Introduction: The Dlx1/Dlx2 double knockout mouse has reduced numbers (33% fewer) of retinal ganglion cells (RGC), due to enhanced apoptosis. Brn3a and Brn3b are closely related members of the Class IV POU-domain gene family, and play functionally interchangeable roles in retinal development. We hypothesized that Brn3a and/or Brn3b are direct DLX transcriptional targets during retinal development. Methods: Chromatin immunoprecipitation (ChIP) assays were performed on E16.5 retinas to identify DLX proteins bound to the Brn3a or Brn3b promoters. Electrophoretic mobility shift assays (EMSA) were used to confirm the specificity of this binding. Luciferase reporter gene assays were performed to confirm the functional significance of DLX binding to the Brn3 or Brn3ba promoters in vitro. In utero retinal electroporation was used to study the effect of DLX gain-of-function on Brn3a/Brn3b expression in vivo. Compound Dlx1/Dlx2/Brn3a and Dlx1/Dlx2/Brn3b knockout mice were generated for analysis of retinal phenotypes. Results: Both DLX1 and DLX2 proteins bound to the Brn3b promoter, but only DLX2 bound to the Brn3a promoter in vivo. Using EMSA, recombinant DLX1 and DLX2 bound to Brn3b and specific supershifted bands resulted from the addition of specific DLX1 or DLX2 antibodies; only recombinant DLX2 bound to Brn3a and the supershifted band resulted from the addition of DLX2 antibody. Both DLX1 and DLX2 binding to the Brn3b promoter activated transcription of a luciferase reporter gene in vitro. Only Dlx2, but not Dlx1, co-transfection with Brn3a activated luciferase reporter gene expression. In utero retinal electroporation showed that ectopic DLX2 expression promoted both Brn3b and Brn3a expression in vivo. Loss of Dlx1/Dlx2 and Brn3b resulted in loss of 90% of RGC and increased cholinergic amacrine cell differentiation. Conclusion: Brn3b is transcriptionally regulated by both DLX1 and DLX2, whereas Brn3a is only regulated by DLX2 in vitro and in vivo. Dlx1/Dlx2 and Brn3b play combinatorial roles in retinogenesis.

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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.

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.

Introduction: The Dlx1/Dlx2 double knockout mouse has reduced numbers (33% fewer) of retinal ganglion cells (RGC), due to enhanced apoptosis. Brn3a and Brn3b are closely related members of the Class IV POU-domain gene family, and play functionally interchangeable roles in retinal development. We hypothesized that Brn3a and/or Brn3b are direct DLX transcriptional targets during retinal development. Methods: Chromatin immunoprecipitation (ChIP) assays were performed on E16.5 retinas to identify DLX proteins bound to the Brn3a or Brn3b promoters. Electrophoretic mobility shift assays (EMSA) were used to confirm the specificity of this binding. Luciferase reporter gene assays were performed to confirm the functional significance of DLX binding to the Brn3 or Brn3ba promoters in vitro. In utero retinal electroporation was used to study the effect of DLX gain-of-function on Brn3a/Brn3b expression in vivo. Compound Dlx1/Dlx2/Brn3a and Dlx1/Dlx2/Brn3b knockout mice were generated for analysis of retinal phenotypes. Results: Both DLX1 and DLX2 proteins bound to the Brn3b promoter, but only DLX2 bound to the Brn3a promoter in vivo. Using EMSA, recombinant DLX1 and DLX2 bound to Brn3b and specific supershifted bands resulted from the addition of specific DLX1 or DLX2 antibodies; only recombinant DLX2 bound to Brn3a and the supershifted band resulted from the addition of DLX2 antibody. Both DLX1 and DLX2 binding to the Brn3b promoter activated transcription of a luciferase reporter gene in vitro. Only Dlx2, but not Dlx1, co-transfection with Brn3a activated luciferase reporter gene expression. In utero retinal electroporation showed that ectopic DLX2 expression promoted both Brn3b and Brn3a expression in vivo. Loss of Dlx1/Dlx2 and Brn3b resulted in loss of 90% of RGC and increased cholinergic amacrine cell differentiation. Conclusion: Brn3b is transcriptionally regulated by both DLX1 and DLX2, whereas Brn3a is only regulated by DLX2 in vitro and in vivo. Dlx1/Dlx2 and Brn3b play combinatorial roles in retinogenesis.

The filtering of light by water is contingent on depth, direction and clarity. Consequently, fish must contend with a much more variable spectral world than terrestrial species. The gene family responsible for light sensitivity, the opsins, has expanded in fish. The duplication events responsible for large fish opsin gene repertoires have been characterized as part of this thesis research. The four-eyed fish, Anableps anableps, swims at the surface with its eyes at the waterline. Among many unusual adaptations, these eyes have two pupils, one above and one below the surface, giving it simultaneous access to broad spectrum aerial light and filtered aquatic light. It also has a nine cone opsin genes including duplications in three of the four cone opsin subfamilies. In situ hybridization was used to localize opsin transcripts in the retina. My data show that A. anableps expresses SWS1, SWS2 and RH2 opsins and has broad spectral sensitivity across its entire retina. In addition, I discovered that the region of the retina exposed to aquatic light expresses LWS and is, therefore, additionally red sensitive to match the longer wavelength available in cloudy water. By comparing this pattern with its normal eyed sister species, Jenynsia onca, I found that this increased red sensitivity is accomplished through the reduction of green sensitive pigments, which in A. anableps (but not J. onca) are expressed only in the ventral region of the retina that is exposed to aerial light. / Graduate

We have studied the factors that are important for producing functional retinal neurons from adult derived retinal stem and progenitor cells (RPCs) investigating the role of substrate stiffness or soluble factors in 2D and 3D culture on the differentiation and survival of RPCs. RPCs were cultured on agarose scaffolds modified with the adhesive peptides of varying stiffness. We observed that cell survival in the 0.75% matrix was greater in 3D than in 2D by a factor of 50% irrespective of the time in culture. We observed the presence of photoreceptors exclusively in 0.75% agarose, while the stiffer matrices (1.75%) led to retinal ganglion cell and glial cell differentiation with no significant difference in the differentiation profiles when cells were cultured in 2D vs. 3D. These data indicate that substrate stiffness, more than growth factors, has a significant impact on both the survival and differentiation profile of retinal precursor cells.

We have studied the factors that are important for producing functional retinal neurons from adult derived retinal stem and progenitor cells (RPCs) investigating the role of substrate stiffness or soluble factors in 2D and 3D culture on the differentiation and survival of RPCs. RPCs were cultured on agarose scaffolds modified with the adhesive peptides of varying stiffness. We observed that cell survival in the 0.75% matrix was greater in 3D than in 2D by a factor of 50% irrespective of the time in culture. We observed the presence of photoreceptors exclusively in 0.75% agarose, while the stiffer matrices (1.75%) led to retinal ganglion cell and glial cell differentiation with no significant difference in the differentiation profiles when cells were cultured in 2D vs. 3D. These data indicate that substrate stiffness, more than growth factors, has a significant impact on both the survival and differentiation profile of retinal precursor cells.

Thesis (Ph. D.)--University of Washington. / Bibliography: l. [232]-254.