Investigative Ophthalmology & Visual Science Cover Image for Volume 66, Issue 5
May 2025
Volume 66, Issue 5
Open Access
Biochemistry and Molecular Biology  |   May 2025
Distinct Transcriptomic Profiles of Cultured Anterior and Posterior Populations of Human Infant Scleral Fibroblasts: Including Dopamine Receptors
Author Affiliations & Notes
  • Jiali Guan
    Center for Biomedical Digital Science, State Key Laboratory of Respiratory Disease, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
    University of Chinese Academy of Sciences, Beijing, China
  • Guangliang Hong
    Guangzhou National Laboratory, Guangzhou, China
  • Zhong Liu
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
  • Yingfeng Zheng
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
    Research Unit of Ocular Development and Regeneration, Chinese Academy of Medical Sciences, Beijing, China
  • Jiangping He
    Guangzhou National Laboratory, Guangzhou, China
    Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
  • Dajiang Qin
    Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
    GuangDong Engineering Technology Research Center of Biological Targeting Diagnosis, Therapy and Rehabilitation, The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
    Guangdong Engineering Research Center of Early Clinical Trials of Biotechnology Drugs, The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
    Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China
    Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, China
  • He Li
    Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
  • Correspondence: He Li, Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, 621 Gangwan Rd., Guangzhou, Guangdong 510700, China; [email protected]
  • Footnotes
     JG, GH, and ZL contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science May 2025, Vol.66, 29. doi:https://doi.org/10.1167/iovs.66.5.29
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      Jiali Guan, Guangliang Hong, Zhong Liu, Yingfeng Zheng, Jiangping He, Dajiang Qin, He Li; Distinct Transcriptomic Profiles of Cultured Anterior and Posterior Populations of Human Infant Scleral Fibroblasts: Including Dopamine Receptors. Invest. Ophthalmol. Vis. Sci. 2025;66(5):29. https://doi.org/10.1167/iovs.66.5.29.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The transcriptomic profiling of scleral fibroblasts remains largely unexplored. To elucidate their heterogeneity, we performed single-cell RNA sequencing (scRNA-seq) on primary infant scleral fibroblasts.

Methods: Primary scleral fibroblasts, cultured at passage 2 from the anterior, equatorial, and posterior regions of infant sclera (3 months to 2 years of age) were subjected to scRNA-seq using the 10x Genomics platform. In-depth analysis revealed distinct transcriptomic profiles between anterior and posterior scleral fibroblasts, including differential expression of dopamine (DA) receptors, which was subsequently validated both in vitro and in situ. Furthermore, the regulatory role of DA in scleral remodeling was assessed using an in vitro collagen gel contraction assay, and the involvement of DA receptor activity and expression in this process was further explored through pharmacological manipulation and gene silencing approaches.

Results: Infant scleral fibroblasts have anterior and posterior subpopulations, each exhibiting distinct transcriptomic profiles. Anterior scleral fibroblasts show increased expression of D1-like receptors, but posterior scleral fibroblasts exhibit elevated expression of D2-like receptors. D1-like receptor activity enhances the inhibitory effect of DA on scleral remodeling in anterior sclera, whereas D2-like receptor activity, particularly that of DRD2 in the posterior sclera, counteracts this effect. Gene silencing of DRD4 significantly enhances DA-mediated inhibition of scleral remodeling in the posterior sclera.

Conclusions: To our knowledge, this study presents the first comprehensive transcriptomic profiling of infant scleral fibroblasts, revealing their heterogeneity. The investigation of the regulatory role of DA receptor activity and expression in DA-mediated inhibition of scleral fibroblast contraction provides new insights into how DA signaling modulates scleral remodeling.

Being the outermost shell of the eye, the sclera supports, protects, and defines the ocular tissue. Scleral fibroblasts are the primary resident cells of the sclera, continuously engaged in matrix remodeling in response to local mechanical stimuli and a cascade of biochemical signals originating from the retina, ultimately driven by visual input. This dynamic process governs tissue-level scleral remodeling and plays a crucial role in determining the final size of the eye.1 
Emerging studies have described fibroblast heterogeneity within individual tissues, where they govern distinct aspects of tissue homeostasis and disease.2 In the sclera, progenitor cells with chondrogenic potential have been identified in the posterior region3 and have been associated with myopia development, where posterior scleral remodeling accompanies abnormal axial elongation of the eye.4 Furthermore, two distinct populations of scleral fibroblasts, which undergo compositional and gene expression changes following myopia induction, have been identified in mice.5 Additionally, a differential anteroposterior pattern of gene expression has been characterized in human infant scleral fibroblasts.6 Meanwhile, variations in cell density, nuclear morphology, and responses to mechanical strain have been observed in human adult scleral fibroblasts isolated from the peripheral and peripapillary regions.79 Collectively, this evidence suggests that scleral fibroblasts exhibit heterogeneity and that cells from different regions of the sclera may perform distinct functions in regulating scleral remodeling. Therefore, elucidating the intra-tissue heterogeneity of scleral fibroblasts may provide deeper insights into scleral remodeling, ultimately contributing to the development of therapeutics for closely associated diseases, such as myopia. 
Dopamine (DA), a key neurotransmitter involved in retinal development, visual signaling, and refractive development, is among the most researched retinal signaling molecules and is recognized as a “STOP” signal for refractive eye growth.10 DA exerts its physiological effects through five G protein–coupled receptor subtypes, classified into D1-like receptors (DRD1 and DRD5) and D2-like receptors (DRD2, DRD3, and DRD4). Although species-specific differences exist in the involvement of D1-like and D2-like receptor pathways, retinal DA receptors play a critical role in regulating refractive eye growth under myopia-inducing conditions. For example, DRD2 activity promotes myopia progression,11,12 whereas DRD1 activity is essential for controlling ocular growth and myopia development in mice.13 Despite the significant role of DA in eye growth regulation, DA signaling has not been directly linked to the sclera, and there is no evidence that DA modulates scleral remodeling. However, among other retina-generated signaling molecules that regulate axial growth, retinoic acid,14,15 adenosine,16 insulin,17 and TGF-β18 all appear to significantly influence scleral remodeling. Moreover, in fibroblasts from other tissues, such as the lungs,19,20 dermis,21 and synovium,22 which express DA receptors, receptor activity plays a critical role in regulating the transition from the profibrotic to fibrosis-resolving phenotypes, as well as in fibroblast-to-myofibroblast transformation and cell migration, suggesting that DA signaling contributes to the modulation of fibroblast-mediated tissue remodeling. Furthermore, a previous study has identified DRD1 expression in human uveoscleral tissue,23 indicating the presence of DA receptors in scleral fibroblasts. More significantly, this finding raises the possibility of direct DA-mediated regulation of scleral remodeling, warranting further investigation. 
In this study, we performed single-cell RNA sequencing (scRNA-seq) analysis on primary human infant scleral fibroblasts cultured from the anterior, equatorial, and posterior regions of scleral tissues donated by individuals 3 months to 2 years old to elucidate their heterogeneity. Subsequently, we identified the differential expression profiles of DA receptors in these cells and verified them at both the gene and protein levels, as well as through in situ hybridization (ISH) in human scleral tissue. Moreover, using a collagen gel contraction model that simulates fibroblast-mediated extracellular matrix (ECM) remodeling in vitro,2426 we demonstrated the direct inhibitory effect of DA on scleral remodeling and explored how DA receptor activation regulates this inhibition, employing specific pharmacological antagonists and agonists, as well as gene silencing methods. The results revealed differences in transcriptomic profiling between anterior and posterior infant scleral fibroblasts and provide new insights into the regulation of scleral remodeling by DA signaling. 
Methods
Ethical Statement
This study adhered to the tenets of the Declaration of Helsinki. Four donated human sclera samples from postmortem children of Chinese ethnicity, ages 3 months (male, donor 14), 4 months (female, donor 12), 1 year (male, donor 13), and 2 years (male, donor 9), and confirmed to be without ocular abnormalities or vision-related diseases, were collected at Zhongshan Ophthalmic Center (ZOC), Sun Yat-Sen University. Human scleral tissue sections from a postmortem donor (female, 76 years old) were also collected at ZOC. The study received approval from the Ethics Committees of both ZOC (2021KYPJ064) and the Fifth Affiliated Hospital of Guangzhou Medical University (KY01-2021-03-03). 
Primary Human Scleral Fibroblast Culture
The preparation of scleral tissues from donated eye samples was carried out as described previously.27 The sclera tissues were maintained at 4°C and processed within 24 hours of receipt. They were incubated in sterile PBS containing 1000 IU/mL Gibco penicillin–streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) for 20 minutes and then dissected into anterior (3–5 mm wide from the limbus), equatorial (the area between the anterior and posterior regions), and posterior (3–5 mm wide from the posterior pole) regions. Each region was cut into small pieces (approximately 2 mm × 2 mm), digested in 0.01% collagenase D (Roche, Basel, Switzerland) in Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher Scientific) for 10 minutes, and subsequently cultured in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific) with 10% fetal bovine serum (Thermo Fisher Scientific) at 37°C in a 5% CO2 incubator. Scleral fibroblasts grew out of the scleral explants within 5 to 7 days. The cells were cultured under the same conditions until passage 2 and collected for scRNA-seq. The remaining cells continued to grow and were cryopreserved at passages 3 to 4 and stored in liquid nitrogen. The cells used for in vitro experiments were no older than passage 8. The purity of the primary scleral fibroblasts was validated using gene expression signatures (Supplementary Fig. S1).28,29 
Single-Cell RNA Sequencing
Primary scleral fibroblasts at passage 2 were harvested (10,000 cells per sample) and processed following the 10x Genomics protocol using the Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics, Pleasanton, CA, USA) and the Chromium Chip G Single Cell Kit (10x Genomics). Cells and reagents were prepared and loaded onto the chip and into the Chromium Controller for droplet generation. Reverse transcription was performed in the droplets, and cDNA was recovered through demulsification and bead purification. The pre-amplified cDNA was used for library preparation, multiplexed, and sequenced on a HiSeq2500 (Illumina, San Diego, CA, USA). 
Data Processing and Analysis
The raw reads were trimmed with TrimGalore30 (https://github.com/FelixKrueger/TrimGalore), then the cleaned reads were aligned to human genomes by STARsolo31 (https://github.com/alexdobin/STAR/blob/master/docs/STARsolo.md). Gene quantification was performed using the scTE pipeline (https://github.com/JiekaiLab/scTE).32 The count matrix was normalized using the NormalizeData function of Seurat (https://satijalab.org/seurat/),33 and the top 2000 most highly variable genes were used for principal component analysis; the first 15 principal components were used. The batch effect was corrected by Seurat. The differential expressed genes were obtained using the rank_genes_groups function of Scanpy (https://github.com/scverse/scanpy).34 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the top marker genes in the anterior, equatorial, and posterior cells were performed using Metascape (https://metascape.org/gp/index.html).35 The GO analysis of the scleral cell clusters was performed using clusterProfiler (https://github.com/YuLab-SMU/clusterProfiler/blob/devel/NEWS.md).36 
Data Availability
All sequencing data generated in this study are available at Genome Sequence Archive37 in National Genomics Data Center,38 China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/gsa-human) under accession number HRA006905. 
Collagen Gel Contraction Assay
The assay was performed as previously described.2426 In brief, scleral fibroblasts were seeded in a 3-mg/mL collagen type-I matrix (Biocoat, Horsham, PA, USA) at a concentration of 1 × 105 cells/mL. Subsequently, the collagen mixture was pipetted into a well of a 35-mm glass-bottom dish (Cellvis, Mountain View, CA, USA) and allowed to set at 37°C with 5% CO2 for 20 minutes. The gels were detached from the edge of the well, and 2 mL of DMEM was added. Gel contraction was monitored daily for 7 days by digital photography. Gel areas were measured using ImageJ (National Institutes of Health, Bethesda, MD, USA),39 and the contraction was plotted as a percentage of gel area normalized to original area (day 0 measurement). In the contraction assay, dopamine hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). The pharmacological agonists and antagonists of DA receptors used included SCH23390 hydrochloride, A68930 hydrochloride, levosulpiride, bromocriptine mesylate, L-741626, PNU-95666E, and L-745870 from MedChemExpress (Monmouth Junction, NJ, USA), and A-412997 from APExBIO Technology (Houston, TX, USA). 
Quantitative Real-Time PCR
Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany), and reverse transcription to cDNA was performed using HiScript III RT SuperMix (Vazyme, Nanjing, China), both following the manufacturers’ instructions. Quantitative real-time PCR (qRT-PCR) was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). The following program was used: 95°C for 5 minutes, followed by 39 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Validation was performed using melting curve analysis. Relative expression levels were normalized to GAPDH and calculated using the 2−∆∆Ct method.40 The primer sequences used are listed in Table 1
Table 1.
 
