Dysfunction of heat shock factor 4 impairs retinal structure and visual function in mice and zebrafish

Dysfunction of heat shock factor 4 impairs retinal structure and visual function in mice and zebrafish | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dysfunction of heat shock factor 4 impairs retinal structure and visual function in mice and zebrafish Baixue Liu, Youfei lang, Meng Jiao Xue, Ming Jun Jiang, Xiao lin Jia, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4220460/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Loss of function of heat shock factor 4(HSF4) causes microphthalmia with lens opacification. The objective of this study is to uncover the regulation of HSF4 on retinal homeostasis. Methods Hsf4 del mutant mice and Hsf4 null zebrafish models were recruited in this study. H&E was used to determine retinal structure. The immunoblot, qRT-PCR and immunofluorescence staining were used to measure the expression of mRNA and protein. AAV2-Hsf4-Flag virus were used to the reconstitution assay. Results The retinal structure of Hsf4 del mice and Hsf4 null zebrafish, which is comparable to wild-type at P10 days old, undergoes atrophy at 7 and 13 months old. Dysfunction of Hsf4 downregulates the expression of visual cycle enzymes (e.g., RPE65, RLBP1 and RDH5 ) and heat shock proteins (e.g., HSP90 and HSP25), and simultaneously activates retinal gliosis (e.g., upregulating the expression of GFAP, GS, CRYAB, inflammatory interleukins, and VEGFA) and the expression of senescent P16 INK4a and P21 cip1 in the retina of postnatal P1- P10 mice and embryonic zebrafish, and those changes are enhanced in 7 and 13 months old mice and zebrafish. Subretinal administration of AAV2-Hsf4b to the retina of one-month Hsf4 del mice partially rescued the expression of changed proteins. ERG results showed that the downregulation of amplitude of a- and b- waves at scotopic response was detected at P15. Overexpression of Flag-Hsf4b in the in vitro cultured primary Hsf4 del RPE cells restores the expression of visual cycle enzymes and heat shock proteins. TUNEL assay shows that there are more apoptotic cells in the ONL and the RPE of 7-and 13-month-Hsf4 del retina than in P10 retina. Conclusion In addition to causing cataracts, the loss of function of HSF4 impairs the visual cycles and activates the gliosis in early postnatal age, which are associated with the retinal atrophy. Heat shock factor 4 Visual cycle Gliosis Retinal degeneration Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The retinal atrophy, which is a leading cause of blindness in the world [ 1 , 2 ], is a predominant pathological process of age-related macular degeneration and congenital pigmental retinitis, characterized by the drusen accumulation, atrophy of photoreceptor and RPE cells, and night vision deterioration [ 3 ]. Multiple risky factors, such as ageing, smoke, alcohol addiction, glaucoma, systemic diseases (hypertension, diabetes, atherosclerosis) and the genetic variations, are associated with retinal degeneration via inducing divergent pathways, e.g. oxidative stress, apoptosis or ferroptosis and inflammation [ 1 , 4 – 6 ]. Missense mutations in visual cycle-restrictive enzymes, like RPE65, RLPB1, RDH5 and LRAT, which impair the visual cycle, are causative for congenital pigmental retinitis characterized by retinal atrophy. In addition, genetic mutations in the transcription factors, such as Pax6, MITF, SOX9, which regulate eye development, results in retinal developmental defect. Hsf4 is a critical transcriptional factor for eye development. However, its regulatory roles on the retinal homeostasis remain unclear. Heat shock factor 4 (HSF4) is a member of family heat shock factors that control the expression of heat shock proteins or none-heat shock proteins in response to heat shock or other cellular proteotoxic stresses at physio-pathological conditions[ 7 , 8 ]. HSF4 gene is located in human chromosome 16q22.1 and constitutively expressed in majority tissues including ocular lens, muscle tissues, cerebral cortex, midbrain, retina, and pancreas, and exhibits diverse functions [ 8 , 9 ].The missense mutations in the DNA binding domain of HSF4 are associated with the hereditary autosomal dominant cataracts in human being[ 10 ]. Germline deletion of Hsf4 gene in mice causes microphthalmias with lens cataracts[ 9 , 11 , 12 ]. The Hsf4-deficent cataracts occur at postnatal age, characterized with lens epithelial cell hyperproliferation, and malfunction of fiber cell terminal differentiation, including the delayed degradation of the mitochondria, ER and nucleus, and down-expression of crystallin proteins[ 9 , 11 , 13 ]. In addition, mice lacking Hsf4 downregulates the tumorigenesis of lymphocytes and hepatocytes in P53-deficient mice[ 14 ]. MEF cells lacking Hsf4 undergo replicative senescence in vitro[ 14 , 15 ]. HSF4 is upregulated in intestine cancer tissue. Those divers functions are associated with HSF4’s dual transcriptional regulations. HSF4 positively regulates the expression of HSP25, CRYAB, γ-crystallins, CP49, DNase II-beta and P53, and negatively controls the expression of P21, FGF4/7, vimentin and HSP70[ 9 , 11 , 16 ]. The hyperproliferation of Hsf4-deleted lens epithelial cells is associated with the down-expression of P53 or upregulation of FGF4/7 signaling[ 9 , 17 ]. However, this phenomenon is not observed in the Hsf4 del mutated mice[ 13 ]. Instead, Hsf4 del lens epithelial cells undergo senescence via upregulating P21 expression[ 13 ]. HSF4 upregulates DNAase II beta expression, which may be associated with fiber cells’ nuclear degradation[ 18 , 19 ]. In addition, HSF4 participates in regulating lysosomal activity by regulating CRYAB-mediated ATP6V1A protein stability, a component of V-ATP-pump complex[ 20 ], and/or the expression of autophagic protein ATP9a in zebrafish and mouse lens[ 21 ], which are postulated to be associated with the degradation of membrane organelles during fiber cell terminal differentiation. In this paper, we studied the regulatory roles of HSF4 on retinal homeostasis by using a Hsf4 del mice [ 21 ] and Hsf4 null zebrafish[ 17 ]. Hsf4 del mice express a short Hsf4 with 42 amino acids-in-frame deletion from amino acids116 to158. We found that HSF4 was expressed in retinal tissues and essential for retinal development. HSF4 mutants impaired the expression of visual cycle enzymes, activated gliosis at the early postnatal retina, and induced retinal disorder in 7 and 13 months old. These results highlighted that HSF4 was an indispensable factor for retinal development. It participates in regulating the visual cycle and glia homeostasis during early postnatal development. 2. Materials and Methods Antibodies and Plasmids HSP70 (6B3) Rat mAb (#4873), P21 Waf1/Cip1 (12D1) Rabbit mAb (#2947), P16 Rabbit mAb (#80772) were purchased from Cell Signaling Technology (Shanghai, China).The rabbit polyclonal antibodies against GAPDH (#10494-1-AP), DYKDDDK (Flag) (#66008-3-lg), HSF4 (#22883-1-AP), GFAP (#16825-1-AP), RLBP1 (#15356-1-AP), GNB1 (#10247-2-AP), Alpha(B) crystallin (#16808-1-AP), alpha-tubulin (#11224-1-AP), ZO1 (#21773-1-AP) were from Proteintech (Wuhan, China). The mouse polyclonal antibody against RDH5 (#sc-377057) and the mouse monoclonal antibodies against Glutamine Synthetase (E-4) (#sc-74430), Opn1sw (1B10) (#sc-517304), Opn1lw/mw (7GB) (#sc-517301) were from Santa Curz Biotechnology (Texas, USA). The mouse monoclonal antibody against Rhodopsin (#ab5417) and the rabbit monoclonal(EPR5477) antibody against HSP25/27 (#ab109376) were from Abcam (Cambridge, MA, USA). The rabbit polyclonal antibody against RPE65 (CQA3629) was from Cohesion Biosciences (London, E14 2DN, UK), The rabbit polyclonal antibody against GNAT2 (PM075) was from MBL (Tokyo, JAPAN ). The mouse monoclonal Antibody against ZO1 ( # 33-9100) was from Invitrogen (Waltham, MA, USA). The HRP-conjugated goat polyclonal anti-rabbit IgG (#SA00001-2), HRP-conjugated goat polyclonal anti-mouse IgG (#SA00001-1) and HRP-conjugated goat polyclonal anti-rat IgG (#SA00001-15) were from proteintech (Wuhan, China). The Alexa Fluor 488 goat anti-mouse IgG antibody (#A32723) and Alexa Fluor 594 goat anti-rabbit IgG antibody (#A32740) were from Invitrogen (Waltham, MA,USA). The Alexa Fluor 594-Goat Anti-Mouse IgG H&L (#ab150116) and Alexa Fluor 488-Goat Anti-Rabbit IgG H&L (#ab150080) were from Abcam (Cambridge, MA, USA). Plasmids: The p3xFlag-CMV7.1-HSF4b: full-length HSF4 cDNA were cloned into the 3x Flag CMV 7.1 vector at EcoRI and BamH1. The AAV2-Flag-HSF4: The human HSF4 cDNA was subcloned into the pTransfer vector at restrictive enzymes sites of Kpn1 and EcoRI. The recombinant plasmids were verified by sequencing. Animals C57/BL6 and C57/BL6/Hsf4 del mice were fed and bred in an SPF-grade animal room. The Hsf4 del mutant mice were established in our lab, in which A 126 bp DNA fragment were deleted from exon 5, resulting in a mutant of Hsf4 with an in-frame deletion of 42 amino acids (Hsf4 del )[ 13 ]. The Hsf4 del mice developed cataracts in the middle postnatal period. HSF4 null zebrafish were presented from Dr. Mugen Liu[ 17 ]. Wild type (Danio rerio) and Hsf4 null zebrafish were maintained at a standard 14 hr light:10 hr dark cycle (LD). The animals were operated following the ARVO statement regarding the use of animals in the field of ophthalmic and visual research. Animal euthanasia and surgical protocols were approved by the Ethics Committee of Zhengzhou University. Reconstitution of Hsf4 into the primary mouse RPE/Hsf4 del cells in vitro For culturing the primary mouse RPE cells, the isolated eyeballs from 2-month wild-type and Hsf4 del mice were recruited. The RPE cells were separated from the neuroretina and the sclera-choroid following the protocols presented in the previous literature[ 22 ]. Briefly, the eyeballs were incubated in 2% (wt/vol) pre-warmed dispase II in a 37°C incubator for 45 min followed by rinsing twice in DEME media. The cornea and the lens were separated and discarded. The left posterior retinal cup were rinsed in DMEM media twice and cut into four flaps. The neuroretina was teared away. The remainder of the eye cup containing RPE was cut and flatted. The RPE tissue was scraped off followed by incubating in 0.25% trypsin-EDTA buffer for 10 minutes. The cells were pelleted by centrifugation at 1000 RPM for five minutes and suspended in DMEM/F12 media containing 20% FBS and antibiotics. 