S100A4, a member of the S100 family of multifunctional calcium-binding proteins, participates in several physiological and pathological processes. In this study, we demonstrate that S100A4 expression is robustly induced in differentiating fiber cells of the ocular lens and that S100A4 (−/−) knockout mice develop late-onset cortical cataracts. Transcriptome profiling of lenses from S100A4 (−/−) mice revealed a robust increase in the expression of multiple photoreceptor- and Müller glia-specific genes, as well as the olfactory sensory neuron-specific gene, S100A5. This aberrant transcriptional profile is characterized by corresponding increases in the levels of proteins encoded by the aberrantly upregulated genes. Ingenuity pathway network and curated pathway analyses of differentially expressed genes in S100A4 (−/−) lenses identified Crx and Nrl transcription factors as the most significant upstream regulators, and revealed that many of the upregulated genes possess promoters containing a high-density of CpG islands bearing trimethylation marks at histone H3K27 and/or H3K4, respectively. In support of this finding, we further documented that S100A4 (−/−) knockout lenses have altered levels of trimethylated H3K27 and H3K4. Taken together, our findings suggest that S100A4 suppresses the expression of retinal genes during lens differentiation plausibly via a mechanism involving changes in histone methylation.
The developing eye represents a fascinating model to interrogate the molecular mechanisms governing the commitment and specification of various ocular tissues, including the retina and lens. During embryogenesis, the retina develops from the neural epithelium of the optic vesicle, whereas the lens develops from the lens vesicle (LV) which is formed upon invagination of the lens placode ectoderm followed by separation of the LV from the surface ectoderm1,2. Once the LV is formed, the posterior cells of the LV elongate and differentiate into lens primary fibers, while the anterior LV becomes the epithelium covering the anterior portion of the lens. After the primary fibers fill the LV, epithelial cells at the equator exit the cell cycle and differentiate into secondary fibers. These fibers undergo terminal differentiation and eventually lose all cellular organelles, including the nuclei, and assume a radial packing organization with perfect hexagonal symmetry. The lens expresses various tissue-preferred and tissue-specific proteins including crystallins, aquaporin-0, gap junctional proteins and beaded filament cytoskeletal proteins required for maintaining lens function1.
Lens morphogenesis and growth are governed by various transcription factors (e.g. Pax6, Sox2, Six3, Maf, Prox1 and Hsf4) and growth factor regulated signaling pathways, including the TGF-β/BMP, FGF, Wnt, integrin, sonic hedgehog and Notch pathways. However, little is known about gene suppressive mechanisms and their role in lineage determination of the lens1. Our current data suggest that one protein potentially involved in these suppressive mechanisms is S100A4, a member of the large S100 family of calcium binding proteins3,4, whose expression is tissue-, cell- and cell cycle-specific3,5.
S100A4, used as a prognostic cancer biomarker3, has been shown to participate in proliferation, differentiation, cell motility, invasion, and immunity3,4,6,7,8. This protein interacts with several intracellular targets, including actin and non-muscle myosin IIA9, and is localized predominantly in the cytosol with some additional distribution to the nucleus3,10. S100A4 can regulate the signaling activity of various growth factors11,12, the secretion of matrix metalloproteinases13, and the epithelial to mesenchymal transition3,7. S100A4 expression is regulated transcriptionally by the Wnt/β-catenin pathway14, and epigenetically by DNA methylation15. Similar to other S100 proteins, Ca2+ binding to S100A4 promotes its interaction with target proteins16.
In this study, we found that S100A4 localizes specifically to mouse (Mus musculus) lens fiber cells, with expression being robustly upregulated in differentiating primary and secondary lens fibers. We further demonstrated that S100A4 knockout mice develop late-onset lens cortical opacification. To understand the role of S100A4 in lens differentiation and function, we evaluated the global gene expression profile of the S100A4 knockout lens using RNA-seq. This analysis revealed that, without S100A4, lens fibers exhibit a robust upregulation of the retinal transcriptome, particularly photoreceptor-specific genes and transcription factors required for specifications of rods, cones and Müller glia. These transcriptome changes are accompanied by chromatin remodeling involving histone-3 lysine (H3K) methylation. Taken together, our findings suggest that S100A4 suppresses the transcription of sensory neuron genes during lens development and differentiation.
S100A4 is robustly expressed in and distributes discretely to differentiating and mature lens fibers
The expression pattern of S100A4 in developing mouse lens was assessed by immunofluorescence staining of eye sections obtained from embryonic, neonatal and weaning stage animals. Figure 1A shows the marked upregulation of S100A4 expression in differentiating and elongating primary fibers of the embryonic lens, starting at E12.5 and continuing through weaning age (analyzed up to P21). Notably, little to no S100A4 was found in the lens epithelium and none was observed in the retina, cornea or other ocular tissues. Despite the previously published suggestion that S100A4 may be expressed in the bovine retina17, we did not detect S100A4 in mouse retina. Whether this discrepancy is related to a species-specific difference requires further evaluation in a future study.