Oligonucleotides Used for qRT-PCR
Table 1.
 
Oligonucleotides Used for qRT-PCR
In Situ Hybridization
ISH was performed using the RNASweAMI in situ hybridization 3,3′-diaminobenzidine (DAB) detection kit (GF001; Servicebio, Hubei, China) according to the manufacturer's instructions. Briefly, after digestion and prehybridization, human scleral tissue sections were incubated individually with specific probes targeting DRD1, DRD2, DRD3, DRD4, or DRD5 at 40°C for 16 hours. The sequences of the probes used are provided in Table 2. The sections were then incubated with digoxin (DIG)-labeled probes, followed by treatment with a horseradish peroxidase (HRP)-conjugated anti-DIG antibody. The signal was visualized using streptavidin–HRP and DAB staining. For imaging, tissue sections were examined using the Tissue FAXS Plus ST system (TissueGnostics, Vienna, Austria) with bright-field illumination and a 20× objective. An automated scanning stage was used to acquire multiple sub-images, which were subsequently stitched using the Grid Stitching plugin in Fiji 2.3.0. ISH signal quantification was performed using the Analyze Particles plugin in Fiji, with 10 regions of interest within each target area acquired and analyzed for signal intensity.41,42 
Table 2.
 
Sequences of Probes Used for ISH
Table 2.
 
Sequences of Probes Used for ISH
Western Blot
The cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) with Roche cOmplete Proteinase Inhibitor Cocktail, followed with a centrifugation at 11,000 rpm at 4°C for 15 minutes. Mini-Protean Tetra Cell Systems (Bio-Rad, Hercules, CA, USA) were used to perform western blot. The gel electrophoresis was performed with 4% to 12% SurePAGE Bis-Tris gel (GenScript, Piscataway, NJ, USA) and Tris-MOPS-SDS Running Buffer (GenScript) at 200 V for 40 minutes. The blotting was performed with a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA) with transfer buffer (GenScript) on ice at 300 mA for 1.5 hours. The membrane was then blocked with Tris-buffered saline with Tween 20 (TBST) buffer containing 5% milk for 1 hour at room temperature and subsequently incubated with the primary antibody (concentrations ranged from 1:1000 to 1:10,000) overnight. The secondary antibody was used at 1:5000 for 2 hours at room temperature. Hypersensitive ECL solution (Zen-Bioscience, Chengdu, China) was used for protein detection on FluorChem E (Bio-Techne, Minneapolis, MN, USA). The results were analyzed using ImageJ software and then normalized against the results of the loading control (GAPDH). The antibodies used were as follows: Dopamine Receptor D1 Rabbit mAb (381747; Zen-Bioscience), DRD2 polyclonal antibody (55084-1-AP; Proteintech, Rosemont, IL, USA), Dopamine Receptor D3 Rabbit mAb (382983; Zen-Bioscience), DRD4 Rabbit polyclonal antibody (28094-1-AP; Proteintech), Dopamine Receptor D5 Rabbit pAb (820522; Zen-Bioscience), GAPDH (7E4) Mouse mAb (200306-7E4; Zen-Bioscience), Beta Tubulin Polyclonal antibody (10094-1-AP; Proteintech), Goat anti-Rabbit IgG(H&L) (HRP conjugate) (511203; Zen-Bioscience), and Goat anti-Mouse IgG (H&L) (HRP conjugate) (511103; Zen-Bioscience). 
Small Interfering RNA Transfection
The small interfering RNA (siRNA) transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) following the manufacturer's instructions. Briefly, scleral fibroblasts were seeded in a six-well plate at a density of 0.5 × 105 cells per well. After 16 hours of incubation, a mixture of siRNA (Tsingke Biotech, Beijing, China) and RNAiMAX was prepared in Gibco Opti-MEM and added dropwise to the cells. The cells were then harvested 48 hours post-transfection. The sequences of siRNA used were as follows: DRD2, (5′–3′) CCAAGAUCUUUGAGAUCCA and (3′–5′) UGGAUCUCAAAGAUCUUGG; DRD4, (5′–3′) GCCCUUCUUCGUGGUGCACAU and (3′–5′) AUGUGCACCACGAAGAAGGGC. 
Statistical Analysis
Data are presented as mean ± standard error of the mean (SEM). Statistical differences were determined using one-way or two-way ANOVA, or a two-tailed Student's t-test, depending on the experimental conditions. Detailed statistical analyses for each experiment are provided in the figure legends. Differences between groups were considered significant at P < 0.05. All statistical analyses were performed using Prism 9 (GraphPad, Boston, MA, USA). 
Results
Human Infant Scleral Fibroblasts Have Distinct Anterior and Posterior Subpopulations
Scleral tissues were obtained from four human infant donors ages 3 months, 4 months, 1 year, and 2 years. The tissues were dissected into the anterior (3–5 mm from the limbus), equatorial (the area between the anterior and posterior regions), and posterior (3–5 mm from the posterior pole) regions. Primary scleral fibroblasts were expended from each specified region under the same condition until passage 2 and then collected for scRNA-seq. Each sample was comprised of 10,000 cells and was processed using the 10x Genomics platform (Fig. 1a). 
Figure 1.
 