10^6 cells were seeded into a 10 cm dish and cultured for 3 or 4 days. The cells were passaged for total three generations. For reconstitution of Flag-Hsf4, the plasmid pCMV-Flag-Hsf4b was transiently transfected into the primary cultured RPE cells by using the lipofectamine 3000 following the protocol with the kit. Quantitative real-time PCR (qRT-PCR) Total RNA extraction was carried out using RNAiso reagents. The cDNA was synthesized following the protocol of reverse transcription kit. An equal amount of cDNA was mixed with Fast-start Universal SYBR Green Master Mix (Roche, San Francisco, CA, USA) and amplified in PCR machine ABI 7500 system (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression in each sample was displayed as 2 −ΔΔCt values, and the data was representative of at least three independent experiments. The used primers were listed in table 1. Immunoblot The retinal tissues, or cultured cells were lysed in RIPA buffer (Sigma-Aldrich, Missouri, USA) containing 1x cocktail protease inhibitor. The protein was separated in sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) followed by electrotransferring to the PVDF membranes. The membranes were blocked in 5% (v/v) BSA at room temperature for 1 h and then incubated with the primary antibody overnight at 4℃. After washing with PBST buffer, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG at room temperature for 1 h. The membranes were developed with SuperLumia enhanced chemiluminescence (ECL) reagents, and the signals were captured with Amersham Imager 680. The Image J was used to quantitate protein intensity in the blots. Gross Histology, immunofluorescence, and whole-mount immunofluorescence staining The C57BL/6 mice were deeply anesthetized with IP administration of ketamine (80–100 mg/kg) and xylazine (5–10 mg/kg), and euthanized by cervical dislocation. The eyeballs of mice were isolated and fixed with FAS solution (Servicebio, China) for 24h, and embedded in paraffin (Servicebio, China). The samples were sliced into 3 µm thickness, and the slices were subjected to H&E staining followed by the protocol provided with kit. For Immunofluorescence staining, the tissue sections were treated with 0.2% (v/v) Triton X-100 for 10 min followed by blocking with 5% BSA for 1 h at room temperature, and then incubated with the primary antibodies at 4℃ overnight. After rinsing in PBST buffer for three times, the sections were incubated with secondary antibodies for 1h at room temperature and counterstained with DAPI for 10min. The fluorescent images were captured with a confocal microscope (Zeiss, German). For whole-mount immunofluorescence, the eyeballs were fixed with 4% paraformaldehyde (PFA) for 40 mins and then dissected in a petri dish containing cold PBS buffered (PH 7.4) to discard the cornea and the lens from the eye. The left eye cups were cut and flatted. The neuroretina was removed from the RPE-choroid-sclera. The left flatted RPE-choroid-sclera were subjected to immunofluorescent staining. For Immunohistochemistry staining, Paraffin sections from the eyes of mice were depigmented by 5% KOH:20% H2O2:ddH2O. The sections were oven dried at 60℃, and dewaxed, hydrated, antigen repair (Tris-EDTA, PH9.0). The immunohistochemistry was performed following the protocol with UltraSensitiveTM SP kit (MXB, Fuzhou, China). Briefly, the sections were put into sequentially in the Reagent 1(an internal peroxidase inhibitor) and the Reagent 2 (a general staining blocker or blocker) for 10 minutes, then incubated with primary antibody at 4°C overnight, after that, the sections were incubated with Reagent 3 (biotin-tagged sheep anti-mouse or rabbit anti-sheep IgG polymers) and the Reagent 4 (streptavidin-antibiotic protein peroxidase) for 10 minutes. The signals was developed by using DAB (MXB, Fuzhou, China). The result of IHC was taken at the light microscope. TUNEL assay TUNEL test was conducted following the protocol of the TMR (Red) Tunel Cell Apoptosis Detection Kit (Servicebio, China). The sections were incubated with proteinase K (20ug/ml) at 37℃ for 20 minutes. After washing in PBS, the section were permeabilized in 0.2% (v/v) Triton X-100 for 10 minutes at room temperature, and then incbubated with freshly prepared TdT buffer at 37℃ in the dark for 1 hour. The sections were then counterstained with DAPI for 10 minutes. The sections were thoroughly washed for 4 to 5 times with PBS, and the signals were taken by using a confocal microscope (Zeiss, German). PAS staining and Oil Red O Staining For PAS staining, the sections were stained with PAS solution following the protocol with kits (Servicebio, Wuhan, China). Briefly, the sections were incubated in solution B (containing 0.5% periodic acid) for 10 to 15 minutes. After rinsing in water, the sections was placed in solution A (containing shiff’s reagent) in the dark for thirty minutes followed by water rinse three times. The sections were counterstained in hematoxylin solution for 3 s and blue-return solution for 3 s to stain the nucleus. After this, the sections were dehydrated covered with coverslips and mounted using Xylens based mounting media. The signals were photographed under microscope with 40 x resolution lens. For Oil-red staining, the frozen sections of the retina were fixed in 4% PFA for 15 minutes, and then stained with Oil Red O staining solution (Servicebio, Wuhan, China) followed by the manufacturer's instructions. Briefly, the slices were incubated with oil red solution for 8 to 10 minutes in the dark. After that, the sections were put into two vats containing 60% isopropyl alcohol for 3 or 5 seconds followed by rinsing in water for 10 seconds. The slices were stained with hematoxylin solution for 3–5 minutes followed by washing in distilled water for 10 seconds. Signals were photographed under the light microscope. Subretinal injection The Hsf4 del mice at P15 were subretinally injected once with two microliter of recombinant AAV2 virus expressing empty vector (AAV2-Vector) or Hsf4-Flag (AAV2-Hsf4-Flag) at MOI 10^8 followed the protocol of published paper [23] . The mice then were allowed to recover at control condition for 2 weeks. And AAV2 virus were also subretinally injected to one month old Hsf4 del mice followed by two months recovery. After that, The mice were measured with ERG, and the retinal tissues were collected for immunofluorescence, immunohistochemistry, immunoblot or qRT-PCR assays. Electroretinogram (ERG). The wild-type, Hsf4 del mices were given the ERG test followed the previously reported protocol[ 24 ]. Briefly, Mice were anesthetized with IP administration of ketamine (80–100 mg/kg) and xylazine (5–10 mg/kg), and maintained on a warm pad at 37℃ to keep body temperature. Animals received tropicamide and phenylephrine chloride eyedrops to dilate the pupil. Every procedure is conducted in dim red light. ERGs was recorded with Reti-MINER-C, a visual electrophysiology system ((AiErXi Medical Equipment Co., Ltd., Chongqing, China). After dark adaption for 12 h, the mice were given a range of stimulus intensities from − 3 and 1 log cd-s/m 2 to reflect optic rod cell function. Mice were light-adapted for 10 mins to a light background set at 30 cd/m 2 . Capturing light-responsive waves in stroboscopic stimuli (0 and 1 log cd-s/m 2 ) as a reflect of optic cone cell function. To maintain consistent responses, a minimum of three tests were conducted at every level of stimulus intensity. Examining the light reaction involves gauging the intensity of the a- and b-waves. Statistical Analysis The densitometry quantitation of proteins in immunoblot was measured by using Image J. SPSS 17.0 and GraphPad Prism 8 were used for data analysis. 2-way ANOVA and two-tailed unpaired t-test were used for statistical analysis. p < 0.05 was considered to be statistically significant. 3. Results A deletion mutation in HSF4 (Hsf4 del ) causes congenital cataracts and retinal degeneration in mice and zebrafish The missense mutations in HSF4’s DNA binding domain and N-terminal hydrophobic region are associated with the inherited autosomal dominant lamellar and all white cataracts[ 10 ]. To determine whether Hsf4 participates in regulating retinal development, the Hsf4 del mice was recruited [ 20 ]. As expected, Hsf4 del mice developed lens opacification after 17 days old (p17) (Fig. S1 A), and the cataractous lens underwent vacuoles, shrink, and fibrosis at 7 and 13 months old (Fig. S1 B). Interestingly, we found that the retinal structure exhibited the disorganized in 7- and 13-month-old Hsf4 del mice (Fig. S1 B). Immunoblot results showed that HSF4 is expressed in the retinal tissues and its expression level decreased with age increases (Fig. S1 C). The immunofluorescent results showed that Hsf4 is predominantly expressed in the cells of ONL and RPE (Fig. S1 D ). The expression of HSF4 protein in Hsf4 del retina was smaller than HSF4 wild-type (Fig. S1 E). These results implied that HSF4 exerts a regulation on retinal homeostasis. To characterize the regulation of Hsf4 del on the retina, we analyzed the retinal structure of Hsf4 del vs. wild-type mice at P10, 7and 13 months old by using H&E, PAS staining and oil-red staining. The results showed that the retinal structure exhibited disordered in 7 and 13 months old Hsf4 del mice as compared to wild-type mice, characterized by disorganization of inner nuclear layer (INL) and inner-outer plexiform layers (IPL and OPL), retinal pigment epithelium (RPE) disconnected (Fig. 1 A and B), lipofuscin deposit underlying RPE (Fig. 1 C), the outer nuclear layer (ONL) atrophy (Fig. 1 A, D-F), and the increase of neovascular vessels (Fig. 1 B). However, this disordered change did not observe in P10 Hsf4 del mice (Fig. 1 A-D). Those results indicated that dysfunction of Hsf4 results in retinal disorder in mice in an age-related manner. To verify this, we recruited Hsf4 null zebrafish model, in which Hsf4 gene was knocked out [ 17 ]. Interestingly the structure of Hsf4 null retina looked normal at 10 dpf except the ONL of Hsf4 null retina became thinner at 13 mpf as compared to that in wild-type (Fig. 1 G). Those results demonstrated that Hsf4 is essential for retinal homeostasis. To determine whether HSF4-mutant affects the visual function, we performed the ERG to Hsf4 del vs. wild-type mice at P15, 3 and 7 months old. The results showed that Hsf4 del mice exhibited low amplitude of a- and b-wave at dark adapted 0.01 cd-s/m 2 or 3.0 cd-s/m 2 as compared to wild-type mice regardless of mouse ages (Fig. 1 H and I). Since no cataract was observed in P15 Hsf4 del mice, we proposed that the changed ERG in Hsf4 del mice was due to the dysfunction of retina but not the cataracts. Taken together, these results demonstrated that the Hsf4 del mutant impaired the vision at postnatal ages, and triggered the retina to undergo age-related atrophy. Hsf4 del mice and Hsf4 null zebrafish attenuate photoreceptor cells in an age-related manner Since Hsf4 del mice exhibited the abnormal ERG at postnatal age, we further studied the regulation of Hsf4 on the photoreceptor cells. we compared the photoreceptor cells of Hsf4 del mice to wild-type mice in postnatal age P10 and adult mice of 7 and 13 months old. The biomarkers of Rhodopsin (RHOD) for rod cells or OPN1/SW and OPN1/MW for cone cells were measured by immunofluorescent staining. The results indicated that the expression of RHOD, OPN1/SW, and OPN1/MW, which exhibited no difference between Hsf4 del and WT retina in P10 old mice, was significantly downregulated in 7 and 13 months Hsf4 del retina as compared to that in the wild-type (Fig. 2 A), and these changes were confirmed again at both protein and mRNA levels by using immunoblotting (Fig. 2 B-E) and qRT-PCR (Fig. 2 F-H). In zebrafish models, Hsf4 null retina downregulated the expression of RHOD, GNB1 and GNAT2 at 7 mpf, and 13mpf as compared to wild-types at both protein (Fig. 2 I-K) and mRNA levels (Fig. 2 L and M). These results are consisted with the results of H&E in Fig. 1 G. Taken together, these results confirmed again that dysfunction of HSF4 results in photoreceptor cells atrophy with age increase. Hsf4 del activates glial cells and inflammation in postnatal retina, and this regulatory effect is enforced with mice ageing Glial cells play an important role in modulating retinal homeostasis. Abnormal activation of the glial cells is associated with the retinal injury by upregulating the inflammation[ 25 ]. To determine whether gliosis is associated with disordered retina of Hsf4 del , we measured the expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) in the retina of HSF4 del mice vs wild-types at P1, P10, 7 and 13 months old. The immunofluorescent results showed that both GS and GFAP proteins were slightly upregulated in P10 HSF4 del retina and this upregulation was enforced in 7- and 13- months HSF4 del retina as compared to that in wild-type (Fig. 3 A). The results of immunoblot and qRT-PCR showed that the expression of GS, which was downregulated in P1 HSF4 del retina at both protein and mRNA levels as compared to wild-types (Fig. 3 B, D and F), was upregulated at protein and mRNA levels in P10 HSF4 del retina, and the upregulation was enforced in 7- and 13-months Hsf4 del retina. (Fig. 3 B, D and F). It is unclear the mechanism underlying the downregulation of GS in P1 Hsf4 del retina. GFAP, which is no difference between HSF4 del and wild-type retina at P1 day, were upregulated at both mRNA and protein levels in HSF4 del retina as compared to wild-types at P10, 7 and 13 months old (Fig. 3 B, C and E). These results suggested that HSF4 del mutation activates gliosis in the postnatal retina, and the activation was enforced with age increase. Since activation of glial cells is associated with non-infectious inflammation, which is the pathological cause of retinopathy and neuronal diseases, we then measured the expression of inflammatory factors using qRT-PCR. The results showed that the expression of IL-1β, IL-6, IL-8 and VEGFA were significantly upregulated in P10, 7 and 13 months HSF4 del retinal tissues as compared to that in wild-types (Fig. 3 G and H). These results suggested that Hsf4 del mice upregulated the expression of inflammatory interleukins in the retina at early postnatal age. In addition to glial cells, the RPE cells were also the resource of inflammatory interleukins in the retina. we then separated the RPE from neuronal tissues of P10 Hsf4 del vs. wild-type mice. The qRT-PCR results showed that the expression of IL-6 and VEGFA were upregulated in both RPE and neuronal tissues of the Hsf4 del retina as compared to that in wild-type counterparts (Fig. 3 I and J). These results suggested that both RPE and glial cells were the resource of inflammatory factors in Hsf4 del mice. To confirm the activation of gliosis is relying on HSF4, we performed a subretinal injection of AAV-Hsf4-Flag to 0.5 month Hsf4 del mice followed by recovery for 2 weeks. The expression of glial proteins and inflammatory cytokines were measured by using immunoblot and qRT-PCR. The results showed that ectopic expression of HSF4b partially downregulated the high expression of GS, GFAP, IL-1β, IL-6 and IL-8 at both protein and mRNA levels (Fig. 3 K-N). To determine whether AAV2-hsf4b-Flag exerts any regulation on retinal structure, we performed a subretinal injection of AAV-Hsf4-Flag to one month Hsf4 del mice followed by recovery for 2 months. The H&E staining results showed that administrated AAV2-Hsf4-Flag could partially reduced the disordered retinal structure of Hsf4 del mice (Fig. S2B). Those results indicated that the activation of gliosis in Hsf4 del retina is due to dysfunction of Hsf4b. In zebrafish models, Hsf4 null mutation slightly upregulated the expression of glial GFAP, GS, and inflammatory interleukins (including IL-1, Il-6 and IL-8) in 10 dpf and 7mpf, and this upregulation was enforced at 13 mpf zebrafish retina as compared to wild-type counterparts (Fig. 3 , O-S). Taken together, these results suggested that dysfunction of HSF4 activates the gliosis in the retina at early postnatal age. In the results of Fig. 1 B, The PAS staining results showed that there were more small blood vessels in the aged Hsf4 del retina than in wild-types. This upregulation was then confirmed by using immunohistochemistry staining with antibodies against CD31 and IB4 (Fig.S2A). Accordingly, we proposed that the upregulation of angiogenesis in the aged Hsf4 del retina is associated with the high expression of VEGFA (Fig. 3 H and I). HSF4 del mice downregulate the expression of visual cycle enzymes in RPE The retina of Hsf4 del mice exhibits lipofuscin deposite in RPE and the abnormal upregulation of cytokines and VEGF expression in RPE cells (Fig. 3 ), implying that HSF4 participates in regulating RPE cell homeostasis. Since RPE’ s predominant function is to modulate visual cycle, we then tested the potential regulatory effect of Hsf4 on the visual cycle by measuring the expression of visual cycles proteins, such as RPE65, RLBP1 and RDH5 in Hsf4 del vs wild-type retina at P1, P10, 7 and 13 months old [ 26 ]. Using the immunofluorescent and RPE whole mount staining, we found that the expression of RPE65, RDH5 and RLBP1 in RPE were downregulated in P1 and P10 HSF4 del retina as compared to that in wild-type, and the downregulation was enforced with age increase (Fig. 4 A and Fig. S3A). This downregulation was also confirmed by the immunoblot results followed by densitometry quantitation (Fig. 4 B-E), and the qRT-PCR (Fig. 4 F-H). To confirm the regulatory role of HSF4, we tested the expression of these visual-cycle proteins in the AAV2-Hsf4b-Flag-reconstituted Hsf4 del retina. AAV2-Hsf4b-Flag were subretinally injected to 0.5 month Hsf4 del retina followed by two weeks recovery. The expression of RPE65, RLBP1 and RDH5 was tested by immunofluorescence, immunoblot and qRT-PCR. The results showed that ectopic expression of HSF4b-Flag rescued the low expression of RPE65, RDH5 and RLBP1 in both protein (Fig. 4 I-K) and mRNA levels (Fig. 4 L) in Hsf4 del retina. the immunofluorescent results showed that the subretinal injected AAV2-Hsf4-Flag was predominantly expressed in RPE cells (Fig. S3B). These results suggested that HSF4 participates in regulating the expression of visual cycle enzymes in RPE. To further confirm the regulatory effect of Hsf4 on the visual cycle-associates enzymes, we isolated and in vitro cultured the primary RPE cells from 2-months-Hsf4 del retina or wild-type. After that, the plasmid pCMV-3xFlag-Hsf4b was transiently transfected into the primary RPE/Hsf4 del cells (Fig. 4 M and N). The immunoblot results showed that the expression of RPE65, RDH5 and RLBP1 was downregulated in RPE/Hsf4 del cells as compared to that in RPE/wt. The overexpression of Hsf4b-Flag restored the expression of those enzymes (Fig. 4 M and N). These results confirmed again that HSF4 participates in regulating the expression of visual cycle proteins in RPE cells. Furthermore, we verified this regulation in Hsf4 null zebrafish. The Hsf4 null lens exhibited transparency at 4 and 10 dpf, and opacification in 7 and 10 mpf (Fig. S3C). Consistent with the results in Hsf4 del mice, the expression of visual cycle proteins RPE65, RDH5 and RLBP1 was downregulated at both mRNA(Fig. S3D) and protein levels (Fig. S3E-H) in 4 dpf, 10dpf, 7mpf and 13 mpf Hsf4 null retina as compared to wild-type. Taken together, those results demonstrated that dysfunction of HSF4 impairs the expression of visual cycle enzymes at very early postnatal age. HSF4 del mice triggers cellular senescence or apoptosis in the retina. The previous data showed that HSF4 del mutation upregulated P21 cip1 expression, which triggered lens epithelial cells to undergo senescence[ 13 ]. In the results of Fig. 3 , Hsf4 del retina expressed a high level of the inflammatory interleukins at the postnatal age, implying that HSF4 del might cause cells to undergo senescence or apoptosis in the retina. To prove this, we immunoblotted the expression of P21 cip1 and P16 INK4a in the retina of Hsf4 del vs. wild-types. P16 INK4a was upregulated at both protein and mRNA in Hsf4 del retina of P10, 7 and 13 months old mice but not in P1 mice (Fig. 5 A, B and D). P21 cip1 is slightly upregulate in P1 Hsf4 del retina as compared to that in wild-type, and aging underpinned the upregulation (Fig. 5 A, C and E). Reconstitution of AAV2-HSF4b-Flag to one-month HSF4 del retina partially downregulated P21 cip1 mRNA expression (Fig. 5 F). In addition, the upregulation of P16 INK4a and P21 cip1 mRNA was also observed in zHsf4 null retina at 4dpf, 10dpf, 7mpf and 13mpf as compared to that in wild-type counterparts (Fig. 5 G and H). These results indicated that dysfunction of Hsf4 upregulated CKIs expression in retinal tissues. To determine whether Hsf4 del retina undergo apoptosis, we then performed TUNEL assay. The TUNEL-positive cells were much more in 7 -and 13- months retina than that in P10 Hsf4 del retina (Fig. 5 I). Few TUNEL- positive cells were observed in the wild-type retina (Fig. 5 I). Those results indicated that dysfunction of HSF4 elevated the cells’ senescence or apoptosis in ONL and RPE. HSF4 del mice deregulate the expression of heat shock proteins in the retina The fundamental function of Hsf4 is to regulate the expression of heat shock proteins. In lens tissue, HSF4 exerts a dual regulation on heat shock protein expression. It upregulates the expression of small heat shock proteins, such as Hsp25, Cryab and γ-crystallins, and simultaneously downregulates the expression of Hsp70, FGF4/7[ 9 , 11 , 16 ]. However, its regulatory effects on the expression of heat shock proteins in the retina remain unclear. The immunoblot results showed that HSF4 del mutation downregulated the expression of Hsp90 and Hsp25, but upregulated CRYAB expression at both protein (Fig. 6 A-D) and mRNA levels (Fig. 6 E-G) in the retina of P1, P10, 7 M and 13 M mice as compared to that in wild-type counterparts. The results of immunofluorescent and RPE whole mount staining assays indicated that CRYAB expression, was significantly upregulated in Hsf4 del retina as compared to wild-type (Fig. S4, A and B). The recapitulation of AAV2-Hsf4b-Flag into one-month Hsf4 del retina restored the changed expression of heat shock proteins at both protein and mRNA levels (Fig. 6 H-J). Since Hsf4b-Flag is predominantly expressed in RPE cells (Fig S3B), we further tested the regulation of Hsf4 on those heat shock proteins’ expression in the primary cultured RPE cells in vitro. As the results indicated in Fig. 6 K, the expression of Hsp90 and Hsp25 was downregulated and CRYAB expression was upregulated in RPE/Hsf4 del cells as compared to that in RPE/wt cells (Fig. 6 K, lanes 1–6 and L). Overexpression of Hsf4b-Flag partially restored the expression change of Hsp90, Hsp25 and CRYAB in the RPE/Hsf4 del cells (Fig. 6 K, lanes 4–9 and L). These results confirmed that Hsf4b exhibits a distinctive regulation on different heat shock proteins’ expression in RPE cells. In addition, we also tested the expression of heat shock proteins in the retina of Hsf4 null vs. wild-type zebrafish. Deficiency of Hsf4 upregulated CRYAB and downregulated Hsp25 in the retina of 4dpf, 10dpf, 7mpf, and 13mpf zebrafish compared to wild-type (Fig. 6 M-O). In contrast, Hsf4 null downregulated Hsp90 only in 7 and 13mpf zebrafish retina, but not in embryonic zebrafish (Fig. 6 M). These results suggested that dysfunction of HSF4 deregulated the expression pattern of heat shock proteins during retinal development. 4. Discussion Mice lacking Hsf4 develop microphthalmia. One of the pathological defects is lens cataracts. However, the regulatory effect of Hsf4 on other ocular tissues’ homeostasis, such as the retina, remains unclear. Using Hsf4 del mice and Hsf4 null zebrafish, we find that in addition to the lens, Hsf4 participates in the regulation of retinal homeostasis. The inframe-deleted mutation of Hsf4 in mice (Hsf4 del ) or removing Hsf4 expression in zebrafish (zHsf4 null ) results in the downregulation of the expression of visual cycle enzymes (RPE65, RDH5 and RLBP1) in RPE (Fig. 4 and Fig. S3), and the activation of gliosis (Fig. 3 and Fig. S2) and cellular senescence (upregulation of P21 cip1 and P16 INK4a , Fig. 5 ), without changing retinal structural (Fig. 1 ) at early postnatal age. With age increase, the retinal structure of Hsf4 del mice and Hsf4 null zebrafish undergoes atrophy, characterized by disorganization of INL and IPL, ONL atrophy, RPE disconnection, lipofuscin accumulation, the increased neovascularization and apoptotic cells. Our results demonstrate that Hsf4 is an indispensable transcription factor for retinal homeostasis during retinal development. The atrophic retina is mainly visible in 7 and 13-month-old HSF4 mutant mice and 13 mpf zebrafish but not in postnatal age. However, Hsf4 del mice and Hsf4 null zebrafish exhibited downregulation of visual cycle enzymes and upregulation of gliosis at postnatal ages. Impairment of visual cycle enzymes are associated with congenital pigmental retinitis and aged-related retinal degeneration. Abnormal activation of Gliosis is the predominantly pathological causative for retinal atrophy. Therefore, we thought that the retinal atrophy of aged Hsf4 del mice and Hsf4 null zebrafish is the consequence of the activation of gliosis and the downregulation of visual cycle enzymes’ expression. The time line of retinal atrophy is overlapped with severe cataracts (Fig. 1 and Fig. S1 ). However, those deteriorations in the retina have not been addressed in the previously reported Hsf4-deleted cataractous mice and zebrafish[ 9 , 11 , 17 ]. The possible reason is that most of previous studies regarding Hsf4 knockout mice focus on lens development at postnatal or rejuvenate ages, in which Hsf4 knockout mice or zebrafish have not developed retinal atrophy based on our data (Fig. 1 ). Although the structure of the retina does not change in the postnatal age, Hsf4 del mice exhibit functional damage in the retina. For example, Hsf4 del mice at postnatal age decrease the dark-adaptive response in ERG assay (Fig. 1 ). We postulate that this downregulation of ERG is associated with the downregulation of visual cycle enzymes, such as RPE65, RDH5 and RLBP1 in Hsf4 del retina (Fig. 4 , Fig. S3 and Fig. 1 ) rather than lens opacification, because the downregulation of those visual cycle-enzymes occurred in the retina as early as P1 mice and 4 dpf zebrafish whereas lens looks normal (Fig. 4 and Fig. S3 and Fig. 1 ). Another supporting evidence is that recapitulation of Hsf4-Flag restores the expression of visual cycle enzymes in Hsf4 del mice in vivo and in REP/Hsf4 del cells in vitro (Fig. 4 I-N and Fig. S3). These results suggest that Hsf4 is an essential transcriptional factor for visual cycle enzymes expression during early retinal development (Fig. 1 – 4 ). Defects of visual cycle turn out the accumulated vitamin A intermediates (e.g., A2E), the inducer of the oxidative stress and retinal degeneration [ 27 ]. Therefore,we proposed that those early changes in Hsf4 del mice and Hsf4 null zebrafish might exert a causative for the retinal degeneration. A number of transcription factors have been reported to participate in regulating retinal development through controlling the expression of visual cycle enzymes, such as Pax9, Otx2, Sox9 and Lhx2 [ 28 – 30 ]. Among these transcription factors, Sox9 can regulate the transcriptional expression of multiple visual cycle enzymes RPE65 and RLBP1 by associating with Otx2 binding to these promoters[ 30 ]. Dysfunctional mutations of those transcriptional factors are associated with congenital retinitis[ 28 ]. Our results in Fig. 4 indicate that Hsf4 acts as a novel transcriptional factor for the expression of visual cycle enzymes during early retinal development. However, the regulatory mechanism is still under investigation. Interestingly, the regulation of Hsf4 on the expression of heat shock proteins differs from lens to retina, especially on Cryab protein. During lens development, Hsf4 upregulates small heat shock protein expression, such as Hsp25, Cryab, γ-crystallin, and simultaneously downregulates Hsp70, FGF and vimentin[ 9 , 11 , 16 ]. However, in the retina of Hsf4 del mice and zHsf4 null zebrafish, we find that dysfunction of Hsf4 downregulates Hsp25 and Hsp90 but upregulates Cryab in the P10 retina, and these regulatory effects are enlarged with age increase. Hsf4 del mice exhibit the upregulation of Cryab predominantly in RPE and glial cells (Fig. 6 and S4). The onset of induction of Cryab is in P10 Hsf4 del retina, but not in P1, and subretinal administration of AAV2-Hsf4b-Flag into Hsf4 del retina or overexpression of Hsf4-Flag in the primary RPE/Hsf4 del cells decreases the high expression of Cryab, which implies that Hsf4 inhibits Cryab expression at least in RPE cells. Cryab regulates diverse signal pathways, including anti-apoptosis, cell migration, myofiber remodeling in skeletal and cardiomyocyte and inflammation[ 31 – 34 ]. The upregulation of Cryab in RPE and Glial cells allow us to postulate that Cryab may be involved in regulating gliosis, inflammation or cell survival, and this hypothesis needs further investigating. Our previous data show that Hsf4 del mice upregulate the expression of P21 cip1 and senescence-associated inflammatory factors in the lens[ 13 ]; The MEF cells lacking Hsf4 undergo replicative senescence[ 14 ], and mice lacking Hsf4 counteract P53-mutation-induced lymphoma and liver cancers[ 14 ]. Those results suggest that the big role of Hsf4 is the anti-cellular senescence. Consistent with its regulatory roles in lens, Hsf4 del mice upregulate senescent biomarkers, such as, P21 cip1 and P16 INK4a (Fig. 5 ) and inflammatory interleukins, IL-1β, Il-6, IL8 and growth factor VEGFA in early postnatal retina (Fig. 3 and Fig. S2). In addition, HSF4 del mutation activate gliosis in the retina of P10 mice (Fig. 3 ). Recapitulation of Hsf4 restores the expression of those changed proteins. These results suggest that Hsf4 plays a role of anti-senescence or anti-gliosis in the retina in addition to regulating visual cycle. As the consequence, the TUNEL results showed that there are more apoptotic cells in RPE and ONL of 7 and 13-month-old Hsf4 del retina than in the P10 retina (Fig. 5 ). Therefore, the up-regulation of cellular senescence and apoptosis may be the rational for retinal atrophy in the aged Hsf4 del mice. Conclusion Hsf4 exerts multiple functions during retinal postnatal development, such as upregulating the expression of the visual cycle enzymes, tuning the expression of heat shock proteins, and downregulating glia-mediated inflammation and the expression of CKIs. Deregulation of the vision cycle and glia-mediated inflammation are early events for Hsf4 del -induced age-related retinal atrophy. Declarations Conflict of interest The authors declare no conflict of interest with respect to the research, authorship, and publication of this article Data available All data supporting the finding of this study are available within the paper and its supplementary information. Acknowledgement This work is supported by grants from the National Nature Science Foundation of China (No.81970785, 81570825, U1604171and 31802314), the Joint Construction Project of Henan Medical Science and Technology Research Plan (Grand number: SBGJ202102157 and SBGJ202103068), and the Key Science and Technology Program of Henan Province (Grant No. 222102310467). Author contributions Baixue Liu and Youfei Lang performed most of experiments and data collection. Mengjiao Xue performed the tissue section and H&E staining assays. Mingjun Jiang assisted in animal breeding and isolation of Hsf4 del RPE cells. Xiaolin Jia and Guiling Zhou assisted in Hsf4 null zebrafish maintaining, breeding and genotyping. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4220460","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289723235,"identity":"3f9b3b3c-8a30-48eb-b06b-9959f91b736c","order_by":0,"name":"Baixue Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Baixue","middleName":"","lastName":"Liu","suffix":""},{"id":289723236,"identity":"732da552-8710-4147-acdd-006e5212014d","order_by":1,"name":"Youfei lang","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Youfei","middleName":"","lastName":"lang","suffix":""},{"id":289723237,"identity":"b7512d01-f15f-47a0-9cd7-23dc5135d681","order_by":2,"name":"Meng Jiao Xue","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"Jiao","lastName":"Xue","suffix":""},{"id":289723238,"identity":"4ce95929-0b12-4e40-84d5-27c11dcce9b4","order_by":3,"name":"Ming Jun Jiang","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"Jun","lastName":"Jiang","suffix":""},{"id":289723239,"identity":"47e30a6b-04f2-4374-939d-b69240d7bc2c","order_by":4,"name":"Xiao lin Jia","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"lin","lastName":"Jia","suffix":""},{"id":289723240,"identity":"a16215dc-d54f-4d0d-a905-75a7178d7518","order_by":5,"name":"Dandan Chen","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Chen","suffix":""},{"id":289723241,"identity":"6fec2f9f-b74b-43e5-bcc5-a0efbc62c700","order_by":6,"name":"Guilinng ZHou","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Guilinng","middleName":"","lastName":"ZHou","suffix":""},{"id":289723242,"identity":"a1036984-043d-45a8-a660-47ed4ac9fea3","order_by":7,"name":"Fengyan Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Fengyan","middleName":"","lastName":"Zhang","suffix":""},{"id":289723243,"identity":"216b66b6-0606-4e87-874c-ef934e01b45b","order_by":8,"name":"Xueyan Peng","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xueyan","middleName":"","lastName":"Peng","suffix":""},{"id":289723244,"identity":"64b8181b-9bd2-4fc2-943f-de032c1123d7","order_by":9,"name":"Yanzhong Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACAwYGxscINpFamI1J1sImTZoWc4n0Z9UFFXcSG9ibt0kw1NwhrMVyRkLa7RlnniU28Bwrk2A49owIh91IOHabt+1wYoNEjpkEY8NhYrQkthXz/gNqkX9DtJZkNmbeBpAtPMRqOfOMWZrn2GHjNp60YouEY8RoOZ7+8DNPzWHZfvbDG298qCFCC4NAAoRmAxEJRGhgYOA/QJSyUTAKRsEoGMkAAFKSOXTZG9dMAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8269-4629","institution":"Henan University","correspondingAuthor":true,"prefix":"","firstName":"Yanzhong","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2024-04-05 03:17:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4220460/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4220460/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54669894,"identity":"4c412d79-21fc-4d2c-88f7-c944301b070d","added_by":"auto","created_at":"2024-04-15 04:38:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5487829,"visible":true,"origin":"","legend":"\u003cp\u003eHsf4\u003csup\u003edel\u003c/sup\u003e mice undergo age-related retinal degeneration. A, H\u0026amp;E staining the retinal structure of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at 10 days (P10), 7 M and 13 M (month) old. GCL: Ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: Outer plexiform layer; OS: Outer segment layer; RPE: retinal pigment epithelium. B, PAS staining the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P10 (a and a’), 7 months\u0026nbsp; (b and b’) and 13 months (c and c’) old. The green arrows indicate small vascular vessels. C, oil-red staining the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P10 and 13 months old. The red arrow indicated the lipo-drusen. D-F, The quantitation of the thickness of retinal ONL of Hsf4\u003csup\u003edel\u003c/sup\u003e vs wild-type at P10, 7 M and 13 M old by using the image J. The shown data represented mean ± SD, n=10, ** p\u0026lt;0.01, ***p\u0026lt;0.001. G, the histology analysis of\u0026nbsp; retina structure of 10dpf and 13mpf zebrafish by H\u0026amp;E staining. H and I, ERG analysis of visual function of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P15, 3 M and 7 M. The quantitation of amplitude of a- and b-waves of the mice accepted 0.01 cd-s/m\u003csup\u003e2\u003c/sup\u003e or 3.0 cd-s/m\u003csup\u003e2\u003c/sup\u003e stimulation at dark-adapted condition. The shown data represent mean ± SD, n=11, ** p\u0026lt;0.01, ***p\u0026lt;0.00, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/f979f1e06f8fbcad07f61147.png"},{"id":54670202,"identity":"9069da02-55ae-4cd7-a051-3516d3c23c58","added_by":"auto","created_at":"2024-04-15 04:46:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2915816,"visible":true,"origin":"","legend":"\u003cp\u003eHsf4\u003csup\u003edel\u003c/sup\u003e mice-induced photoreceptor cells atrophy. A, the immunofluorescent staining of the expression of Rhodopsin, Opn1sw, Opn1mw in Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type at P10, 7 M and 13 M mice. The nuclear was stained with DAPI. B, immunoblot the expression of Rhodopsin, Opn1sw, Opn1mw and a-tubulin (a-tub) in the retina of P10, 7 M and 13 M Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice. C-E, the densitometry quantitation of the fold change of expression of\u0026nbsp; Rhodopsin, Opn1sw, and Opn1mw normalized by a-tubulin in wild-type vs.Hsf4\u003csup\u003edel\u003c/sup\u003e by using image J in B.