Consistent with immunofluorescence results, S100A4 was detected only in lens fiber cell fraction with almost no S100A4 detected in the epithelial fraction (Fig. 1B). At the subcellular level, S100A4 exhibits a cytoplasmic distribution pattern with no obvious presence in the nucleus of lens fiber cells (Fig. 1A, bottom right panel). Additionally, to determine whether S100A4 is present in the nuclei of lens fibers, the nuclear fraction was extracted from P30 lens fibers and subjected to immunoblotting. Whereas some S100A4 was detected in the nuclear fraction, its relative amount did not exceed that of contaminating GAPDH used as a cytosolic marker (Supplementary Fig. S1A), which suggests that S100A4 is essentially a cytosolic protein in the lens. Immunoblot analysis further confirmed that S100A4 is a soluble protein with only a trace amount detected in the membrane-enriched fraction (Fig. 1C). Because S100 proteins can be secreted and interact with various cell surface targets to regulate cellular activities9, we tested whether S100A4 could be secreted from the lens. Lenses from P22 old mice were organ cultured for 48 h and conditioned medium was concentrated and subjected to immunoblotting. However, no detectable S100A4 was observed (Supplementary Fig. S1B).
Additionally, expression of S100A4 and several other members of the S100 family, was reliably detected in RNA-seq analyses of P30 mouse lenses (Supplementary Table S1). The relative levels of S100A6 expression (relative units-RU: 23,779) was found to be higher than that of S100A4 (14,401 RU), while the expression levels of S100A10 (4,056 RU), S100A11 (1,155 RU), S100A16 (923 RU), S100A1 (189 RU), S100A13 (39 RU) and S100B (23 RU) were much lower. Consistent with our findings, the iSyTE database for the gene expression profile of mouse lens also confirms expression of various genes of the S100 family18.
S100A4 deletion induces late-onset lens opacification in mice
Having found that S100A4 is robustly expressed in the wild type (WT) mouse lens, we asked whether S100A4 is required for lens development and function. We examined the ocular phenotype of the well-characterized S100A4−/− mouse, which has been shown to grow and breed normally19. We confirmed absence of S100A4 expression in S100A4 −/− mouse lenses by immunofluorescence analysis (Fig. 2A). These mice displayed bilateral opalescent cataract with onset at ~ 8 months of age (Fig. 2B,C), with the phenotype being much more prevalent in female animals. While there was no difference in lens weight between S100A4−/− (7.15 ± 0.08 mg/lens; n = 10) and WT (7.11 ± 0.04 mg/lens; n = 8) mice, histological analysis of S100A4−/− mouse cataractous lenses revealed swollen and disorganized fibers at the outer cortical region exhibiting abnormal accumulation of vacuoles, relative to those in the nuclear region (Fig. 2D). Additionally, none of the lenses from the S100A4−/− mice exhibited a nuclear cataract as shown in Fig. 2C. These data demonstrate that S100A4 is required for maintenance of lens transparency. Further studies are required to understand the molecular basis of cataract formation and apparent gender-related difference in cataract formation in S100A4−/− mice.
S100A4−/− mouse lens maintains normal actin cytoskeleton organization, calcium level, and gap junctional activity
We next explored whether S100A4 plays a role in actin cytoskeleton organization, calcium homeostasis, and gap junctional activity, all of which are key determinants of lens transparency20,21. These studies did not uncover any significant differences between mature lenses of S100A4−/− and littermate WT mice, either in actin cytoskeletal organization (1 month-old mice; Supplementary Fig. S2A), levels of calcium (2 month-old mice, Supplementary Fig. S2B), and gap junctional coupling activity (2 month-old mice, Supplementary Fig. S2C), suggesting that S100A4 does not influence any of these important characteristics in the mature lens. Whether these characteristics are altered as a function of aging in the S100A4−/− mouse lens needs to be addressed in future studies.
S100A4 deletion induces aberrant gene expression in the mouse lens
To gain insight into the role of S100A4 in lens function, we analyzed gene expression profiles of 1-month-old S100A4−/− and their WT littermates using RNA-seq. Two biological replicates (derived from 6 to 12 pooled lenses/sample) were used per group. The total number of expressed genes was 14,021 in WT (Supplementary Table S1) and 14,925 in S100A4−/− (Supplementary Table S1) lenses. Principal component analysis revealed that the greatest contributor to variance of individual gene counts (by 95%) was the genotype (Fig. 3A). The variation between biological replicates of the same group was much smaller (by 4%) (Fig. 3A). Consistently, heat map construction (z-score normalized) and hierarchical clustering of the differentially expressed genes (DEGs; which we define as genes exhibiting a fold-change ≥ 2 and an adjusted p-value ≤ 0.05) revealed a closer clustering between the two S100A4−/− and two WT biological replicates than among S100A4−/− and WT transcriptomes (Fig. 3B).
This analysis revealed a dramatic upregulation of several DEGs in S100A4−/− lenses (Fig. 3B). A volcano plot in Fig. 3C demonstrates that the number of upregulated genes in the knockout was much higher than the number of downregulated genes. Based on the criteria of fold-change ≥ 2 and adjusted p-value of ≤ 0.05, 383 unique transcripts were upregulated while 5 gene transcripts exhibited downregulation in S100A4−/− lenses (Supplementary Table S2). The fact that ~ 99% of DEGs were upregulated suggests that S100A4 functions to suppress gene expression in the lens. All raw RNA-seq data and normalized expression values are available in the Gene Expression Omnibus with accession number- GSE143909.
Gene Set Enrichment Analysis (GSEA) including gene ontology (Fig. 3D) and curated pathways from MSigDB (containing KEGG and Reactome data) (Fig. 3E), revealed that the genes upregulated in S100A4−/− lenses encode proteins involved in sensory perception, neuronal system processes, rod and cone phototransduction pathways, transport of amino acids, G protein signaling, synaptic transmission and matrisome, and that the promoters of several upregulated genes contained a high density of CpG islands bearing trimethylation marks at histone 3-lysine (H3K27 and H3K4). Figure 3D,E depict the proportion of upregulated vs. downregulated genes within the indicated pathways.
Downregulated genes in S100A4−/− lenses included Rab4a, Pttg1, Cck and Dynlt1b (Supplementary Table S2). GSEA did not reveal significant gene enrichment for any of the downregulated genes in S100A4−/− lenses.
Robust induction of the olfactory sensory neuron gene S100A5 in S100A4−/− lenses
Interestingly, the gene with the highest level of induction in the S100A4−/− lens was S100A5 (~ 275-fold) (Supplementary Table S1). S100A5 is normally expressed in distinct brain regions22, most notably the olfactory bulb and olfactory sensory neurons23, where it is thought to participate in olfactory signaling24. We confirmed increased expression of the S100A5 protein in S100A4−/− lenses by immunostaining and Western blotting analysis. Similar to S100A4 in the normal lens, S100A5 localizes primarily to lens fiber cells (Fig. 4A). Induction of S100A5 expression in the S100A4 knockout lens was observed starting at E16.5 (Fig. 4A). Analysis of P30 (1-month-old) lenses derived from S100A4−/− mice also confirmed a robust increase in S100A5 protein levels by immunofluorescence (Fig. 4A), immunoblotting analyses (Fig. 4B), and qRT-PCR analyses (Fig. 4C) with no signal detected in littermate WT lenses. Neither S100A4 nor S100A5 were detected by either technique in the retinas of WT and S100A4−/− mice (Supplementary Fig. S3), while lens lysate samples from WT and S100A4−/− mice showed positive bands for S100A4 and S100A5, respectively (Supplementary Fig. S3). S100A5 is one of the least studied members of the S100 family25.
Upregulation of retina-specific genes in S100A4−/− lenses
In addition to S100A5, the most significantly upregulated genes in S100A4−/− lenses encode transcription factors regulating retinal and photoreceptor development (Nrl, Crx, Nr2e3, Rax, Vsx2, Otx1 and 2, neurod1 and Rorb) as well as proteins regulating retinogenesis (Mab21l2) and performing various photoreceptor-specific functions26,27. The latter included rhodopsin (Rho), transducin (Gngt1 and Gnat2), peripherin-2 (Prph2), Rom1 (Rom1), arrestin-1 (Sag), rhodopsin kinase (Grk1), cGMP phosphodiesterase (Pde6 a, b and g), recoverin (Rcvrn), guanylate cyclase (Gucy2F), complexins (Cplx) and others (Table 1 and Table S2).
Accordingly, the GSEA analysis revealed an enrichment of genes belonging to the rod and cone phototransduction, “G-alpha1 signaling”, “neuronal system”, “transmission across chemical synapse”, “sensory perception”, “neurological system process” and “response to light stimulus” pathways (Fig. 3D). Figure 5A,B volcano plots showcase the DEGs involved in the phototransduction pathway and sensory perception, respectively. The upregulation of several retina and photoreceptor-specific genes in S100A4−/− lenses was confirmed by qRT-PCR (Fig. 5C). Additionally, immunoblot and immunofluorescence analyses showed increased levels of several proteins encoded by these genes in S100A4−/− lenses (Fig. 5D–F). Having recorded these results in P30 lenses, we also asked whether upregulation of some of these retinal genes begins earlier at the neonatal stage. Using qRT-PCR analysis of RNA derived from pooled S100A4−/− lens samples, we confirmed a significant increase in expression of selected genes including Nrl, Otx2, Rho, Rcvrn and Slc61a in P1 mice (Supplementary Fig. S4). Taken together, these results indicate an aberrant induction of retina-specific genes in S100A4−/− lenses.
Furthermore, to identify upstream regulators involved in altered gene expression in S100A4−/− lenses, we performed network analysis to determine enrichment of downstream genes using the Qiagen Ingenuity Pathway Analysis (IPA) tool. This analysis identified Crx, Rho and Nrl as the top three, most significant upstream regulators (Supplementary Table S3). Crx and Nrl are known crucial transcriptional regulators of photoreceptor development and differentiation27,28, while Rho is a key light-sensitive receptor involved in visual phototransduction29. Of note, induction of photoreceptor-specific differentiation programs has been similarly reported in medulloblastoma under the control of transcription factors Nrl and Crx30. Interestingly, the deficiency of Notch2 and E2F has also been found to be associated with upregulation of certain retinal genes in the mouse lens18.
