Dataset Information

Identification of candidate Klf4 target genes reveals the molecular basis of the diverse regulatory roles of Klf4 in the mouse cornea.

ABSTRACT: Krüppel-like factor 4 (Klf4) plays a crucial role in the development and maintenance of the mouse cornea. In the current study, wild-type (WT) and Klf4-conditional null (Klf4CN) corneal gene expression patterns were examined, to gain understanding of the molecular basis of the Klf4CN corneal phenotype.Expression of more than 22,000 genes in 10 WT and Klf4CN corneas was compared by microarrays, analyzed using BRB ArrayTools (National Cancer Institute, Bethesda, MD) and validated by Q-RT-PCR. Transient cotransfections were used to test whether Klf4 activates the aquaporin-3, Aldh3a1, and TKT promoters.Scatterplot analysis identified 740 and 529 genes up- and downregulated by more than twofold, respectively, in the Klf4CN corneas. Cell cycle activators were upregulated, whereas the inhibitors were downregulated, consistent with the increased Klf4CN corneal epithelial cell proliferation. Desmosomal components were downregulated, consistent with the Klf4CN corneal epithelial fragility. Downregulation of aquaporin-3, detected by microarray, was confirmed by immunoblot and immunohistochemistry. Aquaporin-3 promoter activity was stimulated 7- to 10-fold by cotransfection with pCI-Klf4. The corneal crystallins Aldh3A1 and TKT were downregulated in the Klf4CN cornea, and their respective promoter activities were upregulated 16- and 9-fold by pCI-Klf4 in cotransfections. The expression of epidermal keratinocyte differentiation markers was affected in the Klf4CN cornea. Although the cornea-specific keratin-12 was downregulated, most other keratins were upregulated, suggesting hyperkeratosis.Functionally diverse candidate Klf4 target genes were identified, revealing the molecular basis of the diverse aspects of the Klf4CN corneal phenotype. These results establish Klf4 as an important node in the genetic network of transcription factors regulating the corneal homeostasis.

SUBMITTER: Swamynathan SK

PROVIDER: S-EPMC2774783 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of candidate Klf4 target genes reveals the molecular basis of the diverse regulatory roles of Klf4 in the mouse cornea.

Swamynathan Shivalingappa K SK Davis Janine J Piatigorsky Joram J

Investigative ophthalmology & visual science 20080509 8

<h4>Purpose</h4>Krüppel-like factor 4 (Klf4) plays a crucial role in the development and maintenance of the mouse cornea. In the current study, wild-type (WT) and Klf4-conditional null (Klf4CN) corneal gene expression patterns were examined, to gain understanding of the molecular basis of the Klf4CN corneal phenotype.<h4>Methods</h4>Expression of more than 22,000 genes in 10 WT and Klf4CN corneas was compared by microarrays, analyzed using BRB ArrayTools (National Cancer Institute, Bethesda, MD) ...[more]

PMID: 18469187

Similar Datasets

Project description:Transcription factors (TFs) regulate the expression of target genes, inducing changes in cell morphology or activities needed for cell fate determination and differentiation. The BMP signaling pathway is widely regarded as one of the most important pathways in vertebrate skeletal biology, of which BMP2 is a potent inducer, governing the osteoblast differentiation of bone marrow stromal cells (BMSCs). However, the mechanism by which BMP2 initiates its downstream transcription factor cascade and determines the direction of differentiation remains largely unknown. In this study, we used RNA-seq, ATAC-seq, and animal models to characterize the BMP2-dependent gene regulatory network governing osteoblast lineage commitment. Sp7-Cre; Bmp2fx/fx mice (BMP2-cKO) were generated and exhibited decreased bone density and lower osteoblast number (n > 6). In vitro experiments showed that BMP2-cKO mouse bone marrow stromal cells (mBMSCs) had an impact on osteoblast differentiation and deficient cell proliferation. Osteogenic medium induced mBMSCs from BMP2-cKO mice and control were subjected to RNA-seq and ATAC-seq analysis to reveal differentially expressed TFs, along with their target open chromatin regions. Combined with H3K27Ac CUT&Tag during osteoblast differentiation, we identified 2338 BMP2-dependent osteoblast-specific active enhancers. Motif enrichment assay revealed that over 80% of these elements were directly targeted by RUNX2, DLX5, MEF2C, OASIS, and KLF4. We deactivated Klf4 in the Sp7 + lineage to validate the role of KLF4 in osteoblast differentiation of mBMSCs. Compared to the wild-type, Sp7-Cre; Klf4fx/+ mice (KLF4-Het) were smaller in size and had abnormal incisors resembling BMP2-cKO mice. Additionally, KLF4-Het mice had fewer osteoblasts and decreased osteogenic ability. RNA-seq and ATAC-seq revealed that KLF4 mainly "co-bound" with RUNX2 to regulate downstream genes. Given the significant overlap between KLF4- and BMP2-dependent NFRs and enriched motifs, our findings outline a comprehensive BMP2-dependent gene regulatory network specifically governing osteoblast differentiation of the Sp7 + lineage, in which Klf4 is a novel transcription factor.

Project description:The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8-10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p ? 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation.

Dataset Information

Identification of candidate Klf4 target genes reveals the molecular basis of the diverse regulatory roles of Klf4 in the mouse cornea.

Publications

Identification of candidate Klf4 target genes reveals the molecular basis of the diverse regulatory roles of Klf4 in the mouse cornea.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets