Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells

Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Developmental Biology (Impact Factor: 3.55). 05/2011; 356(1):5-18. DOI: 10.1016/j.ydbio.2011.05.005
Source: PubMed


Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.

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Available from: Shivalingappa Swamynathan, Mar 11, 2014
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    • "Notably, this threshold also appeared to eliminate signals derived from the minimal amount of contamination from neighboring tissues that is, to some extent, inevitable during murine embryonic lens isolation. For example, the expression of the Krüppel-like transcription factors Klf4 and Klf5, which are abundant in the corneal epithelium [14] [15], was detected at 0.48 RPKM and 0.09 RPKM respectively, while those for platelet endothelial cell adhesion molecule 1 (Pecam1), a marker expressed abundantly in blood vessels [16] "
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    ABSTRACT: Next-generation sequencing of the transcriptome (RNA-Seq) is a powerful method that allows for the quantitative determination of absolute gene expression, and can be used to investigate how these levels change in response to an experimental manipulation or disease condition. The sensitivity of this method allows one to analyze transcript levels of all expressed genes, including low abundance transcripts that encode important regulatory molecules, providing valuable insights into the global effects of experimental manipulations. However, this increased sensitivity can also make it challenging to ascertain which expression changes are biologically significant. Here, we describe a novel set of filtering criteria - based on biological insights and computational approaches - that were applied to prioritize genes for further study from an extensive number of differentially expressed transcripts in lenses lacking Smad interacting protein 1 (Sip1) obtained via RNA-Seq by Manthey and colleagues in Mechanisms of Development (Manthey et al., 2014). Notably, this workflow allowed an original list of over 7,100 statistically significant differentially expressed genes (DEGs) to be winnowed down to 190 DEGs that likely play a biologically significant role in Sip1 function during lens development. Focusing on genes whose expression was upregulated or downregulated in a manner opposite to what normally occurs during lens development, we identified 78 genes that appear to be strongly dependent on Sip1 function. From these data (GEO accession number GSE49949), it appears that Sip1 regulates multiple genes in the lens that are generally distinct from those regulated by Sip1 in other cellular contexts, including genes whose expression is prominent in the early head ectoderm, from which the lens differentiates. Further, the analysis criteria outlined here represent a filtering scheme that can be used to prioritize genes in future RNA-Seq investigations performed at this stage of ocular lens development.
    Genomics Data 12/2014; 2:369-374. DOI:10.1016/j.gdata.2014.10.015
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    • "In previous publications using the same expression chips, a N 1.5-fold change combined with P b .05 has been defined as the minimum meaningful fold change in the identification of differentially expressed genes [33] [34] [35]. Application of the same principle to our data resulted in a total of 1197 genes, 569 of which were upregulated and 628 were downregulated (Table S2). "
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    ABSTRACT: Krüppel-like factor 5 (KLF5) regulates multiple biologic processes. Its function in tumorigenesis appears contradictory though, showing both tumor suppressor and tumor promoting activities. In this study, we examined whether and how Klf5 functions in prostatic tumorigenesis using mice with prostate-specific deletion of Klf5 and phosphatase and tensin homolog (Pten), both of which are frequently inactivated in human prostate cancer. Histologic analysis demonstrated that when one Pten allele was deleted, which causes mouse prostatic intraepithelial neoplasia (mPIN), Klf5 deletion accelerated the emergence and progression of mPIN. When both Pten alleles were deleted, which causes prostate cancer, Klf5 deletion promoted tumor growth, increased cell proliferation, and caused more severe morphologic and molecular alterations. Homozygous deletion of Klf5 was more effective than hemizygous deletion. Unexpectedly, while Pten deletion alone expanded basal cell population in a tumor as reported, Klf5 deletion in the Pten-null background clearly reduced basal cell population while expanding luminal cell population. Global gene expression profiling, pathway analysis, and experimental validation indicate that multiple mechanisms could mediate the tumor-promoting effect of Klf5 deletion, including the up-regulation of epidermal growth factor and its downstream signaling molecules AKT and ERK and the inactivation of the p15 cell cycle inhibitor. KLF5 also appears to cooperate with several transcription factors, including CREB1, Sp1, Myc, ER and AR, to regulate gene expression. These findings validate the tumor suppressor function of KLF5. They also yield a mouse model that shares two common genetic alterations with human prostate cancer—mutation/deletion of Pten and deletion of Klf5.
    Neoplasia (New York, N.Y.) 11/2014; 16(11). DOI:10.1016/j.neo.2014.09.006 · 4.25 Impact Factor
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    • "This fold change threshold was chosen based on experimental data comparing relative gene expression between E15.5 lenses from mixed background Sip1 f/f no Cre control mice and inbred C57Bl/6<har> mice (GEO accession GSE49949) which indicated that a large proportion of changes below 2.5-fold are likely due to variation in genetic background, not the Sip1 gene deletion. Further, expression of Klf4 and Klf5, transcription factors abundant in the corneal epithelium (Swamynathan et al., 2007; Kenchegowda et al., 2011), were greatly below the 2 RPKM cutoff for significant expression in both wild type and mutant samples, showing that the samples lacked appreciable corneal contamination arising during the dissection. Similarly, Pecam1, a marker expressed abundantly in blood vessels of the eye (Ferrari et al., 2013 "
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    ABSTRACT: SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes, as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases, where it functionally links TGFβ signaling to the loss of epithelial cell characteristics and gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers, such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation, and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes. Furthermore, 34% of the genes with increased expression in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes with decreased expression in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts.
    Mechanisms of development 10/2013; 131(1). DOI:10.1016/j.mod.2013.09.005 · 2.44 Impact Factor
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