S100A4−/− lenses also displayed an induction of genes encoding various extracellular matrix glycoproteins and collagens, including reelin, interphotoreceptor matrix proteoglycans, Efemp1, fibulins, endostatin producing collagen XVIII (Col18a1) and several others (Supplementary Table S2 and Supplementary Fig. S5A). It is noteworthy that overexpression of endostatin producing collagen XVIII has been previously reported to induce cataracts in transgenic mice31. RT-PCR (Supplementary Fig. S5B), immunoblot (Supplementary Fig. S5C,D) and qRT-PCR (Supplementary Fig. S5E) confirmed a significant increase in the levels of collagen XVIII in S100A4−/− lenses relative to WT controls. Therefore, it is plausible that altered collagen XVIII expression in the S100A4−/− lens is at least partially responsible for the observed lens opacification.
Upregulation of genes responsible for Müller cell differentiation, glutamate metabolism and solute transport in the S100A4−/− lens
The transcriptome of S100A4−/− lenses displayed an upregulation of two transcription factors Lhx2 and Plagl1, required for Müller cell specification and differentiation32,33. These glial cells serve to support retinal neurons and play a crucial role in maintaining the structural and functional stability of the retina34. Other Müller cell-specific or abundant genes upregulated in the S100A4−/− lens included glutamine synthetase (Glul), retinaldehyde binding protein 1 (Rlbp1), glutamate transporters (Slc1a3, Slc17a7, Scl17a8), GABA transporters (Slc6a13, Slc6a1, Slc6a6, Slc6a9), glutamate receptor (Gria3) and glutamate decarboxylase (Gad1) (Supplementary Table S2; Fig. S6A). We independently confirmed the increased expression of glutamine synthetase (Glul) by RT-PCR (Supplementary Fig. S6B), qRT-PCR (Supplementary Fig. S6C) and immunoblotting (Supplementary Fig. S6D, E). These results together with upregulated expression of photoreceptor specific genes in S100A4−/− mouse lens reveal an aberrant induction of retinal differentiation programs in the lens in the absence of S100A4.
S100A4−/− lenses also exhibited upregulated expression of several solute carrier transport genes including Slc4a10, Slc4a1, Slc16a2, Slc12a5, Slc26a4, Slc24a1, Slc7a1, Slc38a8, Slc5a5, Slc22a8, Slc4a5, Slc13a4 and other transporters (Supplementary Table S2; Fig. S7A, B). We not only confirmed increased expression of Slc6a6 which encodes the taurine transporter (TauT) in S100A4−/− lenses (Supplementary Fig. S7B), but also found that levels of this protein were markedly elevated in S100A4−/− lenses (Supplementary Fig. S7C, D). Taurine is a well-characterized osmolyte and has been shown to influence S100A4 expression35.
Interestingly, lenses of older but not young S100A4−/− mice develop cataracts characterized by swelling of lens cortical fibers (Fig. 2). It appears that the presumed osmotic changes resulting from dysregulated expression of various solute carrier transport genes identified above including glutamate, GABA, taurine and others, might not per se lead to cataract formation in young S100A4−/− mice, since dysregulation of solute carrier and extracellular matrix gene expression was evident in P30 S100A4−/− mice. However, the effects of these changes are likely exacerbated by age-related stress factors, leading to cataract development in older S100A4−/− mouse lenses.
Expression of non-lineage genes in the S100A4−/− lens is associated with changes in H3K27 and H3K4 trimethylation
Interestingly, based on gene set enrichment analysis, S100A4−/− lenses upregulated a large number of genes whose promoters contain an intermediate or high density of CpG islands bearing trimethylation marks at histone-3 lysine 27 (H3K27), histone-3 lysine 4 (H3K4) and bivalent H3K27 and H3K4 (Fig. 3E). These histone methylation markings are known to be involved in determining cell state and lineage potential of embryonic stem cells, neural progenitor cells and embryonic fibroblasts36,37. Moreover, examination of individual genes within these gene sets identified many photoreceptor- and retina-specific genes including Nrl, Neurod1, Six6, Rax, Reln, Gnat1, Best2, Rtbdn, Pde6b, Optc, Kcnb1, Slc24a1, Cacng3, Gucy1a2 and several others (Fig. 6A). Since trimethylation of H3K27 and H3K4 has been well-established to regulate gene suppression and activation, respectively38,39, we addressed whether S100A4 expression in the lens may affect H3K methylation. This was accomplished by quantitative ELISA-based analysis of trimethyl H3K27 and H3K4 levels in the total histone fraction extracted from S100A4−/− and WT lenses at P30. While there was a significant decrease in the levels of H3K27me3 in the knockout (Fig. 6B), the levels of H3K4me3 increased significantly (Fig. 6C). Methylated H3K27 and H3K4 were normalized to total histone content in these analyses. The decrease in H3K27me3 was corroborated by immunostaining (Fig. 6D,E).
In this study, we report a novel role for S100A4 in the suppression of non-lineage genes in the developing lens (Fig. 7). We demonstrated that S100A4 is specifically expressed in fiber cells of the lens and that the loss of S100A4 in the lens leads to an aberrant induction of genes involved in differentiation of the retina and eventual lens opacification. Importantly, the induction of retinal genes in the S100A4 knockout lens includes several retina- and photoreceptor-determining transcription factors. Thus, S100A4 appears to play a critical role in lens differentiation by repressing genes responsible for retinal specification at the point when the retina and the lens are separating in development.
