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Games Soccer Sports Games. We have undertaken the present study to provide foundational information for future investigations of iris structure, function, development, and disease. Our approach has been to define the transcriptome profiles of all of the major cell types of the mature murine iris by single nucleus sn RNAseq, to use those profiles to define the full repertoire of those cell types and the relationships among them, and to explore how the transcriptome changes within each iris cell type in response to pupil dilation or constriction.

Using transcription factors as cell type-specific markers, we have also visualized the nuclear deformations that occur in response to pupil dilation and assessed the neural crest origin of different iris cell types. The diversity of cell types in the iris — which includes muscle, epithelial, and stromal cells — suggested that homogenizing the iris and purifying nuclei might provide a more comprehensive and uniform sampling than would enzymatically dissociating the iris into single cells.

Tissue homogenization has the added advantage that regulated RNA synthesis and degradation should largely cease upon cell disruption and dilution of the nuclei into homogenization buffer. By contrast, with enzymatic dissociation preparatory to single-cell sc RNAseq, transcriptome changes can be observed during the 30—60 min required for cell dissociation and processing Lacar et al.

Six and one-half hours was chosen as the duration for dilation or constriction to provide sufficient time for any changes in gene expression to be fully manifest. The data were analyzed using the Seurat platform. Among independent replicates from mice subject to the same treatment, RNAseq read counts showed pairwise Pearson correlations of 0.

Therefore, for all subsequent analyses, each set of replicate samples were merged into a single dataset. Female albino mice were used throughout this study to facilitate visualization of fluorescent antibody and in situ hybridization ISH probes.

Ten iris cell clusters were identified with Seurat, and their identities were subsequently assigned by immunostaining and ISH, as described below.

A Uniform Manifold Approximation and Projection UMAP plot of the snRNAseq data revealed a distinctive distribution of the 10 clusters Figure 1B and C : 1 a comma shape, consisting of the iris pigment epithelium PE and the adjacent and contiguous ciliary body epithelium CBE , 2 a C-shaped arc in which dilator muscle connects to non-epithelial ciliary body cells on one side and sphincter muscle on the other side, 3 two widely separated clusters of stromal cells referred to as stroma 1 and stroma 2 , and 4 small clusters of leukocytes and endothelial cells.

UMAP plots are well matched across treatment conditions and independent replicates Figure 1—figure supplement 2 , with the only visually apparent difference being a small shift in the location of the dilator muscle cell cluster upon pupil dilation. Some cluster identities could be inferred based on existing physiologic data.

For example, classic pharmacologic studies have shown that the dilator muscle is responsive to alpha-adrenergic agonists and the sphincter muscle is responsive to muscarinic agonists. As shown in Figure 1D , transcripts coding for the alpha-1A adrenergic receptor Adra1a are enriched in the dilator cluster, and transcripts coding for muscarinic acetylcholine receptor 3 Chrm3 are enriched in the sphincter cluster. Other transcripts that show high cluster specificity include transcription factors Sox10 stroma 1 , Tfap2b stroma 2 , and Zic1 epithelial cells Figure 1D.

The distinctions between clusters rest on differences in the expression of multiple genes, a small fraction of which are shown in Figure 1F see also Supplementary file 2. That the stroma 1 vs. These data indicate that the mouse iris contains two distinct types of stromal cells and three distinct types of smooth muscle cells. A subset of markers that distinguish the different classes of iris smooth muscle cells were investigated by immunostaining Figure 2A.

In iris cross sections, the strands of dilator muscle typically weave in and out of the plane of section Figure 2B—G , as inferred from SMA immunostaining of iris flat mounts Figure 2H. This region coincides with the region of circumferential muscle fibers visualized by SMA immunostaining, implying that sphincter 1 and sphincter 2 cells are intermingled with each other within the mass of sphincter muscle fibers.

Nitric oxide synthase-1 Nos1 , the NOS isoform enriched in the nervous system and skeletal muscle, is specifically expressed in dilator muscle and localizes in a pattern reciprocal to that of CKM Figure 2H. A Dot plot as described in Figure 1F showing transcript abundances across a subset of iris cell types for transcripts coding for the markers shown in B—H.

Rbfox3 codes for NEUN. Scales are individualized for each transcript to accommodate the large differences in their abundances. B—G Cross sections of untreated iris immunostained as indicated. In this and all other figures showing iris cross sections, the cornea is above and the lens is below the region shown. The pupil is to the right and the region encompassing the sphincter muscle is indicated.

H Flat mounts of untreated iris immunostained as indicated. For each immunostaining analysis in this and subsequent figures, iris cross sections were stained from at least five mice and iris whole mounts were stained from at least two mice. All images are at the same magnification. This analysis localized transcripts for Nos1 to dilator muscle, Ttn to sphincter muscle, and myosin heavy chain Myh11 to both dilator and sphincter muscles Figure 3B and C.

Transcripts coding for synaptotagmin-1 Syt1 , a calcium sensor that regulates neurotransmitter release, localized to sphincter 1 and sphincter 2 cells Figure 3B and C. As predicted from the UMAP analysis, Adra1a transcripts are enriched in dilator muscle and Chrm3 transcripts are enriched in sphincter muscle Figure 3D.

A Dot plot as described in Figure 2A showing transcript abundances across a subset of iris cell types for transcripts coding for markers shown in B—D. B—D Cross sections of untreated iris hybridized with the indicated probes. For each in situ hybridization analysis in this and subsequent figures, iris cross sections were hybridized from at least three mice.

Except for the adrenergic and cholinergic receptors, the physiologic significance of differential gene expression in the different iris smooth muscle subtypes is unclear. A similar elastic role in the sphincter 1 cells appears plausible in light of the 4-fold change in pupil diameter — and, hence, sphincter muscle length — with dilation and constriction.

Why Titin would not similarly be required in dilator muscle is unclear. Alpha-catenins heterodimerize with beta-catenins at adherins junctions and they regulate actin filament assembly, coordinating junctional and cytoskeletal architectures Takeichi, The localization to sphincter cells of NeuN, neuronal activity-regulated pentraxin-2 NPTX2 , and synaptotagmin-1 SYT1 — all of which are enriched in the nervous system — suggests that gene expression in sphincter cells has a partially neuronal character.

The cell-type-specific transcriptomes derived from snRNAseq provide an opportunity to systematically assess signal transduction components that regulate iris smooth muscle function. Our transcriptome analysis shows that of all the genes coding for rhodopsin-like proteins in the mouse genome, Opn4 is the most highly expressed in the iris.

Interestingly, it is expressed in both dilator and constrictor muscles, and it is also expressed in iris PE cells Figure 2—figure supplement 1A. Transcripts for cryptochromes-1 and -2 Cry1 and Cry2 , flavin-based light sensors implicated in circadian rhythms, are also widely expressed in the iris Figure 2—figure supplement 1A. The distribution of adrenergic and muscarinic receptors implies that 1 dilator muscles receive adrenergic input principally via ADRA1A, 2 sphincter muscles receive cholinergic input principally via CHRM3 — consistent with the defect in pupil constriction observed in Chrm3 knockout mice Matsui et al.

The distribution of alpha-adrenergic receptor transcripts also implies that stroma 2 cells are sensitive to adrenergic ligands via ADRA1B receptors Figure 2—figure supplement 1D. Transcription factor ZIC1 localizes exclusively to the nuclei of these two epithelia Figure 4. A Dot plot as described in Figure 2A showing transcript abundances across a subset of iris cell types for the markers shown in B—D. B Flat mounts of untreated iris immunostained as indicated. C,D Cross sections of untreated iris immunostained as indicated.

Stroma 1 cells express multiple melanogenesis-related genes, including endothelin receptor beta Ednrb , microphthalmia-associated transcription factor Mitf , dopachrome tautomerase dopachroma delta-isomerase, Dct , tyrosinase-related protein-1 Tyrp1 , and tyrosinase Tyr , indicating that they serve as a secondary site of melanin synthesis in addition to the principal site of melanin synthesis and accumulation in the iris PE. A Dot plot as described in Figure 2A showing transcript abundances across a subset of iris cell types for the markers shown in B and C.

C Cross sections of untreated iris immunostained as indicated. The distribution of immune cells in the rodent iris — principally macrophages and dendritic cells — has been defined previously by immunocytochemistry of iris flat mounts McMenamin, ; McMenamin, The murine iris is uniformly tiled by irregularly shaped stellate cells, of which the majority are macrophages and the minority are dendritic cells Figure 5—figure supplement 1B.