\u0026nbsp; F-H, the qRT-PCR measured the expression of Rhodopsin, Opn1sw, Opn1mw in the retina of P10, 7M and 13M Hsf4\u003csup\u003edel\u003c/sup\u003e vs.wild-types mice. The shown data represented mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 , ****p\u0026lt;0.0001. I immunoblot the expression of GNB1, GNAT2 and GAPDH in the retina of 7 mpf and 13 mpf Hsf4\u003csup\u003enull\u003c/sup\u003e vs. wild-type zebrafish. J-K, the densitometry quantitation of fold change of the expression of GNB1, GNAT2 normalized by GAPDH in I. The shown data represented mean ± SD, n=30, *p\u0026lt;0.05, **p\u0026lt;0.01. L and M, qRT-PCR measured the expression of Rhodopsin, GNB1, and GNAT2 in the retina of 7 and 13 mpf Hsf4\u003csup\u003enull\u0026nbsp; \u003c/sup\u003ezebrafish vs. wild-type zebrafish. The shown data represented mean ± SD, n=35, *p\u0026lt;0.05, **p\u0026lt;0.01 , ***p\u0026lt;0.001 , ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/945e17089324f79d5b07968e.png"},{"id":54669897,"identity":"cd1d103f-8df9-4e4d-ac7e-2a4a615443c5","added_by":"auto","created_at":"2024-04-15 04:38:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1997093,"visible":true,"origin":"","legend":"\u003cp\u003eActivation of gliosis in Hsf4\u003csup\u003edel\u003c/sup\u003e retina. A, Immunofluorescence staining of GFAP and Glutamine synthesis (GS) in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-types mice at P10, 7 M and 13 M old. The nuclear was stained with DAPI. B, immunoblot of the expression of GFAP, GS and a-tubulin (a-tub) in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P1, P10, 7 M and 13 M old. C-D, the densitometry quantitation of GFAP and GS normalized by a-tub in B by using image J. The shown data represented mean ± SD, n=5, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ns: no statistical significance. E-G,\u0026nbsp; qRT-PCR to determine the mRNA expression levels of GFAP (E), GS(F), IL-1b, IL-6 and IL-8 (G) in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P1, P10, 7 M and 13 M old. The expression of GAPDH mRNA was used as an internal control. H, qRT-PCR to measure the expression of VEGFA mRNA in the in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P1, P10, 7 M and 13 M old. The shown data represented mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ns: no statistical significance. I-J, qRT-PCR to measure the expression of VEGFA\u0026nbsp; and IL-6 in the RPE of P10 Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice. The shown data represented mean ± SD, n=6, **p\u0026lt;0.01, ****p\u0026lt;0.0001. K, immunoblot of the expression of GFAP, GS and a-tub in the retina of\u0026nbsp; wild-type (wt, lanes 1-3) and the Hsf4\u003csup\u003edel\u003c/sup\u003e mice injected with AAV2-vector virus (lanes 4-6) or AAV2-Hsf4b-Flag virus (lanes 7-9). L, the densitometry quantitation of GFAP and GS normalized by a-tub in K by using image J. The shown data represented mean ± SD, n=5, **p\u0026lt;0.01, ***p\u0026lt;0.001. M and N, the qRT-PCR measured the expression of\u0026nbsp; GFAP, GS, IL-1b, IL-6 and IL-8 in the retina of wild-type,\u0026nbsp; Hsf4\u003csup\u003edel\u003c/sup\u003e\u0026nbsp; and AAV2-Hsf4b-Flag-recapitulated Hsf4\u003csup\u003edel\u003c/sup\u003e mice. The data shown represented mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. O-S, qRT-PCR to determine the mRNA expression of GFAP, GS, IL-1b, IL-6 and IL-8 in the retina of\u0026nbsp; 4 dpf, 10 dpf, 7 mpf and 13 mpf wild-type vs Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish. The data shown represented mean ± SD, n=30, *p\u0026lt;0.05, **p\u0026lt;0.01, \u0026nbsp;***p\u0026lt;0.001, ns, no statistical significance.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/a3618efb8357c80ccd4ca0f2.png"},{"id":54670203,"identity":"3276fa03-6bd1-4397-b7ad-9a73b0912ca3","added_by":"auto","created_at":"2024-04-15 04:46:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3754082,"visible":true,"origin":"","legend":"\u003cp\u003eThe downregulation of the expression of visual cycle enzymes in Hsf4\u003csup\u003edel\u003c/sup\u003e retina. A, the immunofluorescent staining the expression of\u0026nbsp; RPE65, RDH5 and RLBP1 in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P1, P10, 7 M and 13 M old. The nuclear was stained with DAPI. B, immunoblot the expression of RPE65, RDH5, RLBP1 and a-tubublin (a -tub) in the retina used in A. C-E, the densitometry quantitation of RPE65, RDH5 and RLBP1 normalized by a-tub in B by using image J. The data shown were mean ± SD, n=5, *P\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u0026nbsp; F-H, the qRT-PCR measures the expression of RPE65, RDH5 and RLBP1 normalized by GAPDH in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P1, P10, 7M and 13M old. The data shown were mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***P\u0026lt;0.001. I, immunofluorescent staining the expression of RPE65, RLBP1 and RDH5 in wild-type retina or Hsf4\u003csup\u003edel\u003c/sup\u003e that were subretinally injected with AAV2-vector virus or AAV2-Hsf4-Flag virus. J, immunoblot the expression of RPE65, RDH5, RLBP1, a-tub and Flag-Hsf4b in wild-type retina (lanes 1-3) or Hsf4\u003csup\u003edel\u003c/sup\u003e retina that was subretinally injected with AAV2 vector virus (lanes 4-6) or AAV2-Hsf4b-Flag virus (lanes 7-9). K, the densitometry quantitation of RPE65, RDH5 and RLBP1 normalized by a-tub in J by using image J. The fold change was calculated by dividing the protein level of wild-types by that of Hsf4\u003csup\u003edel\u003c/sup\u003e or AAV2-Flag-reconstituted Hsf4\u003csup\u003edel\u003c/sup\u003e. The data shown were mean ± SD,\u0026nbsp; n= 6, **p\u0026lt;0.01, ***p\u0026lt;0.001. L, the qRT-PCR measured the expression of RPE65, RDH5 and RLBP1 in wild-type vs. Hsf4\u003csup\u003edel \u003c/sup\u003emice that were treated in the same way as did in J.\u0026nbsp; M, immunoblot the expression of RPE65, RLBP1 and RDH5 in the in vitro cultured primary RPE/wt (lanes 1-3), RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e (lanes 4-6) or Hsf4-Flag-reconstituted RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e (lanes 7-9) cells. N,\u0026nbsp; densitometry quantitation of RPE65, RDH5 and RLBP1 normalized by a-tub in M by using image J. The fold change was calculated by dividing the protein level in RPE/wt cells by that in RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e cells or AAV2-Flag-reconstituted RPE/Hsf4\u003csup\u003edel \u003c/sup\u003e. The data shown were\u0026nbsp; mean ± SD, n= 6, *p\u0026lt;0.05, **p\u0026lt;0.01, ns, no statistical significance.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/b267052616ef0ee8f17495c7.png"},{"id":54669896,"identity":"ca857b3f-c336-49ac-ba60-2b88e1299fa9","added_by":"auto","created_at":"2024-04-15 04:38:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2279959,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulation of P16 \u003csup\u003eINK4a\u003c/sup\u003e and P21\u003csup\u003ecip1 \u003c/sup\u003eand apoptosis in Hsf4\u003csup\u003edel\u003c/sup\u003e retina. A, immunoblot the expression of P16\u003csup\u003eINK4a\u003c/sup\u003e, P21\u003csup\u003ecip1\u003c/sup\u003e and a-tub in the retina of wild-type (wt, lanes 1-3) and Hsf4\u003csup\u003edel\u003c/sup\u003e (lanes 4-6) mice at P1, P10, 7M and 13M old. B and C, The densitometry quantitation of P16\u003csup\u003eINK4a\u003c/sup\u003e and P21\u003csup\u003ecip1\u003c/sup\u003e normalized by a-tub in A by using image-J.\u0026nbsp; The fold change was calculated, and the data were mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, ns, no statistical significance. D-E, qRT-PCR measured the expression of P16\u003csup\u003eINK4a\u003c/sup\u003e and P21\u003csup\u003ecip1\u003c/sup\u003e mRNA in the retina of wild-type and Hsf4\u003csup\u003edel\u003c/sup\u003e mice in the same age as used in A. F, qRT-PCR measured the expression of Hsf4 and P21\u003csup\u003ecip1\u003c/sup\u003e in the retina of wild-type mice or Hsf4\u003csup\u003edel \u003c/sup\u003emice that were subretinally injected with AAV2-vector virus or AAV2-Hsf4b-Flag virus. The data shown represented mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. G and H, the qRT-PCR measured P16\u003csup\u003eINK4a\u003c/sup\u003e and P21\u003csup\u003ecip1\u003c/sup\u003e in the retina of wild-type and Hsf4\u003csup\u003enull \u003c/sup\u003ezebrafish at 4 dpf, 10 dpf, 7 mpf and 13 mpf. The graph data represented mean ± SD, n=30, *p\u0026lt;0.05, **p\u0026lt;0.001. I, Measuring the apoptotic cells in the retina of wild-type vs. Hsf4\u003csup\u003edel\u003c/sup\u003e mice at P10, 7 M and 13 M old by using TUNEL assay. The nuclear was stained by DAPI.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/7553117a984371e8f533020e.png"},{"id":54669900,"identity":"5156a29c-3c70-4dd0-9aef-283546466b60","added_by":"auto","created_at":"2024-04-15 04:38:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1182257,"visible":true,"origin":"","legend":"\u003cp\u003eHsf4\u003csup\u003edel\u003c/sup\u003e mice change the expression pattern of heat shock proteins in the retina. A, immunoblot the expression of Hsp90, Hsp25, Cryab (aB) and a-tub in the retina of wild-type (wt, lanes 1-3) and Hsf4\u003csup\u003edel \u003c/sup\u003e(lanes 4-6) mice at P1, P10, 7 M and 13 M old. B-D, The densitometry quantitation of Hsp90, Hsp25 and aB normalized by a-tub in A by using image-J. The fold change was calculated and the data shown were mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ns, no statistical significance. E-G, qRT-PCR measured the expression of Hsp90a, Hsp25 and aB in the retina of wild-type vs. Hsf4\u003csup\u003edel\u003c/sup\u003e mice at P1, P10, 7 M and 13 M old. The data shown represented mean ± SD, n=6,\u0026nbsp; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. ns: No statistical significance. H and I, immunoblot and densitometry quantitation of the expression of Hsp90, aB and a-tub in the retina of wild-type mice (lanes 1-3) or Hsf4\u003csup\u003edel\u003c/sup\u003e mice that were injected subretinally with AAV2-vector (lanes 4-6) or AAV2-Hsf4b-Flag virus (lanes 7-9). The graph data represented mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ns: no statistical significance. J, qRT-PCR measured the expression of Hsp90a, Hsp90b, Hsp25 and aB normalized by GAPDH in the retina of mice used in H. The data shown represented mean ± SD, n=6, **p\u0026lt;0.01, ***p\u0026lt;0.001. ns: no statistical significance. K, immunoblot the expression of Flag-Hsf4b, Hsp90, Hsp25, aB and a-tub in the in vitro cultured primary RPE/wt cells (lanes 1-3) or RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e cells that were overexpressed empty vector (lane 4-6) or Hsf4b-Flag (lanes 7-9). L, densitometry quantitation of the expression of Hsp90, Hsp25 and aB\u0026nbsp; in K by using image-J. The fold change was calculated and the data shown were mean ± SD, n=6, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ns, no statistical significance. M-O, qRT-PCR measured Hsp90a, Hsp25 and aB in the retina of wild-type and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish at 4 dpf, 10 dpf, 7 mpf and 13 mpf. The graph data represented mean ± SD, n=30, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, ns: no statistical significance.