This novel function of S100A4 in lens resembles its previously reported role to suppress the expression of osteoblast-specific genes and transcription factors in periodontal ligament cells40. Additionally, S100A4 regulates transcriptional programs during the development of mature microfold cells in Peyer’s patches41, suggesting that S100A4 may have a broad role in transcriptional regulation across many tissues. In this study, absence of S100A4 induced expression in the lens of several transcription factors including Crx, Nrl, Rax,Otx2,Vsx2 and Nr2e3, other regulators (Mab21l2) and their target genes which have been demonstrated to be critical for the development and function of retina and photoreceptors26,27,42.
Our findings raise several mechanistic questions regarding S100A4 function. The first is how S100A4 suppresses transcription. Many genes whose expression is altered in S100A4−/− lenses are enriched with promoters containing a medium-to-high density of CpG islands regulated by histone trimethylation, including H3K27me3 and H3K4me3 modifications. Consistently, these two histone trimethylation marks are altered in S100A4−/− lenses, suggesting that abnormal transcription in the S100A4−/− lens is potentially regulated by epigenetic mechanisms. Of note, changes in H3K27 and H3K4 trimethylation have been similarly reported to contribute to retinal development43. Similarly, histone modification regulated by the lysine acetyltransferases (CBP and p300), has been shown to play a crucial role in lens fate determination44. The second question concerns how epigenetic changes are induced by S100A4 deletion. Expression of genes encoding histone and DNA methyltransferases and demethylases, protein components of the polycomb (PRC2) and/or trithorax complexes and nucleosomes, all of which are involved in regulating chromatin remodeling45,46 were not altered significantly in lenses lacking S100A4. Another intriguing question relates to how S100A4 functions in transcriptional repression, given its cytoplasmic localization in lens fibers. Although S100A4 has been found in the nuclei of other cells3,10, we did not observe the protein in the nuclei of lens fiber cells. Interestingly, S100A10, which belongs to the S100 family of proteins has been demonstrated to regulate breast cancer stem cell specification and pluripotency by regulating H3K27me3 chromatin marks47. While S100A5 is robustly upregulated in the absence of S100A4, it remains unclear as to what role (if any) S100A5 plays in the S100A4−/− lens. An observation potentially relevant in this context is that S100A5 expression is upregulated or induced in response to a change from H3K4 dimethylation to trimethylation during embryonic stem cell differentiation48. Overall, there is little evidence for any functional redundancy among members of the S100 family9,25 despite their significant homology. Whether the robust upregulation of S100A5 expression starting from the embryonic stage in S100A4−/− lenses has any role in induction of retinal genes needs to be addressed in future studies.
Interestingly, although the aberrant induction of retina and photoreceptor differentiation programs in lenses lacking S100A4 starts from the early neonatal developmental stages, this abnormality does not appear to, per se, overtly perturb lens morphogenesis and growth. However, it is likely that interactions between age-related stress factors and dysregulated expression of retinal and Müller glia genes, especially those engaged in solute carrier transport and encoding extracellular matrix proteins, secondarily impair lens clarity in older S100A4−/− mice. The impairment of lens clarity likely results from osmotic imbalance and leads to the development of late onset cortical cataract under the absence of S100A4 (Fig. 2).
In conclusion, our study revealed a novel role of S100A4 in gene suppression and cell fate determination in the ocular lens. The goals of future studies are to elucidate this S100A4 function on the mechanistic level and to understand how the absence of S100A4 induces late-onset cortical cataracts.
All experiments using mice were carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Duke University School of Medicine. This study was also carried out in compliance with the ARRIVE guidelines. The S100A4 −/− (null) strain mouse used in this study was maintained on a C57BL/6 J background and has been thoroughly characterized previously19. Both WT (C57BL/6 J strain) and littermate WT mice were used as controls in the S100A4−/− mouse studies. Both male and female mice were used in the described studies. Mice were maintained under a 12 h dark and light cycle with ad libitum food and water as we described earlier49. S100A4−/− and littermate WT mouse genotyping was carried out using PCR analysis of tail DNA and the oligonucleotide primer sets (Supplementary Table S4) described previously49. At required gestational ages, pregnant dams were anesthetized with Euthasol (Virbac AH, Inc., Fort Worth, TX, USA) and fetuses were extracted by hysterectomy.
Mouse eye and lens imaging
Images of cataractous and transparent lenses from live S100A4−/− mice were captured using a Nikon D7500 Wi-Fi 4 K Digital SLR Camera. Dark illuminated dissected lenses of S100A4−/− and WT mice were imaged using a Zeiss Stemi 2000-C stereomicroscope equipped with an AxioCam ERc 5S camera (Carl Zeiss Microscopy, LLC, White Plains, NY, USA).
Enucleated eyes from S100A4−/− and littermate WT mice (8- to 9 month-old) were fixed in 3.7% buffered formalin for 48 h at room temperature. The fixed specimens were subsequently dehydrated, embedded in paraffin, and cut into 5 µm thick sections with a rotary microtome (Leica Biosystems, Buffalo Grove, IL, USA), prior to staining with hematoxylin and eosin as we described earlier49. Images were captured using a Zeiss Axio Imager equipped with a Hamamatsu Orca ER monochrome CCD camera (Carl Zeiss Microscopy, LLC).