Human infant scleral fibroblasts have distinct anterior and posterior subpopulations. (a) Schematic illustration of the experimental design. (b) UMAP clustering of single-cell transcriptomic profiles from primary anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors (12 samples in total) prior to batch effect correction. The color coding represents different cell samples; numbers 9 to 14 indicate individual donors; and the letters A, E, and P denote anterior, equatorial, and posterior scleral fibroblasts, respectively. (c) Spatial grouping of cells by anatomical origin before batch effect correction. (d) UMAP visualization after batch effect correction, incorporating the spatial information of the samples. (eg) The top 15 genes selectively expressed in anterior, equatorial, and posterior scleral fibroblasts, respectively. (hj) The top five biological functions enriched in anterior, equatorial, and posterior scleral fibroblasts, analyzed using the top 100 genes most expressed in each group.
Figure 1.
 
Human infant scleral fibroblasts have distinct anterior and posterior subpopulations. (a) Schematic illustration of the experimental design. (b) UMAP clustering of single-cell transcriptomic profiles from primary anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors (12 samples in total) prior to batch effect correction. The color coding represents different cell samples; numbers 9 to 14 indicate individual donors; and the letters A, E, and P denote anterior, equatorial, and posterior scleral fibroblasts, respectively. (c) Spatial grouping of cells by anatomical origin before batch effect correction. (d) UMAP visualization after batch effect correction, incorporating the spatial information of the samples. (eg) The top 15 genes selectively expressed in anterior, equatorial, and posterior scleral fibroblasts, respectively. (hj) The top five biological functions enriched in anterior, equatorial, and posterior scleral fibroblasts, analyzed using the top 100 genes most expressed in each group.
After quality control, 93,125 single-cell transcriptomes were retained for downstream analysis, including 31,388 anterior cells, 31,748 equatorial cells, and 29,989 posterior cells. The datasets were integrated using the Seurat package. The distribution of the transcriptomic profiles of scleral fibroblasts from all 12 samples was visualized using Uniform Manifold Approximation and Projection (UMAP) prior to batch effect correction (Figs. 1b, 1c). This visualization revealed common profiles shared between anterior and equatorial cells, as well as between equatorial and posterior cells, but not between anterior and posterior cells. Subsequently, we performed batch effect correction on the dataset based on the spatial information of the samples and visualized the results using UMAP (Fig. 1d). The result revealed that infant scleral fibroblasts consist of two distinct cell populations corresponding to anterior and posterior fibroblasts, with each population also containing a subset of equatorial fibroblasts. 
Furthermore, to thoroughly characterize the gene expression profiles of anterior, equatorial, and posterior scleral fibroblasts, we analyzed the top genes selectively expressed by each cell group, highlighting the top 15 for each in Figures 1e to 1g (full gene expression results are provided in Supplementary File S1). In anterior scleral fibroblasts, the top enriched genes are primarily involved in the regulation of the actin cytoskeleton, including genes such as TMSB4X (thymosin beta 4 X-linked), ACTB (actin beta), TPM2 (tropomyosin 2), RHOA (Ras homolog family member A), and RAC1 (Rac family small GTPase 1), as well as ECM remodeling, with genes such as COL1A2 (collagen type I alpha 2 chain), FN1 (fibronectin 1), and TIMP1 (TIMP metallopeptidase inhibitor 1). Additionally, IGFBP3 (insulin-like growth factor binding protein 3), TXN (thioredoxin), and CALU (calumenin) were identified, suggesting active growth and metabolic activities in these cells (Fig. 1e). 
In equatorial scleral fibroblasts, the top expressed genes include actin-regulatory genes such as ARPC5 (actin-related protein 2/3 complex subunit 5) and ENAH (ENAH actin regulator), as well as matrix protein genes such as MGP (matrix Gla protein), which is known to be highly expressed in the sclera, and LAMC1 (laminin subunit gamma 1). Additionally, several enzyme-encoding genes were identified, including GUK1 (guanylate kinase 1), PRDX6 (peroxiredoxin 6), CAPN2 (calpain 2), and PTGIS (prostaglandin I2 synthase), which are involved in various physiological processes within the cells, such as signal transduction, nucleotide metabolism, and cell proliferation (Fig. 1f). 
In posterior scleral fibroblasts, in addition to cytoskeleton-related genes such as TUBA1B (tubulin alpha 1b) and TPM1 (tropomyosin 1), several transcription factors were found to be enriched. These include HIF1A (hypoxia-inducible factor 1 subunit alpha), which regulates the transcriptional response to hypoxia and may play a crucial role in myogenesis,5,43 as well as NR2F2 (nuclear receptor subfamily 2 group F member 2) and SIX1 (SIX homeobox 1). Also, proteinases and kinases that play important regulatory roles in signal transduction, glycolysis, cell proliferation, differentiation, and survival were enriched, including PRSS23 (serine protease 23), CAPN12 (calpain 12), PFKP (phosphofructokinase, platelet), MAP2K3 (mitogen-activated protein kinase kinase 3), and SPHK1 (sphingosine kinase 1). Furthermore, we identified TNFRSF12A (TNF receptor superfamily member 12A) and SMAD7 (SMAD family member 7), suggesting potential activation of TNF and TGF-β signaling pathways in these cells (Fig. 1g). 
Moreover, we analyzed the biological functions enriched in anterior, equatorial, and posterior scleral fibroblasts using the top 100 most-expressed genes in each cell group via Metascape,35 and the top five enriched GO and KEGG pathways for each cell group are shown in Figures 1h to 1j. In anterior and equatorial scleral cells, both “VEGFA–VEGFR2 signaling” and “actin cytoskeleton organization” were enriched. Additional pathways enriched in anterior scleral cells included “Parkinson's disease,” “signaling by receptor tyrosine kinases,” and “Wnt signaling,” whereas, in equatorial scleral cells, “RNA metabolism,” “muscle structure development,” and “diseases of signal transduction by growth factor receptors” were enriched. In posterior scleral cells, metabolism-related pathways, such as “aerobic respiration and respiratory electron transport,” “purine ribonucleoside triphosphate metabolism,” and “aerobic glycolysis,” as well as developmental pathways, including “axon guidance” and “Eph/ephrin signaling,” were also enriched. 
Transcriptomic Profiling of Anterior and Posterior Scleral Fibroblast Subpopulations
Subsequently, graph-based Leiden clustering was employed to delineate the transcriptomic profiles in these scleral fibroblasts, revealing 11 distinct clusters characterized by divergent gene expression profiles (Fig. 2a). The anterior subpopulation included clusters 1, 2, 3, 5, 9, and 11, and the posterior subpopulation included clusters 0, 4, 6, 8, and 10. We analyzed the genes selectively expressed in each cluster (full gene expression results are provided in Supplementary File S2), and the three most-expressed genes for each cluster of the anterior and posterior subpopulations are shown in Figures 2b and 2c. In the anterior subpopulation, the top expressed genes were strongly associated with ECM structure and organization, as observed in cluster 1, which expressed KRT19 (keratin 19), POSTN (periostin), and TAGLN (transgelin). Cluster 2 expressed FGF7 (fibroblast growth factor 7), CTSK (cathepsin K), and FBLN5 (fibulin 5), and a response to growth factor stimulus was demonstrated by cluster 3, which expressed IGFBP5 (insulin-like growth factor binding protein 5), CLEC3B (C-type lectin domain family 3 member B), and PTGIS (prostaglandin I2 synthase). DNA replication was seen in Cluster 5, which expressed TYMS (thymidylate synthetase), UBE2T (ubiquitin conjugating enzyme E2 T), and MAD2L1 (mitotic arrest deficient 2 like 1) (Fig. 2b). 
Figure 2.
 
Transcriptomic profiling of anterior and posterior scleral fibroblast subpopulations. (a) Leiden community-detection clustering identified 11 cell clusters in the anterior and posterior cell populations. (b, c) The top three genes selectively expressed in the clusters of anterior and posterior subpopulations, respectively. (d) Dot plot illustrating the main gene functions expressed in each cell cluster of the anterior and posterior cell populations.
Figure 2.
 