\u0026nbsp;\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/d342eaf83fa9341f42f302fe.png"},{"id":62307946,"identity":"9f51dc20-81c2-48d9-bbae-6368ef7639d1","added_by":"auto","created_at":"2024-08-12 19:06:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20901158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/292e4415-a774-4806-84e0-9257f818110f.pdf"},{"id":54669893,"identity":"0f7c2ed6-4571-4683-8945-b0fdfeacc007","added_by":"auto","created_at":"2024-04-15 04:38:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15953,"visible":true,"origin":"","legend":"","description":"","filename":"KeyMessages.docx","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/3e6fc64c0860e6f7c79bf0ab.docx"},{"id":54669901,"identity":"94ddc813-2000-418b-9ab3-8f3447cc1360","added_by":"auto","created_at":"2024-04-15 04:38:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17847656,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaldata.docx","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/83f220f166bbf6dfc1077440.docx"},{"id":54769203,"identity":"3f31c4c2-95c4-4e7a-b8e1-cfcde724ed4e","added_by":"auto","created_at":"2024-04-16 13:45:59","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":111540,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4220460/v1/3cdde015a4a71402380ad87a.pdf"}],"financialInterests":"","formattedTitle":"Dysfunction of heat shock factor 4 impairs retinal structure and visual function in mice and zebrafish","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe retinal atrophy, which is a leading cause of blindness in the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], is a predominant pathological process of age-related macular degeneration and congenital pigmental retinitis, characterized by the drusen accumulation, atrophy of photoreceptor and RPE cells, and night vision deterioration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Multiple risky factors, such as ageing, smoke, alcohol addiction, glaucoma, systemic diseases (hypertension, diabetes, atherosclerosis) and the genetic variations, are associated with retinal degeneration via inducing divergent pathways, e.g. oxidative stress, apoptosis or ferroptosis and inflammation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Missense mutations in visual cycle-restrictive enzymes, like RPE65, RLPB1, RDH5 and LRAT, which impair the visual cycle, are causative for congenital pigmental retinitis characterized by retinal atrophy. In addition, genetic mutations in the transcription factors, such as Pax6, MITF, SOX9, which regulate eye development, results in retinal developmental defect. Hsf4 is a critical transcriptional factor for eye development. However, its regulatory roles on the retinal homeostasis remain unclear.\u003c/p\u003e \u003cp\u003eHeat shock factor 4 (HSF4) is a member of family heat shock factors that control the expression of heat shock proteins or none-heat shock proteins in response to heat shock or other cellular proteotoxic stresses at physio-pathological conditions[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. HSF4 gene is located in human chromosome 16q22.1 and constitutively expressed in majority tissues including ocular lens, muscle tissues, cerebral cortex, midbrain, retina, and pancreas, and exhibits diverse functions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].The missense mutations in the DNA binding domain of HSF4 are associated with the hereditary autosomal dominant cataracts in human being[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Germline deletion of Hsf4 gene in mice causes microphthalmias with lens cataracts[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The Hsf4-deficent cataracts occur at postnatal age, characterized with lens epithelial cell hyperproliferation, and malfunction of fiber cell terminal differentiation, including the delayed degradation of the mitochondria, ER and nucleus, and down-expression of crystallin proteins[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, mice lacking Hsf4 downregulates the tumorigenesis of lymphocytes and hepatocytes in P53-deficient mice[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. MEF cells lacking Hsf4 undergo replicative senescence in vitro[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. HSF4 is upregulated in intestine cancer tissue. Those divers functions are associated with HSF4\u0026rsquo;s dual transcriptional regulations. HSF4 positively regulates the expression of HSP25, CRYAB, γ-crystallins, CP49, DNase II-beta and P53, and negatively controls the expression of P21, FGF4/7, vimentin and HSP70[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The hyperproliferation of Hsf4-deleted lens epithelial cells is associated with the down-expression of P53 or upregulation of FGF4/7 signaling[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, this phenomenon is not observed in the Hsf4\u003csup\u003edel\u003c/sup\u003e mutated mice[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Instead, Hsf4\u003csup\u003edel\u003c/sup\u003e lens epithelial cells undergo senescence via upregulating P21 expression[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. HSF4 upregulates DNAase II beta expression, which may be associated with fiber cells\u0026rsquo; nuclear degradation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, HSF4 participates in regulating lysosomal activity by regulating CRYAB-mediated ATP6V1A protein stability, a component of V-ATP-pump complex[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and/or the expression of autophagic protein ATP9a in zebrafish and mouse lens[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which are postulated to be associated with the degradation of membrane organelles during fiber cell terminal differentiation.\u003c/p\u003e \u003cp\u003eIn this paper, we studied the regulatory roles of HSF4 on retinal homeostasis by using a Hsf4\u003csup\u003edel\u003c/sup\u003e mice [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Hsf4\u003csup\u003edel\u003c/sup\u003e mice express a short Hsf4 with 42 amino acids-in-frame deletion from amino acids116 to158. We found that HSF4 was expressed in retinal tissues and essential for retinal development. HSF4 mutants impaired the expression of visual cycle enzymes, activated gliosis at the early postnatal retina, and induced retinal disorder in 7 and 13 months old. These results highlighted that HSF4 was an indispensable factor for retinal development. It participates in regulating the visual cycle and glia homeostasis during early postnatal development.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cem\u003eAntibodies and Plasmids\u003c/em\u003e \u003c/p\u003e \u003cp\u003eHSP70 (6B3) Rat mAb (#4873), P21 Waf1/Cip1 (12D1) Rabbit mAb (#2947), P16 Rabbit mAb (#80772) were purchased from Cell Signaling Technology (Shanghai, China).The rabbit polyclonal antibodies against GAPDH (#10494-1-AP), DYKDDDK (Flag) (#66008-3-lg), HSF4 (#22883-1-AP), GFAP (#16825-1-AP), RLBP1 (#15356-1-AP), GNB1 (#10247-2-AP), Alpha(B) crystallin (#16808-1-AP), alpha-tubulin (#11224-1-AP), ZO1 (#21773-1-AP) were from Proteintech (Wuhan, China). The mouse polyclonal antibody against RDH5 (#sc-377057) and the mouse monoclonal antibodies against Glutamine Synthetase (E-4) (#sc-74430), Opn1sw (1B10) (#sc-517304), Opn1lw/mw (7GB) (#sc-517301) were from Santa Curz Biotechnology (Texas, USA). The mouse monoclonal antibody against Rhodopsin (#ab5417) and the rabbit monoclonal(EPR5477) antibody against HSP25/27 (#ab109376) were from Abcam (Cambridge, MA, USA). The rabbit polyclonal antibody against RPE65 (CQA3629) was from Cohesion Biosciences (London, E14 2DN, UK), The rabbit polyclonal antibody against GNAT2 (PM075) was from MBL (Tokyo, JAPAN ). The mouse monoclonal Antibody against ZO1 ( # 33-9100) was from Invitrogen (Waltham, MA, USA). The HRP-conjugated goat polyclonal anti-rabbit IgG (#SA00001-2), HRP-conjugated goat polyclonal anti-mouse IgG (#SA00001-1) and HRP-conjugated goat polyclonal anti-rat IgG (#SA00001-15) were from proteintech (Wuhan, China). The Alexa Fluor 488 goat anti-mouse IgG antibody (#A32723) and Alexa Fluor 594 goat anti-rabbit IgG antibody (#A32740) were from Invitrogen (Waltham, MA,USA). The Alexa Fluor 594-Goat Anti-Mouse IgG H\u0026amp;L (#ab150116) and Alexa Fluor 488-Goat Anti-Rabbit IgG H\u0026amp;L (#ab150080) were from Abcam (Cambridge, MA, USA). Plasmids: The p3xFlag-CMV7.1-HSF4b: full-length \u003cem\u003eHSF4\u003c/em\u003e cDNA were cloned into the 3x Flag CMV 7.1 vector at EcoRI and BamH1. The AAV2-Flag-HSF4: The human HSF4 cDNA was subcloned into the pTransfer vector at restrictive enzymes sites of Kpn1 and EcoRI. The recombinant plasmids were verified by sequencing.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnimals\u003c/em\u003e \u003c/p\u003e \u003cp\u003eC57/BL6 and C57/BL6/Hsf4\u003csup\u003edel\u003c/sup\u003e mice were fed and bred in an SPF-grade animal room. The Hsf4\u003csup\u003edel\u003c/sup\u003e mutant mice were established in our lab, in which A 126 bp DNA fragment were deleted from exon 5, resulting in a mutant of Hsf4 with an in-frame deletion of 42 amino acids (Hsf4\u003csup\u003edel\u003c/sup\u003e)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The Hsf4\u003csup\u003edel\u003c/sup\u003e mice developed cataracts in the middle postnatal period. HSF4\u003csup\u003enull\u003c/sup\u003e zebrafish were presented from Dr. Mugen Liu[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Wild type (Danio rerio) and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish were maintained at a standard 14 hr light:10 hr dark cycle (LD). The animals were operated following the ARVO statement regarding the use of animals in the field of ophthalmic and visual research. Animal euthanasia and surgical protocols were approved by the Ethics Committee of Zhengzhou University.\u003c/p\u003e \u003cp\u003e \u003cem\u003eReconstitution of Hsf4 into the primary mouse RPE/Hsf4\u003c/em\u003e \u003csup\u003e \u003cem\u003edel\u003c/em\u003e \u003c/sup\u003e \u003cem\u003ecells in vitro\u003c/em\u003e\u003c/p\u003e \u003cp\u003eFor culturing the primary mouse RPE cells, the isolated eyeballs from 2-month wild-type and Hsf4\u003csup\u003edel\u003c/sup\u003e mice were recruited. The RPE cells were separated from the neuroretina and the sclera-choroid following the protocols presented in the previous literature[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, the eyeballs were incubated in 2% (wt/vol) pre-warmed dispase II in a 37\u0026deg;C incubator for 45 min followed by rinsing twice in DEME media. The cornea and the lens were separated and discarded. The left posterior retinal cup were rinsed in DMEM media twice and cut into four flaps. The neuroretina was teared away. The remainder of the eye cup containing RPE was cut and flatted. The RPE tissue was scraped off followed by incubating in 0.25% trypsin-EDTA buffer for 10 minutes. The cells were pelleted by centrifugation at 1000 RPM for five minutes and suspended in DMEM/F12 media containing 20% FBS and antibiotics. 10^6 cells were seeded into a 10 cm dish and cultured for 3 or 4 days. The cells were passaged for total three generations.\u003c/p\u003e \u003cp\u003eFor reconstitution of Flag-Hsf4, the plasmid pCMV-Flag-Hsf4b was transiently transfected into the primary cultured RPE cells by using the lipofectamine 3000 following the protocol with the kit.\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuantitative real-time PCR (qRT-PCR)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTotal RNA extraction was carried out using RNAiso reagents. The cDNA was synthesized following the protocol of reverse transcription kit. An equal amount of cDNA was mixed with Fast-start Universal SYBR Green Master Mix (Roche, San Francisco, CA, USA) and amplified in PCR machine ABI 7500 system (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression in each sample was displayed as 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e values, and the data was representative of at least three independent experiments. The used primers were listed in table 1.\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunoblot\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe retinal tissues, or cultured cells were lysed in RIPA buffer (Sigma-Aldrich, Missouri, USA) containing 1x cocktail protease inhibitor. The protein was separated in sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) followed by electrotransferring to the PVDF membranes. The membranes were blocked in 5% (v/v) BSA at room temperature for 1 h and then incubated with the primary antibody overnight at 4℃. After washing with PBST buffer, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG at room temperature for 1 h. The membranes were developed with SuperLumia enhanced chemiluminescence (ECL) reagents, and the signals were captured with Amersham Imager 680. The Image J was used to quantitate protein intensity in the blots.