RNA extraction, library construction and RNA-seq analysis
For RNA extraction, P30 lenses derived from S100A4−/− and littermate WT mice were stored in RNAlater at − 80 °C until extraction. Six to twelve lenses were pooled per sample for the WT1, WT2, S100A4−/− 1 and 2 samples. RNA was isolated using the RNeasy Micro kit (Qiagen, Inc., Valencia, CA, USA) with a minor modification. Tissue homogenates prepared in RLT buffer containing β-mercaptoethanol were incubated with proteinase K solution (20 mg/ml for 15 min at room temperature) prior to following the extraction protocol provided in the manufacturer’s instructions.
RNA was quantified using a Qubit instrument (Thermo Fisher Scientific, Waltham, MA USA). RNA quality was analyzed on an Agilent TapeStation Model 2200 (Agilent Technologies, Santa Clara, CA, USA). All samples yielded RIN numbers > 7. Stranded mRNA-seq libraries were constructed using the Kapa kit (Kapa Biosystems, Inc, Wilmington, MA, USA) and following manufacturer's instructions. Libraries were indexed using a single 6 bp index allowing for multiple libraries to be pooled. RNA-Seq libraries were then normalized to a concentration of 10 nM, and pooled in equimolar ratio. The final pool of libraries was sequenced in one lane of an Illumina HiSeq 4000 sequencing flow cell (Illumina, Inc, San Diego, CA) with a 50 bp single end read length. Resultant raw bcl files were converted into fastq files and sequences demultiplexed using Illumina bcl2fastq conversion software version 2 (Illumina, Inc).
RNA-seq transcriptome-based analysis of differential gene expression
RNA-seq raw data were processed using the Trim Galore toolkit which employs Cutadapt to trim low quality bases and Illumina sequencing adapters from the 3′ end of the reads. Only reads that were 20nt or longer after trimming were used for further analysis. Reads were mapped to the mouse genome and transcriptome (GRCm38v73 version) using the STAR RNA-seq alignment tool50,51. Gene counts were compiled using the HTSeq tool for reads that mapped to a single genomic locus. Only those genes that had at least 10 reads in any given library were used in subsequent analysis. Normalization and analysis of differential expression was carried out using the DESeq2, Bioconductor package with the R statistical programming environment52,53. We considered a gene differentially expressed if exhibits a greater than or equal to two-fold difference in expression between the S100A4−/− and littermate controls, and an adjusted P value of less than or equal to 0.05. Gene Set Enrichment Analysis (GSEA)54 was performed to identify differentially regulated pathways and gene ontology terms. The false discovery rate (FDR) was used to correct for multiple hypothesis testing, and pathways were considered significant if the FDR ≤ 5%. Network analysis was performed using the Qiagen Ingenuity Pathway Analysis (IPA) tool to identify upstream regulators of differentially regulated genes in S100A4−/− mouse lenses.
To confirm the results of RNA-seq based identification of DEGs in S100A4−/− lenses, qRT-PCR analysis was performed for selected DEGs. For this, RNA was extracted from pooled P1 or P30 S100A4−/− and littermate WT mouse lenses as described above using an RNeasy Micro kit and reverse transcribed using the Advantage RT for PCR Kit (Clontech Laboratories, Inc., Mountain View, CA, USA). Reverse-transcribed single-stranded cDNA and gene-specific forward and reverse oligonucleotide PCR primers (Table S4) were used for qRT-PCR analyses as we described previously49. A few of the selected gene transcripts were also amplified by RT-PCR analyses and the amplified DNA products were separated on an agarose gel and visualized with GelRed Nucleic Acid Stain (Biotium, Hayward, CA, USA) and imaged using Chemidoc Touch (Bio-Rad, Hercules, CA, USA). The amplified DNA products were excised, purified using a gel extraction kit (Qiagen, Inc., Valencia, CA, USA) and sequenced to confirm gene identity.
Histone extraction and histone-3K trimethylation analysis
The EpiQuik Total Histone Extraction Kit (Epigentek Group Inc. Farmingdale, NY) was used to extract total histone from P30 S100A4−/− and littermate WT lenses as per manufacturer’s instructions. Total histone protein isolated from the S100A4−/− and littermate WT lenses were used to quantify H3K27 and H3K4 global trimethylation using EpiQuik Global H3K27 and H3K4 Tri-Methylation Assay Kits (Epigentek Group Inc.). Briefly, the levels of histone proteins tri-methylated at K27 or K4 were quantified by colorimetric analysis at 450 nm using high-affinity antibodies specific to trimethylated H3K27 or H3K4 in conjunction with horse radish peroxidase conjugated secondary antibody and a SpectraMax M5 microplate reader (Molecular Devices LLC. San Jose, CA, USA).