Transcriptomic profiling of anterior and posterior scleral fibroblast subpopulations. (a) Leiden community-detection clustering identified 11 cell clusters in the anterior and posterior cell populations. (b, c) The top three genes selectively expressed in the clusters of anterior and posterior subpopulations, respectively. (d) Dot plot illustrating the main gene functions expressed in each cell cluster of the anterior and posterior cell populations.
In the posterior subpopulation, BDNF (brain-derived neurotrophic factor), CEMIP (cell migration inducing hyaluronidase 1), and THBS1 (thrombospondin 1), selectively expressed by cluster 0, were associated with anti-apoptosis and the maintenance of homeostasis in scleral ECM remodeling.44 AKR1C1 (aldo-keto reductase family 1 member C1), AKR1C2 (aldo-keto reductase family 1 member C2), and MGP in cluster 4 were related to steroid hormone metabolism and calcification regulation. PCLAF (PCNA clamp associated factor), CDKN3 (cyclin-dependent kinase inhibitor 3), and BIRC5 (baculoviral IAP repeat containing 5) in cluster 6 were involved in cell-cycle regulation, whereas HELLS (helicase, lymphoid specific), CLSPN (claspin), and GINS2 (GINS complex subunit 2) in cluster 7 were associated with DNA replication. PPP1R14A (protein phosphatase 1 regulatory inhibitor subunit 14A), MMP1 (matrix metallopeptidase 1), and YIF1B (Yip1 interacting factor homolog B, membrane trafficking protein) in cluster 8 were linked to cytoskeletal dynamics and vesicular trafficking, and GOLGA8B (golgin A8 family member B), NPIPB5 (nuclear pore complex interacting protein family member B5), and MEG3 (maternally expressed 3) in cluster 10 were implicated in nucleocytoplasmic transport (Fig. 2c). 
Furthermore, functional annotations of gene sets expressed in each cluster were analyzed to elucidate their biological roles and were visualized with dot plots (Fig. 2d, Supplementary File S3). Consistent with previous analyses, clusters in the anterior population were enriched with functions related to active actin cytoskeletal organization, oxidative phosphorylation, corticosteroid response, glycosphingolipid metabolism, ossification, and proteolysis. Potentially activated signaling pathways included TGF-β and Wnt signaling. In contrast, clusters in the posterior population showed a strong association with responses to TNF and TGF-β stimuli, regulation of organ morphogenesis, ECM organization, and oxygen level responses. Additionally, collagen metabolic processes, bone morphogenetic protein (BMP) signaling, and chondrocyte proliferation were observed, reflecting the chondrogenic potential of the human posterior sclera.45 Both anterior and posterior populations shared functional annotations linked to cell-cycle regulation and DNA replication, indicating a commonality in active cell growth and proliferation. 
Differential DA Receptor Expression in Anterior and Posterior Scleral Fibroblasts
scRNA-seq analysis revealed distinct expression patterns of DA receptors in anterior and posterior scleral fibroblasts. The posterior subpopulation exhibited elevated expression of D2-like receptors and reduced expression of D1-like receptors compared to the anterior subpopulation, which showed higher expression of D1-like receptors and lower expression of D2-like receptors (Fig. 3k). Subsequently, we validated the gene expression profiles of DA receptors in anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors using qRT-PCR. Our findings confirmed the expression of all five DA receptors (DRD1 to DRD5) across these fibroblast populations. Specifically, posterior scleral fibroblasts exhibited lower mRNA levels of D1-like receptors and higher levels of D2-like receptors, particularly DRD2 and DRD4. Conversely, anterior scleral fibroblasts showed higher mRNA expression of D1-like receptors and lower expression of D2-like receptors (Figs. 3a–e). These results are consistent with the scRNA-seq analysis, in which the absence of DRD3 may be attributed to its low expression levels. Moreover, protein expression of DA receptors in these cells was confirmed by western blot. Whereas anterior cells expressed significantly higher levels of D1-like receptors compared to posterior cells—consistent with the gene expression profile—the protein expression levels of D2-like receptors did not show a statistically significant difference between anterior and posterior cells (Figs. 3f–j). In addition, the gene expression of DA receptors was further verified using ISH in human scleral tissue sections from an adult donor (Figs. 3l–p). The results demonstrated enhanced expression of D1-like receptors in the anterior sclera and increased expression of D2-like receptors in the posterior sclera, consistent with the scRNA-seq results and qRT-PCR validation. These findings also suggest that the differential gene expression of DA receptors in the sclera persists from infancy to adulthood. 
Figure 3.
 
Differential DA receptor expression in anterior and posterior scleral fibroblasts. (ae) The mRNA levels of DA receptors in all scleral fibroblast samples, validated by qRT-PCR. Relative expression levels were normalized to those of anterior cells. One experiment was conducted for each biological sample (total n = 4). *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). (fj) Protein expression levels of DA receptors in all scleral fibroblast samples, quantified by western blot. The blot image below represents one of the experimental repeats. Protein band quantification was first normalized to GAPDH and then to the value of anterior fibroblasts. Two experiments were performed per biological sample (total n = 8; for the DRD5 blot, one experiment was performed, n = 4). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (k) Gene expression profiles of DA receptors in anterior and posterior cell populations, as revealed by scRNA-seq analysis. (lp) ISH results for DRD1 to DRD5 in human scleral tissue sections from an adult donor. The intensity of the brown color indicates the expression level of each DA receptor. The right panel images show magnified views of the boxed areas in the left panel. Scale bars: 200 µm (left panel); 50 µm (right panel). Images of full tissue sections are shown below each ISH panel, with the regions corresponding to the anterior and posterior sclera marked in blue and pink, respectively. Signal intensity was quantified from 10 regions of interest (ROIs) per image. ****P < 0.0001 (unpaired t-test).
Figure 3.
 
Differential DA receptor expression in anterior and posterior scleral fibroblasts. (ae) The mRNA levels of DA receptors in all scleral fibroblast samples, validated by qRT-PCR. Relative expression levels were normalized to those of anterior cells. One experiment was conducted for each biological sample (total n = 4). *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). (fj) Protein expression levels of DA receptors in all scleral fibroblast samples, quantified by western blot. The blot image below represents one of the experimental repeats. Protein band quantification was first normalized to GAPDH and then to the value of anterior fibroblasts. Two experiments were performed per biological sample (total n = 8; for the DRD5 blot, one experiment was performed, n = 4). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (k) Gene expression profiles of DA receptors in anterior and posterior cell populations, as revealed by scRNA-seq analysis. (lp) ISH results for DRD1 to DRD5 in human scleral tissue sections from an adult donor. The intensity of the brown color indicates the expression level of each DA receptor. The right panel images show magnified views of the boxed areas in the left panel. Scale bars: 200 µm (left panel); 50 µm (right panel). Images of full tissue sections are shown below each ISH panel, with the regions corresponding to the anterior and posterior sclera marked in blue and pink, respectively. Signal intensity was quantified from 10 regions of interest (ROIs) per image. ****P < 0.0001 (unpaired t-test).
DA Inhibits Scleral Fibroblast-Mediated ECM Remodeling, Particularly in Posterior Cells
The expression of DA receptors in scleral fibroblasts suggests a potential regulatory role of DA signaling in scleral remodeling. To investigate this, we applied an in vitro collagen gel contraction assay24,46 to assess the ECM remodeling activity of these cells by measuring the gel contraction rate (Fig. 4a). Additionally, DA was added to the culture medium to evaluate its regulatory effect on ECM remodeling in these cells. Under normal physiological conditions, DA concentrations in the eye (measured in the vitreous) change dynamically with light, increasing in light-adapted eyes and decreasing in dark-adapted eyes, following a rising gradient from the vitreous to the layer of dopaminergic cells in the retina.47 Although DA concentrations in the human sclera have not been reported, its levels in the vitreous body and aqueous humor of the rabbit eye have been measured at approximately 10 µM and 60 µM, respectively.48 Therefore, we estimated that DA concentrations in the human eye would fall within a similar range. Accordingly, we initially tested a DA concentration range of 1 to 100 µM in the collagen contraction assay using one group of infant anterior, equatorial, and posterior scleral fibroblasts (HSF13; age, 1 year old) (Figs. 4b–d). The results showed that 1-µM and 10-µM DA did not affect cell contraction activity during the 7-day assay, whereas 50-µM DA noticeably inhibited cell contraction, and 100-µM DA completely suppressed it. Notably, 50-µM DA induced greater inhibition in posterior cells compared to anterior and equatorial cells. To confirm this observation, we repeated the collagen gel contraction assay using anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors (Fig. 4e). The results demonstrate that DA can directly suppress ECM remodeling mediated by scleral fibroblasts, with significantly greater inhibition observed in posterior cells (Figs. 4f, 4g). This suggests that suppression of scleral remodeling, particularly in the posterior sclera, may be one of the mechanisms through which DA regulates eye growth. Furthermore, the anterior cells exhibited slightly higher contractile activity than the posterior cells, consistent with predictions from the previous scRNA-seq analysis. 
Figure 4.
 
