Table_1_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.docx

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.</p

Image_2_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.tif

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.</p

Image_1_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.tif

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.</p

Table_2_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.xlsx

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.</p

Image_4_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.tif

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.</p

Image_5_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.tif

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.</p

Image_3_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.tif

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.</p

Investigation of Islet2a function in zebrafish embryos: Mutants and morphants differ in morphologic phenotypes and gene expression

<div><p>Zebrafish primary motor neurons differ from each other with respect to morphology, muscle targets and electrophysiological properties. For example, CaP has 2-3-fold larger densities of both inward and outward currents than do other motor neurons. We tested whether the transcription factor Islet2a, uniquely expressed in CaP, but not other primary motor neurons, plays a role in specifying its stereotypic electrophysiological properties. We used both TALEN-based gene editing and antisense morpholino approaches to disrupt Islet2a function. Our electrophysiology results do not support a specific role for Islet2a in determining CaP’s unique electrical properties. However, we also found that the morphological phenotypes of CaP and a later-born motor neuron differed between <i>islet2a</i> mutants and morphants. Using microarrays, we tested whether the gene expression profiles of whole embryo morphants, mutants and controls also differed. Morphants had 174 and 201 genes that were differentially expressed compared to mutants and controls, respectively. Further, <i>islet2a</i> was identified as a differentially expressed gene. To examine how mutation of <i>islet2a</i> affected <i>islet</i> gene expression specifically in CaPs, we performed RNA <i>in situ</i> hybridization. We detected no obvious differences in expression of <i>islet1</i>, <i>islet2a</i>, or <i>islet2b</i> in CaPs of mutant versus sibling control embryos. However, immunolabeling studies revealed that an Islet protein persisted in CaPs of mutants, albeit at a reduced level compared to controls. While we cannot exclude requirement for some Islet protein, we conclude that differentiation of the CaP’s stereotypic large inward and outward currents does not have a specific requirement for Islet2a.</p></div

Relative levels of <i>islet</i> and downstream gene transcripts in morphants vs. mutants.

<p>qPCR was used to compare expression levels of <i>islet1</i>, <i>islet2a</i> and <i>islet2b</i> between wildtype (wt), mutant (mut), morphant (morph) and Ctl MO-injected (Ctl MO) embryos. In <i>islet2a</i> mutants, <i>islet1</i> levels were significantly increased by ~ 25%; no changes in the levels of other <i>islet</i> transcripts were detected in mutants. Both morphants and Ctl MO-injected embryos had significantly decreased levels of <i>islet1</i>. The largest change detected was a ~ 4-fold increase in <i>islet2a</i> levels in morphant compared to wildtype or mutant. ***, p<0.001; **, p<0.003; *, p<0.005; ANOVA with post hoc Bonferroni. To facilitate comparisons, for each gene, expression levels were normalized to that of the control group.</p

qPCR gene assays.

<p>qPCR gene assays.</p