Measurements of intracellular calcium in lens
Lens intracellular calcium levels were measured as we described earlier21. Briefly, lenses dissected from P60 S100A4−/− and littermate WT mice were placed in a Sylgard Petri dish containing normal Tyrode solution (137.7 mM NaCl, 2.3 mM NaOH, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, and 10 mM glucose, pH 7.4.). A small volume of 2 mM FURA2 solution was injected into the fiber cells at different depths in the lens. FURA2 was dissolved in the pipette solution containing 83 mM K-aspartic acid, 17 mM KCl, 10 mM NaCH3OSO3, and 5 mM Hepes, and pH adjusted to 6.9 with KOH. Images were acquired and the ratios of fluorescence emission calculated from 360 and 380 nm excitation curves. The ratios were converted into [Ca2+]i using the [Ca2+] calibration curve corresponding to the appropriate lens depth as described previously21.
Measurements of gap junction-coupling conductance
Gap junction coupling conductance was measured as we previously described55. Briefly, one microelectrode was placed in a central fiber cell into which a wide-band stochastic current was injected. The induced voltage was recorded by a second microelectrode that was placed in a peripheral fiber cell r (cm) from the center of a lens. The impedance (induced voltage ÷ injected current) of the lens was recorded in real time using a fast Fourier analyzer (Hewlett Packard, Palo Alto, CA). The high frequency asymptote of the magnitude of the impedance is proportional to gap junction coupling resistance. The voltage recording microelectrode was advanced from the surface to the center of the lens, recording coupling at five or six locations. Data from 5 or 6 lenses were pooled to obtain the final resistance curve.
Tissue homogenates and immunoblotting
Lens epithelium and fiber mass samples derived from P21 mice and lenses derived from P30 S100A4−/− and littermate WT mice were homogenized at 4 °C using a glass homogenizer and ice cold hypotonic buffer (10 mM Tris buffer, pH 7.4, containing 0.2 mM MgCl2, 5 mM N-ethylmaleimide, 2.0 mM sodium orthovanadate, 10 mM sodium fluoride, 60 μM phenylmethylsulfonyl fluoride, 0.4 mM iodoacetamide, protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (one each/10 ml buffer; obtained from Roche, Manheim, Germany)). Tissue lysates were spun at 800 g for 10 min at 4 °C, and the protein content of the supernatant measured using a Micro BCA Protein assay kit (Thermo Fisher Scientific). For the separation of lens cytosolic (100,000 g supernatant) and membrane-enriched fractions (100,000 g pellet), the 800 g lysates prepared above were subjected to ultracentrifugation by spinning at 100,000 g for 1 h at 4 °C. Pellets were suspended in 8 M urea buffer, and protein was estimated using Pierce 660 nm protein assay reagent (Thermo Fisher Scientific).
Lens fiber cytosolic and nuclear fraction proteins of P30 WT mice were extracted using nuclear fraction extraction kit from BioVision (BioVision, Inc, Milpitas, CA, USA) by following the manufacturer’s protocol. Briefly, freshly extracted lenses were dissected to remove the capsule/epithelium and the fiber mass was used. The protein content of cytosolic and nuclear fractions was determined using BCA Protein Assay Reagent Kit. GAPDH and Histone H2B antibodies were used to confirm the purity of cytosolic and nuclear protein fractions, respectively.
For preparation of retinal lysates, mouse eyes were dissected along the Ora serrata to remove and discard the anterior chamber along with lens. The eyecup was further washed with cold PBS to detach the retina, and retinas homogenized in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing both protease and phosphatase inhibitors prior to determining protein content using Pierce BCA protein Assay Kit (Thermo Fisher Scientific).
Equal amounts of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE, using an appropriate percent of acrylamide depending on the test protein analyzed), followed by electrophoretic transfer to nitrocellulose membrane. The membranes were subsequently blocked for 2 h in 5% blocking grade milk protein in TBST buffer (Tris buffered saline containing 0.1% Tween 20) followed by incubation with primary antibodies (details are provided in Supplementary Table S5) overnight at 4 °C. After washing thoroughly, blots were incubated with appropriate secondary antibodies for 2 h at room temperature and developed by enhanced chemiluminescence using Chemidoc Touch system (Bio-Rad). Immunostained protein bands were quantified using ImageJ Software. For normalizing protein loading for membrane-rich proteins, protein fractions were resolved on SDS-PAGE, stained with Gel code blue protein stain (Thermo Fisher Scientific), and destained with distilled water prior to densitometric quantification using ImageJ software. Protein bands detected at 37 kDa and 15 kDa were used for normalization. For analysis of soluble proteins, GAPDH was immunoblotted as a loading control using anti-GAPDH antibody after stripping the respective blots.
