Expression data in PAX6 depleted corneal epithelial cells
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ABSTRACT:
PROVIDER: PRJNA817442 | ENA |
REPOSITORIES: ENA
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Project description:PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates thecell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify thefunction of PAX6 in human CECs using gene knockout via the clustered regularly interspaced shortpalindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAsfor different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but becamelarger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specificand included keratin 12, keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1(ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes wereprimarily epidermis-related and included keratin 10, keratin 1, involucrin (IVL), filaggrin (FLG). Thesefindings suggest that PAX6 maintains CEC identity by regulating differentiation.
2022-07-27 | GSE198924 | GEO
Project description:PURPOSE. Limbal stem/progenitor cells (LSC) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. Previous study revealed that Paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CEC). However, the molecular events downstream of PAX6 remain largely unknown. In this study, we aimed to clarify the regulation network of PAX6 in driving CEC differentiation. METHODS. An air-liquid culture system was used to differentiate LSC into mature CEC. Specific targeting PAX6 short-hairpin shRNAs were used to knock down PAX6 in LSC. RNA-seq was used to analyze shPAX6-transfected CEC and CEC differentiation-associated genes to identify the potential downstream targets of PAX6. RNA-seq analysis, quantitative real-time PCR, and immunofluorescence staining were performed to clarify the function of WNK lysine deficient protein kinase 2 (WNK2), a downstream target of PAX6, and its relationship with corneal diseases. RESULTS. WNK2 expression increased during CEC differentiation and decreased upon PAX6 depletion. The distribution of WNK2 was specifically limited to the central corneal epithelium and suprabasal layer of the limbus. Knockdown of WNK2 impaired the expression of CEC-specific markers (KRT12, ALDH3A1, and CLU), disrupted the corneal differentiation process, and activated the terms of keratinization, inflammation, and cell proliferation, consistent with PAX6-depleted CEC and published microbial keratitis. Thus, aberrant expression of WNK2 was linked to corneal ulcers. CONCLUSIONS. As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.
2024-10-30 | GSE272034 | GEO
Project description:PAX6-WNK2 axis governs corneal epithelial homeostasis
| PRJNA1134753 | ENA
Project description:Lineage-specific transcription factors (TFs) play key roles in maintaining the unique properties of cells, but the molecular mechanism that regulates the homeostasis of human corneal epithelial cells (CECs) is still poorly understood. We aimed to modulate KLF4 and PAX6 in human CECs using gene knockout system to clarify the regulatory network of KLF4, and then elucidate how KLF4 regulates the transcriptional genes of human CECs compared with PAX6. We performed a functional analysis of KLF4 via gene knockout using a lentivirus vector that carries both Cas9 and guide RNAs. We designed guide RNAs targeted for KLF4 and PAX6, and created KLF4-, PAX6-, and both KLF4- and PAX6-depleted CECs (KLF4-KO, PAX6-KO, and DKO, respectively). An empty vector was used as a control. The morphology of KLF4-KO CECs displayed an epithelial-mesenchymal transition (EMT)-like change, including upregulation of mesenchymal genes and downregulation of epithelial genes, as well as downregulation of keratin (KRT) 3 and KRT12. Global analyses using NGS revealed that the downregulated genes in KLF4-KO CECs were enriched in more widely keratin-related genes than PAX6-KO CECs. DKO cells showed disruption of the epithelial barrier due to downregulation of epithelial genes and showed more increase of KRT1 and KRT10 than PAX6-KO and KLF4-KO CECs, respectively. In conclusion, KLF4 modulates keratin-related genes as well as EMT-related genes and, together with PAX6, co-regulates the human CEC identity.
2022-07-27 | GSE198977 | GEO
Project description:The corneal epithelium is maintained by limbal stem cells (LSCs) that differentiate into mature epithelial cells to ensure transparency and barrier function. Epigenetic regulation is critical for corneal homeostasis, yet the role of histone methyltransferase DOT1L, the sole enzyme catalyzing H3K79 methylation, remains unexplored. Here, we show that DOT1L is indispensable for corneal epithelial differentiation, postnatal development, and wound healing. Using conditional knockout mice (Dot1lflox/flox; K14-Cre), we find that DOT1L deficiency causes progressive corneal opacification, squamous metaplasia, and pathological angiogenesis. Immunofluorescence and histochemical analyses reveal that DOT1L deficiency impedes corneal epithelial differentiation, consequently inhibiting both postnatal corneal development and wound-induced corneal repair. Single-cell RNA sequencing of WT and Dot1l-KO corneas demonstrates that DOT1L loss disrupts the LSC-to-superficial-cell differentiation trajectory, and identifies Pax6, the master regulator of corneal epithelial identity, as one of the differentially expressed genes. Mechanistically, DOT1L directly deposits H3K79me2 at the Pax6 promoter to activate its transcription. Concurrently, DOT1L deficiency represses interferon signaling genes, coinciding with pathological angiogenesis. Collectively, our findings establish DOT1L as a critical guardian of corneal epithelial identity through regulation of epithelial differentiation, positioning DOT1L as a potential therapeutic target for corneal disorders.
