting that these DLX transcription factors may repress CRX expression. OTX2 expression is increased in the Dlx1/Dlx2 knockout retina at E16.5 suggesting that DLX2 negatively regulates OTX2 expression. The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina along with increased OTX2 expression. Our data suggests that both CRX and OTX2 are transcriptional targets directly repressed by the DLX1 and DLX2.