Tissue processing, immunofluorescence and imaging
Tissue specimens from WT mice (heads of E12.5; E13.5; E14.5 and E17.5 and eyes of postnatal (P21), and S100A4−/− and littermate WT mice (head of E16.5 and eyes of P30) were fixed for cryosectioning in 4% buffered paraformaldehyde for 24 h at 4 °C, transferred into 5% and 30% sucrose in PBS (Phosphate-buffered saline) on consecutive days as we described previously49. After embedding in optimal cutting temperature media (Tissue-Tek, Torrance, CA, USA), samples were cut into 10 μm-thick sagittal sections using a Microm HM550 Cryostat (GMI, Ramsey, MN, USA) and stored at − 80 °C. Additionally, P30 mouse eyes were cut at the sagittal and equatorial plane. Air-dried tissue cryosections were treated with Image-iT FX signal enhancer (Invitrogen, Eugene, OR, USA) and blocked in blocking buffer (5% globulin-free bovine serum albumin and 5% filtered goat serum in 0.3% Triton X-100 containing PBS) for 30 min each. Tissue sections were then incubated overnight at 4 °C with primary antibodies (details in Supplementary Table S5). After washing, sections were incubated with appropriate Alexa Fluor (488 or 594) –conjugated secondary antibodies for 2 h at room temperature. For F-actin staining, preblocked equatorial sections were labeled with tetra rhodamine isothiocyanate–conjugated phalloidin (TRITC; Sigma Aldrich) for 2 h at a 1:500 dilution, washed, and mounted as described above. All representative immunofluorescence data reported in this study are based on analysis of a minimum of four tissue sections derived from four independent specimens (biological replicates). Images were captured using an Eclipse 90i confocal laser scanning microscope (Nikon Instruments, Inc., Melville, NY, USA).
For paraffin sectioning and immunohistochemistry, P30 day-old eyes from S100A4−/− and littermate WT mice were fixed and sectioned as we described previously56. Paraffin sections were deparaffinized with three changes of Xylene and hydrated with absolute alcohol and antigen retrieval was performed as we described earlier56. Tissue sections were blocked for 10 min in a humidified chamber with medical Background Sniper reducing solution (Biocare Medical, Concord, CA, USA), prior to incubation for 24 h at 4 °C with H3K27ME3 or S100A4 rabbit polyclonal antibodies. Tissue sections were washed in TBS (Tris-buffered saline) buffer and incubated with Alexa Fluor 594 and 488–conjugated secondary antibodies (Invitrogen, Grand Island, NY, USA; at a 1: 200 dilution) in a dark humidified chamber for 2 h at room temperature. After this, sections were washed again with Tris buffered saline buffer, mounted on slides using Vectamount and nail polish, then imaged using a Nikon Eclipse 90i confocal laser scanning microscope to obtain single optical images.
For staining retinal sections, P30 S100A4−/− and littermate WT eyes were enucleated and fixed for 30 min with 4% paraformaldehyde in PBS buffer, pH 7.5. The anterior chambers along with lenses were removed, and the eyecups were further fixed overnight at 4 °C. The following day, eyecups were rinsed three times for 5 min each with 1X PBS, and embedded in 4% agarose (Invitrogen). One hundred micron-thick sections from the central portion of the retina were collected into 24 well plates using a vibratome (HM 650 V, Microm). The floating sections were then incubated on an orbital shaker for 4 h with blocking solution containing 3% donkey serum and 0.1% Triton X-100 in PBS buffer. These sections were further incubated overnight with either S100A4 or S100A5 rabbit polyclonal antibodies, washed three times with PBS, and incubated for 2 h with goat anti-rabbit Alexa Fluor 488 secondary antibody (1:500). After thoroughly washing with PBS for several times, sections were stained with Hoechst (1:1000) for 15 min. Following a final wash with PBS, sections were mounted under coverslips using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA), then imaged using a Nikon Eclipse 90i confocal microscope.
Statistical analyses were performed using GraphPad Prism version 7. Mann Whitney U test was used to compare 2 groups. Data from immunoblot quantification experiments and qRT-PCR were analyzed by the Student’s t-test, a P < 0.05 was considered to define statistically significant differences between S100A4−/− and littermate controls. Values are presented as mean ± standard deviation (SD) for qRT-PCR data and mean ± standard error of the mean (S.E.M) for immunoblots. All the immunoblots were representative of 3–5 biological replicates.
All raw RNA-seq data and normalized expression values are available in the Gene Expression Omnibus with accession number- GSE143909. All other data of this study are included in the article and supplementary information files.
Bicinchoninic acid assay
Differentially expressed genes
Enzyme-linked immunosorbent assay
False discovery rate
Glyceraldehyde 3-phosphate dehydrogenase
Gene set enrichment analysis
Histone 3 lysine
Ingenuity pathway analysis
Institutional review board
Kyoto encyclopedia of genes and genomes
Molecular signatures database
Reverse transcription polymerase chain reaction
Quantitative reverse transcription polymerase chain reaction
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
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This work was supported by the National Institutes of Health: R21EY027053 to R.M., R01EY025096 to P.V.R., EY029929 to T.R.L. and P30EY5722 (Core grant). We thank Kelly Buddin and Jainming Qiu for their technical help in qRT-PCR, tissue processing, animal maintenance and immunoblotting, and Nicolas Devos, Ph.D and David Corcoran, Ph.D from Duke Center for Genomic and Computational Biology Core service for their help in RNA-seq and genomic and bioinformatics analysis, respectively. We thank Jeremy Kay, Ph.D for sharing some of the antibodies used in this study.
The authors declare no competing interests.
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Maddala, R., Gao, J., Mathias, R.T. et al. Absence of S100A4 in the mouse lens induces an aberrant retina-specific differentiation program and cataract. Sci Rep 11, 2203 (2021). https://doi.org/10.1038/s41598-021-81611-y
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