One of the most striking properties of the iris is its mechanical flexibility. These large-scale changes in tissue structure suggested the possibility that iris dilation might also produce changes in gene expression. A Images of isolated eyes from mice with dilated pupils, constricted pupils, or no treatment. Red arrowheads mark the edge of the pupil. B Flat mounts of dilated and untreated irises showing the pattern of nerve fibers left and blood vessels right.

All images are at the same magnification and are oriented with the pupil to the right. C Scatter plots of single nucleus sn RNAseq read counts log 10 average normalized expression and R 2 values for pairwise comparisons of all transcripts for dilated vs. D Summary plot showing the difference in R 2 values between the dilated vs. As noted in connection with Figure 1E and Figure 1—figure supplements 1 and 2 , the transcriptomes of constricted and untreated irises are virtually indistinguishable across all cell types, presumably because the untreated pupil diameter is relatively small, so that pharmacologic constriction produces only a modest additional reduction in its diameter.

Therefore, comparisons between the transcriptomes of constricted and untreated irises can be used to estimate technical variability. Scatter plots of snRNAseq read counts for the constricted vs.

For seven of the ten iris cell types, there was more scatter in the dilated vs. The dilator muscle showed the largest transcriptome changes, with a reduction in R 2 from 0. The three cell types that showed little change in R 2 or a small trend in the opposite direction were endothelial cells, CB epithelial cells, and CB cells.

Examples of transcripts that increased or decreased with dilation defined as a log 2 -fold change greater than 0. The dot plots in Figure 6—figure supplement 1 also illustrate the near identity of constricted and untreated snRNAseq read counts.

To independently assess the changes seen with snRNAseq, we focused on three dilator muscle transcripts that were among the most strongly induced by dilation: Egr1 , a zinc-finger transcription factor that is an immediate-early response gene in diverse cell types; Slc26a4 , a broad-specificity anion exchanger that is expressed in multiple epithelia; and Tmem , a membrane protein of unknown function Figure 7A.

Similarly, ISH shows the accumulation of Slc26a4 and Tmem transcripts in dilator muscle with pupil dilation, whereas the abundance of Adrala transcripts remains unchanged Figure 7C , quantified in Figure 7—figure supplement 1.

Confirming the snRNAseq data, quantification of the ISH signals showed accumulation of Anks1b , Btg2 , Junb , and Pde10a transcripts in dilator muscle and Tmem transcripts in sphincter muscle with pupil dilation Figure 7—figure supplement 1. For each cell type, the symbols for the dilated iris blue are above the symbols for the untreated iris red. B Cross sections of dilated left and untreated right irises immunostained as indicated. C Cross sections of dilated left and untreated right irises hybridized with the indicated probes.

D Immunostaining of irises harvested 30 min left and 90 min right after the onset of dilation. Other cyclic nucleotide phosphodiesterase transcripts — including Pde1c , Pde3b , Pde4d , Pde7b — are also upregulated, suggesting that negative regulation of cyclic nucleotide signaling is a general feature of iris dilation Supplementary file 3.

In dilator muscle, the second most highly induced transcript is Btg2 , and, unlike Pde10a , Btg2 induction is specific to dilator muscle Supplementary file 3. BTG2 could play a regulatory role in the iris dilator muscle analogous to its role in cardiac muscle. Changes in iris structure at the macroscopic level must have counterparts at the cellular level.

Early electron micrographic studies visualized ultrastructural responses to dilation or constriction on the plasma membrane morphology of iris PE cells and the surface morphology of sphincter and dilator muscles Lim and Webber, a ; Lim and Webber, b ; Murata et al. Still largely unexplored are the effects of changes in iris structure on nuclear morphology. Current evidence suggests that changes in nuclear morphology alter chromatin organization, gene expression, and intracellular signals Kirby and Lammerding, ; Lele et al.

The identification of 1 transcription factors that mark defined iris cell type and 2 antibodies that can be used to immunolocalize those proteins in iris whole mounts presented an opportunity to visualize nuclear morphology in defined cell types in response to changes in iris structure Figure 8A. For untreated, dilated, and constricted irises, the length:width ratio was quantified for fluorescently stained nuclei in Z-stacked confocal images of iris flat mounts Figure 8B and.