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGross Histology, immunofluorescence, and whole-mount immunofluorescence staining\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe C57BL/6 mice were deeply anesthetized with IP administration of ketamine (80\u0026ndash;100 mg/kg) and xylazine (5\u0026ndash;10 mg/kg), and euthanized by cervical dislocation. The eyeballs of mice were isolated and fixed with FAS solution (Servicebio, China) for 24h, and embedded in paraffin (Servicebio, China). The samples were sliced into 3 \u0026micro;m thickness, and the slices were subjected to H\u0026amp;E staining followed by the protocol provided with kit.\u003c/p\u003e \u003cp\u003eFor Immunofluorescence staining, the tissue sections were treated with 0.2% (v/v) Triton X-100 for 10 min followed by blocking with 5% BSA for 1 h at room temperature, and then incubated with the primary antibodies at 4℃ overnight. After rinsing in PBST buffer for three times, the sections were incubated with secondary antibodies for 1h at room temperature and counterstained with DAPI for 10min. The fluorescent images were captured with a confocal microscope (Zeiss, German).\u003c/p\u003e \u003cp\u003eFor whole-mount immunofluorescence, the eyeballs were fixed with 4% paraformaldehyde (PFA) for 40 mins and then dissected in a petri dish containing cold PBS buffered (PH 7.4) to discard the cornea and the lens from the eye. The left eye cups were cut and flatted. The neuroretina was removed from the RPE-choroid-sclera. The left flatted RPE-choroid-sclera were subjected to immunofluorescent staining.\u003c/p\u003e \u003cp\u003eFor Immunohistochemistry staining, Paraffin sections from the eyes of mice were depigmented by 5% KOH:20% H2O2:ddH2O. The sections were oven dried at 60℃, and dewaxed, hydrated, antigen repair (Tris-EDTA, PH9.0). The immunohistochemistry was performed following the protocol with UltraSensitiveTM SP kit (MXB, Fuzhou, China). Briefly, the sections were put into sequentially in the Reagent 1(an internal peroxidase inhibitor) and the Reagent 2 (a general staining blocker or blocker) for 10 minutes, then incubated with primary antibody at 4\u0026deg;C overnight, after that, the sections were incubated with Reagent 3 (biotin-tagged sheep anti-mouse or rabbit anti-sheep IgG polymers) and the Reagent 4 (streptavidin-antibiotic protein peroxidase) for 10 minutes. The signals was developed by using DAB (MXB, Fuzhou, China). The result of IHC was taken at the light microscope.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTUNEL assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTUNEL test was conducted following the protocol of the TMR (Red) Tunel Cell Apoptosis Detection Kit (Servicebio, China). The sections were incubated with proteinase K (20ug/ml) at 37℃ for 20 minutes. After washing in PBS, the section were permeabilized in 0.2% (v/v) Triton X-100 for 10 minutes at room temperature, and then incbubated with freshly prepared TdT buffer at 37℃ in the dark for 1 hour. The sections were then counterstained with DAPI for 10 minutes. The sections were thoroughly washed for 4 to 5 times with PBS, and the signals were taken by using a confocal microscope (Zeiss, German).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePAS staining and Oil Red O Staining\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFor PAS staining, the sections were stained with PAS solution following the protocol with kits (Servicebio, Wuhan, China). Briefly, the sections were incubated in solution B (containing 0.5% periodic acid) for 10 to 15 minutes. After rinsing in water, the sections was placed in solution A (containing shiff\u0026rsquo;s reagent) in the dark for thirty minutes followed by water rinse three times. The sections were counterstained in hematoxylin solution for 3 s and blue-return solution for 3 s to stain the nucleus. After this, the sections were dehydrated covered with coverslips and mounted using Xylens based mounting media. The signals were photographed under microscope with 40 x resolution lens.\u003c/p\u003e \u003cp\u003eFor Oil-red staining, the frozen sections of the retina were fixed in 4% PFA for 15 minutes, and then stained with Oil Red O staining solution (Servicebio, Wuhan, China) followed by the manufacturer's instructions. Briefly, the slices were incubated with oil red solution for 8 to 10 minutes in the dark. After that, the sections were put into two vats containing 60% isopropyl alcohol for 3 or 5 seconds followed by rinsing in water for 10 seconds. The slices were stained with hematoxylin solution for 3\u0026ndash;5 minutes followed by washing in distilled water for 10 seconds. Signals were photographed under the light microscope.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSubretinal injection\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe Hsf4\u003csup\u003edel\u003c/sup\u003e mice at P15 were subretinally injected once with two microliter of recombinant AAV2 virus expressing empty vector (AAV2-Vector) or Hsf4-Flag (AAV2-Hsf4-Flag) at MOI 10^8 followed the protocol of published paper\u003csup\u003e[23]\u003c/sup\u003e. The mice then were allowed to recover at control condition for 2 weeks. And AAV2 virus were also subretinally injected to one\u003c/p\u003e \u003cp\u003emonth old Hsf4\u003csup\u003edel\u003c/sup\u003e mice followed by two months recovery. After that, The mice were measured with ERG, and the retinal tissues were collected for immunofluorescence, immunohistochemistry, immunoblot or qRT-PCR assays.\u003c/p\u003e \u003cp\u003e \u003cem\u003eElectroretinogram (ERG).\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe wild-type, Hsf4\u003csup\u003edel\u003c/sup\u003e mices were given the ERG test followed the previously reported protocol[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, Mice were anesthetized with IP administration of ketamine (80\u0026ndash;100 mg/kg) and xylazine (5\u0026ndash;10 mg/kg), and maintained on a warm pad at 37℃ to keep body temperature. Animals received tropicamide and phenylephrine chloride eyedrops to dilate the pupil. Every procedure is conducted in dim red light. ERGs was recorded with Reti-MINER-C, a visual electrophysiology system ((AiErXi Medical Equipment Co., Ltd., Chongqing, China). After dark adaption for 12 h, the mice were given a range of stimulus intensities from \u0026minus;\u0026thinsp;3 and 1 log cd-s/m\u003csup\u003e2\u003c/sup\u003e to reflect optic rod cell function. Mice were light-adapted for 10 mins to a light background set at 30 cd/m\u003csup\u003e2\u003c/sup\u003e. Capturing light-responsive waves in stroboscopic stimuli (0 and 1 log cd-s/m\u003csup\u003e2\u003c/sup\u003e) as a reflect of optic cone cell function. To maintain consistent responses, a minimum of three tests were conducted at every level of stimulus intensity. Examining the light reaction involves gauging the intensity of the a- and b-waves.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe densitometry quantitation of proteins in immunoblot was measured by using Image J. SPSS 17.0 and GraphPad Prism 8 were used for data analysis. 2-way ANOVA and two-tailed unpaired t-test were used for statistical analysis. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cem\u003eA deletion mutation in HSF4 (Hsf4\u003c/em\u003e \u003csup\u003e \u003cem\u003edel\u003c/em\u003e \u003c/sup\u003e \u003cem\u003e) causes congenital cataracts and retinal degeneration in mice and zebrafish\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe missense mutations in HSF4\u0026rsquo;s DNA binding domain and N-terminal hydrophobic region are associated with the inherited autosomal dominant lamellar and all white cataracts[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To determine whether Hsf4 participates in regulating retinal development, the Hsf4\u003csup\u003edel\u003c/sup\u003e mice was recruited [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As expected, Hsf4\u003csup\u003edel\u003c/sup\u003e mice developed lens opacification after 17 days old (p17) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), and the cataractous lens underwent vacuoles, shrink, and fibrosis at 7 and 13 months old (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Interestingly, we found that the retinal structure exhibited the disorganized in 7- and 13-month-old Hsf4\u003csup\u003edel\u003c/sup\u003e mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Immunoblot results showed that HSF4 is expressed in the retinal tissues and its expression level decreased with age increases (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). The immunofluorescent results showed that Hsf4 is predominantly expressed in the cells of ONL and RPE (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD ). The expression of HSF4 protein in Hsf4\u003csup\u003edel\u003c/sup\u003e retina was smaller than HSF4 wild-type (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). These results implied that HSF4 exerts a regulation on retinal homeostasis.\u003c/p\u003e \u003cp\u003eTo characterize the regulation of Hsf4\u003csup\u003edel\u003c/sup\u003e on the retina, we analyzed the retinal structure of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P10, 7and 13 months old by using H\u0026amp;E, PAS staining and oil-red staining. The results showed that the retinal structure exhibited disordered in 7 and 13 months old Hsf4\u003csup\u003edel\u003c/sup\u003e mice as compared to wild-type mice, characterized by disorganization of inner nuclear layer (INL) and inner-outer plexiform layers (IPL and OPL), retinal pigment epithelium (RPE) disconnected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B), lipofuscin deposit underlying RPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), the outer nuclear layer (ONL) atrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, D-F), and the increase of neovascular vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, this disordered change did not observe in P10 Hsf4\u003csup\u003edel\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). Those results indicated that dysfunction of Hsf4 results in retinal disorder in mice in an age-related manner. To verify this, we recruited Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish model, in which Hsf4 gene was knocked out [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Interestingly the structure of Hsf4\u003csup\u003enull\u003c/sup\u003e retina looked normal at 10 dpf except the ONL of Hsf4\u003csup\u003enull\u003c/sup\u003e retina became thinner at 13 mpf as compared to that in wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Those results demonstrated that Hsf4 is essential for retinal homeostasis. To determine whether HSF4-mutant affects the visual function, we performed the ERG to Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice at P15, 3 and 7 months old. The results showed that Hsf4\u003csup\u003edel\u003c/sup\u003e mice exhibited low amplitude of a- and b-wave at dark adapted 0.01 cd-s/m\u003csup\u003e2\u003c/sup\u003e or 3.0 cd-s/m\u003csup\u003e2\u003c/sup\u003e as compared to wild-type mice regardless of mouse ages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and I). Since no cataract was observed in P15 Hsf4\u003csup\u003edel\u003c/sup\u003e mice, we proposed that the changed ERG in Hsf4\u003csup\u003edel\u003c/sup\u003e mice was due to the dysfunction of retina but not the cataracts. Taken together, these results demonstrated that the Hsf4\u003csup\u003edel\u003c/sup\u003e mutant impaired the vision at postnatal ages, and triggered