DA inhibits scleral fibroblast-mediated ECM remodeling, especially in posterior cells. (a) Schematic illustration of the in vitro collagen gel contraction assay. (bd) Seven-day contraction curves of anterior, equatorial, and posterior infant scleral fibroblasts (HSF13) treated with 1-µM, 10-µM, 50-µM, and 100-µM DA. One experiment with triplicate wells was performed. (e) Seven-day contraction curves of anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors treated with 50-µM DA. One experiment with triplicate wells was performed for each biological sample (n = 12 total). Statistical differences between sample groups: F(5, 66) = 13.03, ****P < 0.0001 (two-way ANOVA). (f) Comparison of contraction percentages on day 7. *P < 0.05, ****P < 0.0001 (two-way ANOVA). (g) Percentage of contraction reduced by DA on day 7, calculated as (% control – % DA)/% control × 100. **P < 0.01, ***P < 0.001 (one-way ANOVA).
Figure 4.
 
DA inhibits scleral fibroblast-mediated ECM remodeling, especially in posterior cells. (a) Schematic illustration of the in vitro collagen gel contraction assay. (bd) Seven-day contraction curves of anterior, equatorial, and posterior infant scleral fibroblasts (HSF13) treated with 1-µM, 10-µM, 50-µM, and 100-µM DA. One experiment with triplicate wells was performed. (e) Seven-day contraction curves of anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors treated with 50-µM DA. One experiment with triplicate wells was performed for each biological sample (n = 12 total). Statistical differences between sample groups: F(5, 66) = 13.03, ****P < 0.0001 (two-way ANOVA). (f) Comparison of contraction percentages on day 7. *P < 0.05, ****P < 0.0001 (two-way ANOVA). (g) Percentage of contraction reduced by DA on day 7, calculated as (% control – % DA)/% control × 100. **P < 0.01, ***P < 0.001 (one-way ANOVA).
D1-Like Receptor Activity Inhibits Cell Contraction and Enhances the Effect of DA in Anterior Cells But D2-Like Receptors—Particularly DRD2 in Posterior Cells—Exert an Opposite Effect
To further investigate the role of DA receptor activity in scleral cell contraction and the inhibitory effect of DA, we administered specific DA receptor antagonists and agonists into the contraction medium of infant scleral fibroblasts derived from two randomly selected donors (HSF12 and HSF13), 1 hour prior to the addition of 50 µM DA. We focused on anterior and posterior scleral fibroblasts, as previous results indicated that they predominantly account for the differences between scleral cell populations. The drugs we used included D1-like receptor antagonist SCH23390 (10 µM), D1-like receptor agonist A68930 (10 µM), D2-like receptor antagonist levosulpiride (20 µM), D2-like receptor agonist bromocriptine (10 µM), selective DRD2 antagonist L741626 (10 µM), selective DRD2 agonist PNU-95666E (10 µM), selective DRD4 antagonist L745870 (10 µM), and selective DRD4 agonist A412997 (10 µM). All drugs, including DA, were maintained in the culture medium for 24 hours. Gel sizes were then monitored, and contraction curves were subsequently plotted. 
The results demonstrated that, in anterior scleral fibroblasts, antagonizing D1-like receptors significantly enhanced cell contraction and reversed the inhibitory effect of DA on contraction (Fig. 5a). Conversely, activation of D1-like receptors significantly reduced cell contraction and showed a tendency toward the inhibitory effect of DA (Fig. 5b). By contrast, neither antagonizing nor agonizing D2-like receptors had a significant effect on cell contraction or on the inhibitory action of DA (Figs. 5c, 5d). Moreover, activation of DRD2 or DRD4 reduced the inhibitory effect of DA on cell contraction (Figs. 5f, 5h), whereas antagonizing either receptor alone had no impact on cell contraction or on the inhibitory effect of DA (Figs. 5e, 5g). These results suggest that D1-like receptor activity reduces scleral cell contraction and enhances the inhibitory effect of DA in the anterior sclera, whereas D2-like receptor activity counteracts this inhibitory effect. 
Figure 5.
 
D1-like receptor activity promotes the inhibitory effect of DA on cell contraction, whereas D2-like receptor activity, particularly that of DRD2 in the posterior sclera, impedes it. (ap) The cell contraction rates at 24 hours for anterior (ah) and posterior (ip) scleral fibroblasts from HSF12 and HSF13 were measured following treatment with a range of DA receptor antagonists and agonists, administered 1 hour prior to the addition of DA. One experiment was conducted with three replicate wells for each compound tested, using two biological samples (HSF12 and HSF13; total n = 6). Results from the two cell lines were averaged and presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA).
Figure 5.
 
D1-like receptor activity promotes the inhibitory effect of DA on cell contraction, whereas D2-like receptor activity, particularly that of DRD2 in the posterior sclera, impedes it. (ap) The cell contraction rates at 24 hours for anterior (ah) and posterior (ip) scleral fibroblasts from HSF12 and HSF13 were measured following treatment with a range of DA receptor antagonists and agonists, administered 1 hour prior to the addition of DA. One experiment was conducted with three replicate wells for each compound tested, using two biological samples (HSF12 and HSF13; total n = 6). Results from the two cell lines were averaged and presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA).
In posterior scleral fibroblasts, neither antagonizing nor agonizing D1-like receptors had a significant impact on cell contraction or the inhibitory effect of DA on contraction (Figs. 5i, 5j). Similarly, modulation of D2-like receptors—either through antagonism or agonism—did not significantly alter cell contraction or the inhibitory action of DA (Fig. 5k, 5l). Notably, antagonizing DRD2 significantly reduced cell contraction and enhanced the inhibitory effect of DA (Fig. 5m), whereas agonizing DRD2, as well as either antagonizing or agonizing DRD4, had minimal impact on both cell contraction and the inhibitory effect of DA (Figs. 5n–p). These findings suggest that DRD2 activity promotes scleral cell contraction and attenuates DA-mediated suppression of contraction in the posterior sclera, whereas pharmacological modulation of D1-like receptor activity does not significantly affect either cell contraction or the inhibitory effect of DA. The results of the pharmacological modulation of DA receptors and their effects on anterior and posterior scleral fibroblasts are shown in Table 3
Table 3.
 
Summary of DA Receptor Modulation on Cell Contraction and DA-Mediated Inhibition
Table 3.
 
Summary of DA Receptor Modulation on Cell Contraction and DA-Mediated Inhibition
Silencing DRD4 Significantly Enhances DA-Mediated Contraction Inhibition, Particularly in Posterior Scleral Fibroblasts
To investigate the regulatory roles of D2-like receptors, DRD2 and DRD4, in DA-mediated contraction inhibition of scleral fibroblasts, we employed siRNA to silence the expression of DRD2 and DRD4 in anterior and posterior scleral cells from HSF12 and HSF13, respectively. The 7-day gel contraction activity of these cells, both with and without the addition of 50-µM DA, was measured. The results showed that knockdown of DRD2 led to a slight increase in cell contraction and enhanced the inhibitory effect of DA on contraction in both anterior and posterior scleral fibroblasts, with a more pronounced effect observed in posterior cells (Figs. 6a–f). In contrast, knockdown of DRD4 significantly reduced cell contraction and markedly amplified DA-mediated inhibition of contraction, especially in posterior cells (Figs. 6g–l). These results were further validated by comparing the percentage reduction in contraction on day 7 following DRD2 or DRD4 knockdown in anterior and posterior scleral fibroblasts, which revealed that DRD2 silencing slightly increased contraction, whereas DRD4 silencing led to a notable decrease (Figs. 6m, 6o). Furthermore, in a comparison among groups treated with DA alone, DRD2 knockdown plus DA, and DRD4 knockdown plus DA demonstrated that silencing DRD2 or DRD4—particularly DRD4—significantly enhanced DA-mediated inhibition of contraction in posterior scleral fibroblasts. These findings suggest that DRD4 expression plays a key role in modulating DA-induced suppression of scleral fibroblast contraction in the posterior sclera (Fig. 6n, 6p). 
Figure 6.
 