2026-07-03 | GSE336843 | GEO
Project description:Forkhead box C1 (FOXC1) is required for neural crest and ocular development, and mutations in FOXC1 lead to inherited Axenfeld–Rieger syndrome. Here, we find that FOXC1 and paired box 6 (PAX6) are co-expressed in the human limbus and central corneal epithelium. Deficiency of FOXC1 and alternation in epithelial features occur in patients with corneal ulcers. FOXC1 governs the fate of the corneal epithelium by directly binding to lineage-specific open promoters or enhancers marked by H3K4me2. FOXC1 depletion not only activates the keratinization pathway and reprograms corneal epithelial cells into skin-like epithelial cells but also disrupts the collagen metabolic process and interferon signaling pathways. Loss of interferon regulatory factor 1 (IRF1) and PAX6 induced by FOXC1 dysfunction is linked to the corneal ulcer. Collectively, our results reveal a FOXC1-mediated regulatory network responsible for corneal epithelial homeostasis and provide a potential therapeutic target for corneal ulcer.
2021-07-22 | GSE155773 | GEO
Project description:Purpose: We find that Wnt7a-PAX6 axis determine corneal epithelial cell fate. To obtain global evidence for successful cell fate conversion, we performed gene expression profiling by RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation. Methods: Under 3-D culture condition, limbal stem cell (LSCs) can be differentiated to Cornea epithelial cells (CECs), and skin epithelial stem cells (SESCs) can be differentiated to skin epithelial cells (SECs). Total RNA was isolated from CECs, SECs, and LSCs after knocking down PAX6 (3-D shPAX6 LSCs) and on SESCs transduced with PAX6 (3-D PAX6+ SESCs) upon 3-D differentiation. Libraries were prepared following published standard protocol (Fox-Walsh K et al., 2011, genomics, 266-71). mRNA profiles were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. Results: Following optimized decoding and mapping workfollow, we mapped about 5 million sequence reads to the human genome and identified more than 23659 transcripts per sample. Conclusions: Hierarchical clustering analysis of differentially expressed gene signatures revealed that the gene expression pattern of SESCs with PAX6 transduction was strikingly similar to that of CECs, whereas the profile of LSCs with PAX6 knockdown was highly related to that in SECs upon differentiation. These data therefore provided global evidence for a decisive role of the WNT7A/PAX6 axis in cell fate conversion from SESCs to CECs. RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation, using Illumina HiSeq 2000
2014-07-11 | E-GEOD-54322 | biostudies-arrayexpress
Project description:We successfully induced corneal epithelial cells from human iPSCs. Then, we perfomed global expression analysis using microarray to compare the character of hiPSC-derived corneal epithelial cells with that of the other kinds of cells. Total RNA was obtained from human iPSCs (hiPSCs), human iPSC-derived corneal epithelial cells (hiCECs), human corneal limbal epithelial cells (HCECs), human oral keratinocytes (HOKs), human dermal fibroblasts (HDFs) and six weeks-differentiated hiPSCs (hiPSC-derived ocular surface ectoderm, OSE) using the QIAZol reagent. A microarray analysis using Sure Print G3 human 8x60K slides (Agilent technologies) was performed at Takara Bio (Shiga, Japan).
2016-03-09 | E-GEOD-73971 | biostudies-arrayexpress
Project description:We successfully induced corneal epithelial cells from human iPSCs. Then, we perfomed global expression analysis using microarray to compare the character of hiPSC-derived corneal epithelial cells with that of the other kinds of cells.
2016-03-09 | GSE73971 | GEO
Project description:Purpose: We find that Wnt7a-PAX6 axis determine corneal epithelial cell fate. To obtain global evidence for successful cell fate conversion, we performed gene expression profiling by RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation. Methods: Under 3-D culture condition, limbal stem cell (LSCs) can be differentiated to Cornea epithelial cells (CECs), and skin epithelial stem cells (SESCs) can be differentiated to skin epithelial cells (SECs). Total RNA was isolated from CECs, SECs, and LSCs after knocking down PAX6 (3-D shPAX6 LSCs) and on SESCs transduced with PAX6 (3-D PAX6+ SESCs) upon 3-D differentiation. Libraries were prepared following published standard protocol (Fox-Walsh K et al., 2011, genomics, 266-71). mRNA profiles were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. Results: Following optimized decoding and mapping workfollow, we mapped about 5 million sequence reads to the human genome and identified more than 23659 transcripts per sample. Conclusions: Hierarchical clustering analysis of differentially expressed gene signatures revealed that the gene expression pattern of SESCs with PAX6 transduction was strikingly similar to that of CECs, whereas the profile of LSCs with PAX6 knockdown was highly related to that in SECs upon differentiation. These data therefore provided global evidence for a decisive role of the WNT7A/PAX6 axis in cell fate conversion from SESCs to CECs.
2014-07-11 | GSE54322 | GEO