As the 2D projection of a 3D ellipsoid exhibits a length:width ratio less than or equal to the actual 3D ratio, our analysis may underrepresent the elongation of some nuclei.

A Flat mounts of untreated, dilated, or constricted irises, immunostained for the indicated nuclear markers. Cell types are defined by the indicated marker. Each data point is an individual nucleus. The data for each condition in each plot are derived from three mice. The biological effects of these changes in nuclear morphology remain to be determined. Lineage tracing experiments in avian embryos provided the first evidence that some anterior ocular structures were derived, at least partially, from the neural crest via its contribution to the peri-ocular mesenchyme Johnston et al.

Neural crest lineage tracing in mice using Cre-LoxP to mark cells has produced conflicting results, with one laboratory describing no contribution of neural crest cell to the iris with a Wnt1-Cre driver Gage et al.

We have revisited this issue using SoxCre Matsuoka et al. SoxCre labels migratory neural crest cells and Wnt1-Cre2 labels multiple cells within the embryonic CNS, including premigratory neural crest Debbache et al. For each Cre driver line, the fraction of cells of a given type that expressed the H2B-GFP reporter was determined from iris flat mounts and is summarized in Figure 9A. As seen in the iris flat mount and cross-sectional images in Figure 9 and Figure 9—figure supplement 1 , co-labeling with H2B-GFP was widespread with the Wnt1-Cre2 driver but limited to stroma 1 and stroma 2 cells with the SoxCre driver.

The data are consistent with a model in which 1 a subset of migratory neural crest cells, defined by SoxCre expression, contribute exclusively to the stromal cell populations and provide all or nearly all of the stromal cell progenitors, and 2 migratory neural crest cells that express Wnt1-Cre2 but not SoxCre contribute the majority of the progenitors for iris PE, dilator, and sphincter 2 cells, but contribution minimally to the sphincter 1 population.

The vast majority of sphincter 1 cells presumably arise either from an unlabeled neural crest population or from local ocular progenitors. Iris cell types were identified by immunostaining for the nuclear-localized markers listed.

The intensities of GFP and the cell type markers vary among nuclei. A nucleus was considered to exhibit co-localization if both signals were present, regardless of intensity.

For sphincter muscle, the number of cells scored for each Cre driver ranged from to For each of the other cell types, the number of cells scored for each Cre driver ranged from to The work described here is presented as a resource for future investigations of the mammalian iris.

We have 1 used snRNAseq to define all of the major cell types in the mouse iris, which has led to the discovery of two types of stromal cells and two types of sphincter cells; 2 validated a series of molecular markers that can be used to visualize each of the major iris cell types; 3 identified transcriptome changes and distortions in nuclear morphology associated with iris dilation; and 4 clarified the neural crest contribution to the iris by showing that Wnt1-Cre -expressing progenitors contribute to nearly all iris cell types, whereas SoxCre -expressing progenitors contribute only to stromal cells.

As described more fully below, this work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation. Going forward, it will be of interest to obtain and compare similar snRNAseq data from the irises of other species, most especially from humans. The present work defines the molecular heterogeneity among different classes of iris smooth muscle cells.

Smooth muscle cells are present in multiple organs and they control a wide range of physiological functions, including vascular tone, airway resistance, gastrointestinal GI motility, and pupil diameter. The comprehensive determination of the abundances of transcripts for all known receptors, channels, and signaling components for sphincter 1, sphincter 2, and dilator muscles constrains the possible ligand-receptor systems that control contraction and relaxation in each of these muscle types.

The iris PE cell transcriptome provides a foundation for strategies to genetically engineer these cells and monitor their state of trans-differentiation in cell culture. Over the past 30 years, the iris PE has been studied as a potential source of cells for autologous transplantation to replace dying or dysfunctional retinal pigment epithelial RPE cells in individuals with age-related macular degeneration. These studies were motivated by the surgical accessibility of human iris PE cells, which can be harvested from a small iridectomy sample and expanded in culture, and by earlier work showing that the iris PE in non-mammalian vertebrates can transdifferentiate into other ocular cell types Hu et al.

In three clinical trials of sub-retinal transplantation of iris PE in age-related macular degeneration patients, there have been minimal complications but also minimal effect on the clinical course of the disease Lappas et al. Why do I want to? AI versus NetHack! At least on April Fool's Day. We even got a screenshot. The abstract is available online.

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