Silencing DRD4 significantly enhances DA-mediated contraction inhibition, particularly in posterior scleral fibroblasts. (a, b) Seven-day gel contraction curves of DRD2-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA. Each condition was tested in triplicate for cells derived from two donors (n = 6). The control group was transfected with non-targeting siRNA. (c, e) Western blot validation of DRD2 knockdown in anterior and posterior fibroblasts from two donors. (d, f) Comparison of contraction rates at day 7 for DRD2-knockdown anterior and posterior cells, respectively. Statistical analysis: ***P < 0.001, ****P < 0.0001 (two-way ANOVA). (g, h) Seven-day gel contraction curves of DRD4-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA (n = 6). (i, k) Western blot validation of DRD4 knockdown in anterior and posterior fibroblasts from two donors. (j, l) Comparison of contraction rates at day 7 for DRD4-knockdown anterior and posterior cells, respectively. Statistical analysis: *P < 0.05, **P < 0.01, ****P < 0.0001 (two-way ANOVA). (m, o) Percentage reduction in gel contraction on day 7 resulting from siRNA-knockdown of DRD2 or DRD4, compared to non-targeting siRNA controls, in anterior and posterior scleral fibroblasts, respectively. Statistical analysis: *P < 0.05, ****P < 0.0001 (t-test). (n, p) Percentage reduction in gel contraction on day 7 caused by DA treatment alone (compared to control), and by DRD2 or DRD4 knockdown combined with DA (compared to DRD2 or DRD4 knockdown without DA), in anterior and posterior fibroblasts, respectively (n = 6). Whiskers represent minimum to maximum values. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way ANOVA).
Figure 6.
 
Silencing DRD4 significantly enhances DA-mediated contraction inhibition, particularly in posterior scleral fibroblasts. (a, b) Seven-day gel contraction curves of DRD2-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA. Each condition was tested in triplicate for cells derived from two donors (n = 6). The control group was transfected with non-targeting siRNA. (c, e) Western blot validation of DRD2 knockdown in anterior and posterior fibroblasts from two donors. (d, f) Comparison of contraction rates at day 7 for DRD2-knockdown anterior and posterior cells, respectively. Statistical analysis: ***P < 0.001, ****P < 0.0001 (two-way ANOVA). (g, h) Seven-day gel contraction curves of DRD4-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA (n = 6). (i, k) Western blot validation of DRD4 knockdown in anterior and posterior fibroblasts from two donors. (j, l) Comparison of contraction rates at day 7 for DRD4-knockdown anterior and posterior cells, respectively. Statistical analysis: *P < 0.05, **P < 0.01, ****P < 0.0001 (two-way ANOVA). (m, o) Percentage reduction in gel contraction on day 7 resulting from siRNA-knockdown of DRD2 or DRD4, compared to non-targeting siRNA controls, in anterior and posterior scleral fibroblasts, respectively. Statistical analysis: *P < 0.05, ****P < 0.0001 (t-test). (n, p) Percentage reduction in gel contraction on day 7 caused by DA treatment alone (compared to control), and by DRD2 or DRD4 knockdown combined with DA (compared to DRD2 or DRD4 knockdown without DA), in anterior and posterior fibroblasts, respectively (n = 6). Whiskers represent minimum to maximum values. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way ANOVA).
Discussion
Cells from the human eye have been extensively profiled using scRNA-seq, with the aim of constructing a comprehensive cell atlas of the entire human eye.28,49 However, most existing studies did not focus on the sclera or typically used samples from adult eyes. Profiling human scleral fibroblasts—particularly during early ages, when their ECM remodeling activity is closely associated to the rapid phase of axial growth50—offers important insights into the regulation of scleral remodeling. 
By analyzing genes that were both highly and ubiquitously expressed in anterior and posterior scleral fibroblast populations, we identified several key GO functional pathways that were uniquely enriched in each region. For example, anterior cells showed strong signatures of actin cytoskeleton organization, supported by broad and high expression of actin-regulating genes and matrix proteins (Figs. 1e, 1h). This was further validated by our in vitro contraction assay, which demonstrated that anterior fibroblasts possess stronger contractile capacity than posterior fibroblasts (Fig. 4e). In contrast, posterior cells were enriched for ECM remodeling activities, with elevated expression of metalloproteinases and hyaluronidases involved in matrix degradation (Figs. 2c, 2d). This may reflect ongoing morphogenesis and proliferation in the posterior region—as indicated by GO analysis—which likely demands continuous ECM remodeling. These findings are consistent with observations in young animals, where the posterior sclera exhibits lower collagen fibril content and reduced elastic modulus compared to the anterior and equatorial regions,50 supporting the in vivo relevance of our results. Moreover, in humans, axial length increases by approximately 25% during the first 2 to 3 years after birth, compared to only 1% over the subsequent 10 years.51 This highlights a period of rapid scleral growth and expansion that aligns with the age range of our samples. Therefore, the enrichment of functional clusters related to DNA replication, cell proliferation, and adhesion (Figs. 2b–d) is expected and should not be attributed solely to culture conditions. 
Furthermore, our analysis revealed activation of Wnt signaling in anterior cells and chondrogenic features in posterior cells—both previously implicated in the regulation of axial growth and in the development of myopia when dysregulated.4,52 We also identified TGF-β signaling activity across both anterior and posterior populations, underscoring its critical role in scleral development. In vivo studies have shown that ablation of TGF-β signaling significantly alters ECM structure in the posterior sclera,53 supporting its key involvement in scleral remodeling. Notably, we observed elevated expression of HIF1A and enrichment of the GO term “aerobic glycolysis” in posterior cells. As hypoxia-induced glycolysis in scleral fibroblasts has been shown to drive myopia progression by promoting fibroblast-to-myofibroblast transformation,17 our findings suggest that HIF1A expression and glycolytic activity may contribute to the physiological regulation of posterior scleral growth. Further investigation is needed to determine how this regulatory mechanism is maintained or disrupted, potentially leading to pathological remodeling. In addition, we identified significant enrichment of insulin-like growth factor binding proteins (IGFBPs), calcium-binding proteins, proteinases, kinases, and transcription factors, as well as pathways related to VEGF signaling, TNF signaling, and steroid and lipid metabolism in both anterior and posterior fibroblast populations. Recent studies have proposed that calcium homeostasis, oxidative stress, and lipid metabolism play critical roles in regulating scleral remodeling.54,55 Our data further support the involvement of these processes, highlighting promising targets for future investigation in both in vitro and in vivo models. 
Moreover, fibroblast heterogeneity has been observed across species, organs, and developmental stages. Within individual organs, fibroblast subtypes have been identified in the dermis, lung, heart, and colon of both humans and mice, where they perform distinct regulatory functions—including, but not limited to, ECM remodeling, myofibroblast transformation, immune modulation, and response to injury,2,56,57 supporting the notion that fibroblast diversity is a conserved and functionally significant feature of connective tissue biology. Although our study focuses on infant scleral samples, similar regional differences in gene expression and cell behavior have also been observed in mice,5 guinea pigs,58 and human adult scleral fibroblasts,7,8 suggesting that the scleral fibroblast heterogeneity persists throughout life. We also show that the differential expression of DA receptors persists into adulthood. This heterogeneity likely arises from the complex microenvironment of the sclera, which generates diverse extracellular cues—such as mechanical strain and tissue stiffness—that regulate cytoskeletal organization, cell morphology, and gene expression in region-specific fibroblasts.7,8 Recognizing this cellular diversity is essential for advancing our understanding of scleral biology and supporting future research into the mechanisms underlying pathological scleral remodeling. 
Importantly, we identified differential expression pattern of five DA receptor subtypes in human scleral fibroblasts, a finding not previously reported. Combined with our in vitro contraction assay results—demonstrating that DA directly suppresses fibroblast-mediated contraction, particularly in posterior scleral cells—these findings suggest that inhibition of scleral remodeling, especially in the posterior region, may be one mechanism through which DA regulates eye growth. Although the pathway by which DA is transported from the retina to the sclera remains unclear, current studies show a correlation between retinal and choroidal DA levels, indicating that retinal DA signaling may be relayed through the choroid.59,60 It is therefore plausible that the choroid mediates DA signaling to the sclera to influence remodeling. Further investigation is needed to clarify the mechanisms of DA transmission to the sclera and how this process is regulated. 
Furthermore, our findings indicate that both the activation and expression levels of DA receptors modulate DA-mediated inhibition of scleral fibroblast contraction. Specifically, D1-like receptor activity enhances the inhibitory effect of DA in the anterior sclera, whereas D2-like receptor activity—particularly that of DRD2 in the posterior sclera—attenuates this effect on scleral remodeling. These findings are consistent with reports that D1-like receptor agonism suppresses pulmonary fibroblast-mediated fibrosis and that DRD2 antagonism protects against hepatic macrophage-mediated liver fibrosis—both through inhibition of YAP/TAZ signaling19,61—suggesting that similar signaling pathways may operate downstream of DA receptor activity in scleral fibroblasts. Moreover, endoplasmic reticulum (ER) stress has also been reported to be associated with scleral remodeling during myopia development, and DA receptors have been implicated in the regulation of ER stress. However, signaling through D1-like receptors has been linked to the induction of ER stress,62,63 whereas DRD2 activation has been shown to attenuate it.64 This pattern contrasts with the roles these receptors appear to play in regulating scleral remodeling; therefore, we speculate that DA signaling modulates scleral remodeling through pathways independent of ER stress. 
Interestingly, we observed that DA inhibited contraction in both anterior and posterior scleral fibroblasts, with a more pronounced effect in the posterior cells, which express higher levels of D2-like receptors—whose activity typically attenuates the inhibitory effect of DA. Although this may seem counterintuitive, it could be explained by the fact that the net biological outcome depends on the relative expression levels, cellular localization, and downstream signaling balance between D1-like and D2-like receptors. In posterior fibroblasts, DA-mediated inhibition may occur not only through D1-like receptors but also via mechanisms such as D2-like receptor desensitization, internalization, or crosstalk with other signaling pathways.10,65 Moreover, D1-like and D2-like receptors can converge to mediate cooperative functional effects, particularly through phosphoinositide-linked DA signaling pathways.66 In our study, the inhibitory effect observed with 50-µM DA—a concentration likely to activate both receptor families—may have overridden receptor-specific signaling preferences, resulting in a net suppression of ECM remodeling in both anterior and posterior scleral fibroblast populations. These findings highlight the importance of the intrinsic balance between D1-like and D2-like receptor activity. Additionally, in vivo studies have shown that, although retinal DA levels are significantly reduced under myopic conditions,67 DA levels in the sclera remain unchanged.68 This suggests that an altered balance of DA receptor activity in the sclera may contribute to pathological changes in myopia—a hypothesis that warrants further investigation. 
In addition, our results indicate that DA receptor expression, particularly DRD4 on posterior scleral fibroblasts, significantly regulates cell contraction and DA-mediated inhibition of contraction. This regulatory effect is much stronger than that of receptor activity. This suggests that DRD4 expression may play an important role in the signaling pathways involved in both scleral remodeling and the inhibitory effect of DA on this process. It also introduces the possibility of controlling scleral remodeling through targeted deletion of DRD4. The absence of Drd4 in mice has been found to result in more myopic refractions during normal refractive development,69 and the downstream signaling pathways of DRD4 may be associated with PDGFRβ, ERK1/2, mTOR, and the autophagy–lysosomal pathways.70 By contrast, the regulation of DRD2 expression on both cell contraction and DA-mediated suppression of contraction is less pronounced than that of DRD4 silencing. Silencing DRD2 led to a slight increase in cell contraction, which differs from its antagonism, which significantly reduced contraction in posterior scleral fibroblasts. This suggests that different signaling pathways are involved in DRD2 expression and activation in regulating scleral remodeling. This may explain why fibroblast-specific Drd2 knockout does not effectively intervene in myopia progression in the myopic mouse model.12 Nevertheless, future studies should focus on elucidating the downstream signaling pathways of specific DA receptors to better understand how to modulate scleral remodeling. Targeting these pathways—either directly or indirectly—may offer a way to regulate DA signaling specifically within the sclera, providing potential therapeutic strategies for diseases associated with scleral deformation, such as myopia. For example, previous studies have explored the use of topical eye drops containing dopaminergic compounds to slow myopia progression in chicks.71,72 However, targeting downstream pathways rather than the receptors themselves may be more feasible and effective for human applications. 
Limitations of the Study
scRNA-seq was performed on cultured primary infant scleral fibroblasts, which represents a limitation of the study. Although the cells were cultured for a relatively short period, this in vitro environment may still influence the expression of certain genes—particularly those involved in proliferation, migration, and cell–environment interactions—compared to their native three-dimensional, in vivo context. However, the observed differential transcriptomic profiles between anterior and posterior scleral fibroblasts should remain unaffected. Importantly, the differential expression of DA receptors was validated in situ using human scleral tissue sections, supporting the reliability of the findings. Despite this limitation, our study provides valuable insights into the intra-tissue heterogeneity of the human infant sclera, which will inform and support future research in this field. Moreover, although the heterogeneity between anterior and posterior scleral fibroblasts may persist throughout life, their transcriptomic profiles are likely to change across different developmental stages. Future transcriptomic studies on scleral fibroblasts from other life stages, such as adolescence, and comparative analyses of these datasets would provide deeper insights. 
Furthermore, our findings on the regulatory role of DA receptor activity and expression in scleral remodeling are based on in vitro experiments using scleral fibroblasts derived from two infant donors. Although the results were highly consistent between the two samples, the relatively small sample size may limit the statistical power and obscure significance in cases where biological variation exists between individuals. To strengthen the robustness of our conclusions, future studies should include larger sample sizes and incorporate in vivo models to further validate the role of DA signaling in scleral remodeling. For example, the use of a fibroblast-specific DRD4 knockout animal model could help elucidate the functional role of DRD4 in scleral remodeling during both normal refractive development and myopia progression. 
Acknowledgments
Supported by grants from the Science and Technology Projects in Guangzhou (202102080156, 2025A03J3233), Guangzhou Municipal Science and Technology Bureau; the Open Research Program of CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (KLRB202113); the National Natural Science Foundation of China (32200461, 32370856, 82471386); CAMS Innovation Fund for Medical Sciences (2019-I2M-5-005), the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University; Key Laboratory of Guangdong Higher Education Institutes (2021KSYS009); the Research Funds from Health@InnoHK Program launched by Innovation Technology Commission of the Hong Kong SAR, P. R. China; and the Major Project of Guangzhou National Laboratory (GZNL2024A03012). 
Author Contributions: Validation and Investigation, J.G.; Z.L.; Formal Analysis, J.H.; G.H.; Resources, Y.Z.; Z.L.; Supervision, D.Q.; Conceptualization, Investigation and Writing, H.L. 
Disclosure: J. Guan, None; G. Hong, None; Z. Liu, None; Y. Zheng, None; J. He, None; D. Qin, None; H. Li, None 
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Figure 1.
 
Human infant scleral fibroblasts have distinct anterior and posterior subpopulations. (a) Schematic illustration of the experimental design. (b) UMAP clustering of single-cell transcriptomic profiles from primary anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors (12 samples in total) prior to batch effect correction. The color coding represents different cell samples; numbers 9 to 14 indicate individual donors; and the letters A, E, and P denote anterior, equatorial, and posterior scleral fibroblasts, respectively. (c) Spatial grouping of cells by anatomical origin before batch effect correction. (d) UMAP visualization after batch effect correction, incorporating the spatial information of the samples. (eg) The top 15 genes selectively expressed in anterior, equatorial, and posterior scleral fibroblasts, respectively. (hj) The top five biological functions enriched in anterior, equatorial, and posterior scleral fibroblasts, analyzed using the top 100 genes most expressed in each group.
Figure 1.
 
Human infant scleral fibroblasts have distinct anterior and posterior subpopulations. (a) Schematic illustration of the experimental design. (b) UMAP clustering of single-cell transcriptomic profiles from primary anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors (12 samples in total) prior to batch effect correction. The color coding represents different cell samples; numbers 9 to 14 indicate individual donors; and the letters A, E, and P denote anterior, equatorial, and posterior scleral fibroblasts, respectively. (c) Spatial grouping of cells by anatomical origin before batch effect correction. (d) UMAP visualization after batch effect correction, incorporating the spatial information of the samples. (eg) The top 15 genes selectively expressed in anterior, equatorial, and posterior scleral fibroblasts, respectively. (hj) The top five biological functions enriched in anterior, equatorial, and posterior scleral fibroblasts, analyzed using the top 100 genes most expressed in each group.
Figure 2.
 
Transcriptomic profiling of anterior and posterior scleral fibroblast subpopulations. (a) Leiden community-detection clustering identified 11 cell clusters in the anterior and posterior cell populations. (b, c) The top three genes selectively expressed in the clusters of anterior and posterior subpopulations, respectively. (d) Dot plot illustrating the main gene functions expressed in each cell cluster of the anterior and posterior cell populations.
Figure 2.
 
Transcriptomic profiling of anterior and posterior scleral fibroblast subpopulations. (a) Leiden community-detection clustering identified 11 cell clusters in the anterior and posterior cell populations. (b, c) The top three genes selectively expressed in the clusters of anterior and posterior subpopulations, respectively. (d) Dot plot illustrating the main gene functions expressed in each cell cluster of the anterior and posterior cell populations.
Figure 3.
 
Differential DA receptor expression in anterior and posterior scleral fibroblasts. (ae) The mRNA levels of DA receptors in all scleral fibroblast samples, validated by qRT-PCR. Relative expression levels were normalized to those of anterior cells. One experiment was conducted for each biological sample (total n = 4). *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). (fj) Protein expression levels of DA receptors in all scleral fibroblast samples, quantified by western blot. The blot image below represents one of the experimental repeats. Protein band quantification was first normalized to GAPDH and then to the value of anterior fibroblasts. Two experiments were performed per biological sample (total n = 8; for the DRD5 blot, one experiment was performed, n = 4). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (k) Gene expression profiles of DA receptors in anterior and posterior cell populations, as revealed by scRNA-seq analysis. (lp) ISH results for DRD1 to DRD5 in human scleral tissue sections from an adult donor. The intensity of the brown color indicates the expression level of each DA receptor. The right panel images show magnified views of the boxed areas in the left panel. Scale bars: 200 µm (left panel); 50 µm (right panel). Images of full tissue sections are shown below each ISH panel, with the regions corresponding to the anterior and posterior sclera marked in blue and pink, respectively. Signal intensity was quantified from 10 regions of interest (ROIs) per image. ****P < 0.0001 (unpaired t-test).
Figure 3.
 
Differential DA receptor expression in anterior and posterior scleral fibroblasts. (ae) The mRNA levels of DA receptors in all scleral fibroblast samples, validated by qRT-PCR. Relative expression levels were normalized to those of anterior cells. One experiment was conducted for each biological sample (total n = 4). *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). (fj) Protein expression levels of DA receptors in all scleral fibroblast samples, quantified by western blot. The blot image below represents one of the experimental repeats. Protein band quantification was first normalized to GAPDH and then to the value of anterior fibroblasts. Two experiments were performed per biological sample (total n = 8; for the DRD5 blot, one experiment was performed, n = 4). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (k) Gene expression profiles of DA receptors in anterior and posterior cell populations, as revealed by scRNA-seq analysis. (lp) ISH results for DRD1 to DRD5 in human scleral tissue sections from an adult donor. The intensity of the brown color indicates the expression level of each DA receptor. The right panel images show magnified views of the boxed areas in the left panel. Scale bars: 200 µm (left panel); 50 µm (right panel). Images of full tissue sections are shown below each ISH panel, with the regions corresponding to the anterior and posterior sclera marked in blue and pink, respectively. Signal intensity was quantified from 10 regions of interest (ROIs) per image. ****P < 0.0001 (unpaired t-test).
Figure 4.
 
DA inhibits scleral fibroblast-mediated ECM remodeling, especially in posterior cells. (a) Schematic illustration of the in vitro collagen gel contraction assay. (bd) Seven-day contraction curves of anterior, equatorial, and posterior infant scleral fibroblasts (HSF13) treated with 1-µM, 10-µM, 50-µM, and 100-µM DA. One experiment with triplicate wells was performed. (e) Seven-day contraction curves of anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors treated with 50-µM DA. One experiment with triplicate wells was performed for each biological sample (n = 12 total). Statistical differences between sample groups: F(5, 66) = 13.03, ****P < 0.0001 (two-way ANOVA). (f) Comparison of contraction percentages on day 7. *P < 0.05, ****P < 0.0001 (two-way ANOVA). (g) Percentage of contraction reduced by DA on day 7, calculated as (% control – % DA)/% control × 100. **P < 0.01, ***P < 0.001 (one-way ANOVA).
Figure 4.
 
DA inhibits scleral fibroblast-mediated ECM remodeling, especially in posterior cells. (a) Schematic illustration of the in vitro collagen gel contraction assay. (bd) Seven-day contraction curves of anterior, equatorial, and posterior infant scleral fibroblasts (HSF13) treated with 1-µM, 10-µM, 50-µM, and 100-µM DA. One experiment with triplicate wells was performed. (e) Seven-day contraction curves of anterior, equatorial, and posterior scleral fibroblasts derived from four infant donors treated with 50-µM DA. One experiment with triplicate wells was performed for each biological sample (n = 12 total). Statistical differences between sample groups: F(5, 66) = 13.03, ****P < 0.0001 (two-way ANOVA). (f) Comparison of contraction percentages on day 7. *P < 0.05, ****P < 0.0001 (two-way ANOVA). (g) Percentage of contraction reduced by DA on day 7, calculated as (% control – % DA)/% control × 100. **P < 0.01, ***P < 0.001 (one-way ANOVA).
Figure 5.
 
D1-like receptor activity promotes the inhibitory effect of DA on cell contraction, whereas D2-like receptor activity, particularly that of DRD2 in the posterior sclera, impedes it. (ap) The cell contraction rates at 24 hours for anterior (ah) and posterior (ip) scleral fibroblasts from HSF12 and HSF13 were measured following treatment with a range of DA receptor antagonists and agonists, administered 1 hour prior to the addition of DA. One experiment was conducted with three replicate wells for each compound tested, using two biological samples (HSF12 and HSF13; total n = 6). Results from the two cell lines were averaged and presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA).
Figure 5.
 
D1-like receptor activity promotes the inhibitory effect of DA on cell contraction, whereas D2-like receptor activity, particularly that of DRD2 in the posterior sclera, impedes it. (ap) The cell contraction rates at 24 hours for anterior (ah) and posterior (ip) scleral fibroblasts from HSF12 and HSF13 were measured following treatment with a range of DA receptor antagonists and agonists, administered 1 hour prior to the addition of DA. One experiment was conducted with three replicate wells for each compound tested, using two biological samples (HSF12 and HSF13; total n = 6). Results from the two cell lines were averaged and presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA).
Figure 6.
 
Silencing DRD4 significantly enhances DA-mediated contraction inhibition, particularly in posterior scleral fibroblasts. (a, b) Seven-day gel contraction curves of DRD2-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA. Each condition was tested in triplicate for cells derived from two donors (n = 6). The control group was transfected with non-targeting siRNA. (c, e) Western blot validation of DRD2 knockdown in anterior and posterior fibroblasts from two donors. (d, f) Comparison of contraction rates at day 7 for DRD2-knockdown anterior and posterior cells, respectively. Statistical analysis: ***P < 0.001, ****P < 0.0001 (two-way ANOVA). (g, h) Seven-day gel contraction curves of DRD4-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA (n = 6). (i, k) Western blot validation of DRD4 knockdown in anterior and posterior fibroblasts from two donors. (j, l) Comparison of contraction rates at day 7 for DRD4-knockdown anterior and posterior cells, respectively. Statistical analysis: *P < 0.05, **P < 0.01, ****P < 0.0001 (two-way ANOVA). (m, o) Percentage reduction in gel contraction on day 7 resulting from siRNA-knockdown of DRD2 or DRD4, compared to non-targeting siRNA controls, in anterior and posterior scleral fibroblasts, respectively. Statistical analysis: *P < 0.05, ****P < 0.0001 (t-test). (n, p) Percentage reduction in gel contraction on day 7 caused by DA treatment alone (compared to control), and by DRD2 or DRD4 knockdown combined with DA (compared to DRD2 or DRD4 knockdown without DA), in anterior and posterior fibroblasts, respectively (n = 6). Whiskers represent minimum to maximum values. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way ANOVA).
Figure 6.
 
Silencing DRD4 significantly enhances DA-mediated contraction inhibition, particularly in posterior scleral fibroblasts. (a, b) Seven-day gel contraction curves of DRD2-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA. Each condition was tested in triplicate for cells derived from two donors (n = 6). The control group was transfected with non-targeting siRNA. (c, e) Western blot validation of DRD2 knockdown in anterior and posterior fibroblasts from two donors. (d, f) Comparison of contraction rates at day 7 for DRD2-knockdown anterior and posterior cells, respectively. Statistical analysis: ***P < 0.001, ****P < 0.0001 (two-way ANOVA). (g, h) Seven-day gel contraction curves of DRD4-knockdown anterior and posterior infant scleral fibroblasts, with or without 50-µM DA (n = 6). (i, k) Western blot validation of DRD4 knockdown in anterior and posterior fibroblasts from two donors. (j, l) Comparison of contraction rates at day 7 for DRD4-knockdown anterior and posterior cells, respectively. Statistical analysis: *P < 0.05, **P < 0.01, ****P < 0.0001 (two-way ANOVA). (m, o) Percentage reduction in gel contraction on day 7 resulting from siRNA-knockdown of DRD2 or DRD4, compared to non-targeting siRNA controls, in anterior and posterior scleral fibroblasts, respectively. Statistical analysis: *P < 0.05, ****P < 0.0001 (t-test). (n, p) Percentage reduction in gel contraction on day 7 caused by DA treatment alone (compared to control), and by DRD2 or DRD4 knockdown combined with DA (compared to DRD2 or DRD4 knockdown without DA), in anterior and posterior fibroblasts, respectively (n = 6). Whiskers represent minimum to maximum values. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way ANOVA).
Table 1.
 
Oligonucleotides Used for qRT-PCR
Table 1.
 
Oligonucleotides Used for qRT-PCR
Table 2.
 
Sequences of Probes Used for ISH
Table 2.
 
Sequences of Probes Used for ISH
Table 3.
 
Summary of DA Receptor Modulation on Cell Contraction and DA-Mediated Inhibition
Table 3.
 
Summary of DA Receptor Modulation on Cell Contraction and DA-Mediated Inhibition
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