the retina to undergo age-related atrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eHsf4\u003c/em\u003e \u003csup\u003e \u003cem\u003edel\u003c/em\u003e \u003c/sup\u003e \u003cem\u003emice and Hsf4\u003c/em\u003e\u003csup\u003e\u003cem\u003enull\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ezebrafish attenuate photoreceptor cells in an age-related manner\u003c/em\u003e\u003c/p\u003e \u003cp\u003eSince Hsf4\u003csup\u003edel\u003c/sup\u003e mice exhibited the abnormal ERG at postnatal age, we further studied the regulation of Hsf4 on the photoreceptor cells. we compared the photoreceptor cells of Hsf4\u003csup\u003edel\u003c/sup\u003e mice to wild-type mice in postnatal age P10 and adult mice of 7 and 13 months old. The biomarkers of Rhodopsin (RHOD) for rod cells or OPN1/SW and OPN1/MW for cone cells were measured by immunofluorescent staining. The results indicated that the expression of RHOD, OPN1/SW, and OPN1/MW, which exhibited no difference between Hsf4\u003csup\u003edel\u003c/sup\u003e and WT retina in P10 old mice, was significantly downregulated in 7 and 13 months Hsf4\u003csup\u003edel\u003c/sup\u003e retina as compared to that in the wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and these changes were confirmed again at both protein and mRNA levels by using immunoblotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E) and qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H). In zebrafish models, Hsf4\u003csup\u003enull\u003c/sup\u003e retina downregulated the expression of RHOD, GNB1 and GNAT2 at 7 mpf, and 13mpf as compared to wild-types at both protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-K) and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and M). These results are consisted with the results of H\u0026amp;E in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG. Taken together, these results confirmed again that dysfunction of HSF4 results in photoreceptor cells atrophy with age increase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eHsf4\u003c/em\u003e \u003csup\u003e \u003cem\u003edel\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eactivates glial cells and inflammation in postnatal retina, and this regulatory effect is enforced with mice ageing\u003c/em\u003e\u003c/p\u003e \u003cp\u003eGlial cells play an important role in modulating retinal homeostasis. Abnormal activation of the glial cells is associated with the retinal injury by upregulating the inflammation[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To determine whether gliosis is associated with disordered retina of Hsf4\u003csup\u003edel\u003c/sup\u003e, we measured the expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) in the retina of HSF4\u003csup\u003edel\u003c/sup\u003e mice vs wild-types at P1, P10, 7 and 13 months old. The immunofluorescent results showed that both GS and GFAP proteins were slightly upregulated in P10 HSF4\u003csup\u003edel\u003c/sup\u003e retina and this upregulation was enforced in 7- and 13- months HSF4\u003csup\u003edel\u003c/sup\u003e retina as compared to that in wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results of immunoblot and qRT-PCR showed that the expression of GS, which was downregulated in P1 HSF4\u003csup\u003edel\u003c/sup\u003e retina at both protein and mRNA levels as compared to wild-types (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D and F), was upregulated at protein and mRNA levels in P10 HSF4\u003csup\u003edel\u003c/sup\u003e retina, and the upregulation was enforced in 7- and 13-months Hsf4\u003csup\u003edel\u003c/sup\u003e retina. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D and F). It is unclear the mechanism underlying the downregulation of GS in P1 Hsf4\u003csup\u003edel\u003c/sup\u003e retina. GFAP, which is no difference between HSF4\u003csup\u003edel\u003c/sup\u003e and wild-type retina at P1 day, were upregulated at both mRNA and protein levels in HSF4\u003csup\u003edel\u003c/sup\u003e retina as compared to wild-types at P10, 7 and 13 months old (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C and E). These results suggested that HSF4\u003csup\u003edel\u003c/sup\u003e mutation activates gliosis in the postnatal retina, and the activation was enforced with age increase. Since activation of glial cells is associated with non-infectious inflammation, which is the pathological cause of retinopathy and neuronal diseases, we then measured the expression of inflammatory factors using qRT-PCR. The results showed that the expression of IL-1β, IL-6, IL-8 and VEGFA were significantly upregulated in P10, 7 and 13 months HSF4\u003csup\u003edel\u003c/sup\u003e retinal tissues as compared to that in wild-types (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H). These results suggested that Hsf4\u003csup\u003edel\u003c/sup\u003e mice upregulated the expression of inflammatory interleukins in the retina at early postnatal age. In addition to glial cells, the RPE cells were also the resource of inflammatory interleukins in the retina. we then separated the RPE from neuronal tissues of P10 Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-type mice. The qRT-PCR results showed that the expression of IL-6 and VEGFA were upregulated in both RPE and neuronal tissues of the Hsf4\u003csup\u003edel\u003c/sup\u003e retina as compared to that in wild-type counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and J). These results suggested that both RPE and glial cells were the resource of inflammatory factors in Hsf4\u003csup\u003edel\u003c/sup\u003e mice. To confirm the activation of gliosis is relying on HSF4, we performed a subretinal injection of AAV-Hsf4-Flag to 0.5 month Hsf4\u003csup\u003edel\u003c/sup\u003e mice followed by recovery for 2 weeks. The expression of glial proteins and inflammatory cytokines were measured by using immunoblot and qRT-PCR. The results showed that ectopic expression of HSF4b partially downregulated the high expression of GS, GFAP, IL-1β, IL-6 and IL-8 at both protein and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-N). To determine whether AAV2-hsf4b-Flag exerts any regulation on retinal structure, we performed a subretinal injection of AAV-Hsf4-Flag to one month Hsf4\u003csup\u003edel\u003c/sup\u003e mice followed by recovery for 2 months. The H\u0026amp;E staining results showed that administrated AAV2-Hsf4-Flag could partially reduced the disordered retinal structure of Hsf4\u003csup\u003edel\u003c/sup\u003e mice (Fig. S2B). Those results indicated that the activation of gliosis in Hsf4\u003csup\u003edel\u003c/sup\u003e retina is due to dysfunction of Hsf4b.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn zebrafish models, Hsf4\u003csup\u003enull\u003c/sup\u003e mutation slightly upregulated the expression of glial GFAP, GS, and inflammatory interleukins (including IL-1, Il-6 and IL-8) in 10 dpf and 7mpf, and this upregulation was enforced at 13 mpf zebrafish retina as compared to wild-type counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, O-S). Taken together, these results suggested that dysfunction of HSF4 activates the gliosis in the retina at early postnatal age.\u003c/p\u003e \u003cp\u003eIn the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, The PAS staining results showed that there were more small blood vessels in the aged Hsf4\u003csup\u003edel\u003c/sup\u003e retina than in wild-types. This upregulation was then confirmed by using immunohistochemistry staining with antibodies against CD31 and IB4 (Fig.S2A). Accordingly, we proposed that the upregulation of angiogenesis in the aged Hsf4\u003csup\u003edel\u003c/sup\u003e retina is associated with the high expression of VEGFA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and I).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHSF4\u003c/em\u003e \u003csup\u003e \u003cem\u003edel\u003c/em\u003e \u003c/sup\u003e \u003cem\u003emice downregulate the expression of visual cycle enzymes in RPE\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe retina of Hsf4\u003csup\u003edel\u003c/sup\u003e mice exhibits lipofuscin deposite in RPE and the abnormal upregulation of cytokines and VEGF expression in RPE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), implying that HSF4 participates in regulating RPE cell homeostasis. Since RPE\u0026rsquo; s predominant function is to modulate visual cycle, we then tested the potential regulatory effect of Hsf4 on the visual cycle by measuring the expression of visual cycles proteins, such as RPE65, RLBP1 and RDH5 in Hsf4\u003csup\u003edel\u003c/sup\u003e vs wild-type retina at P1, P10, 7 and 13 months old [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Using the immunofluorescent and RPE whole mount staining, we found that the expression of RPE65, RDH5 and RLBP1 in RPE were downregulated in P1 and P10 HSF4\u003csup\u003edel\u003c/sup\u003e retina as compared to that in wild-type, and the downregulation was enforced with age increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig. S3A). This downregulation was also confirmed by the immunoblot results followed by densitometry quantitation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E), and the qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H). To confirm the regulatory role of HSF4, we tested the expression of these visual-cycle proteins in the AAV2-Hsf4b-Flag-reconstituted Hsf4\u003csup\u003edel\u003c/sup\u003e retina. AAV2-Hsf4b-Flag were subretinally injected to 0.5 month Hsf4\u003csup\u003edel\u003c/sup\u003e retina followed by two weeks recovery. The expression of RPE65, RLBP1 and RDH5 was tested by immunofluorescence, immunoblot and qRT-PCR. The results showed that ectopic expression of HSF4b-Flag rescued the low expression of RPE65, RDH5 and RLBP1 in both protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-K) and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL) in Hsf4\u003csup\u003edel\u003c/sup\u003e retina. the immunofluorescent results showed that the subretinal injected AAV2-Hsf4-Flag was predominantly expressed in RPE cells (Fig. S3B). These results suggested that HSF4 participates in regulating the expression of visual cycle enzymes in RPE. To further confirm the regulatory effect of Hsf4 on the visual cycle-associates enzymes, we isolated and in vitro cultured the primary RPE cells from 2-months-Hsf4\u003csup\u003edel\u003c/sup\u003e retina or wild-type. After that, the plasmid pCMV-3xFlag-Hsf4b was transiently transfected into the primary RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM and N). The immunoblot results showed that the expression of RPE65, RDH5 and RLBP1 was downregulated in RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e cells as compared to that in RPE/wt. The overexpression of Hsf4b-Flag restored the expression of those enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM and N). These results confirmed again that HSF4 participates in regulating the expression of visual cycle proteins in RPE cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we verified this regulation in Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish. The Hsf4\u003csup\u003enull\u003c/sup\u003e lens exhibited transparency at 4 and 10 dpf, and opacification in 7 and 10 mpf (Fig. S3C). Consistent with the results in Hsf4\u003csup\u003edel\u003c/sup\u003e mice, the expression of visual cycle proteins RPE65, RDH5 and RLBP1 was downregulated at both mRNA(Fig. S3D) and protein levels (Fig. S3E-H) in 4 dpf, 10dpf, 7mpf and 13 mpf Hsf4\u003csup\u003enull\u003c/sup\u003e retina as compared to wild-type. Taken together, those results demonstrated that dysfunction of HSF4 impairs the expression of visual cycle enzymes at very early postnatal age.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHSF4\u003c/em\u003e \u003csup\u003e \u003cem\u003edel\u003c/em\u003e \u003c/sup\u003e \u003cem\u003emice triggers cellular senescence or apoptosis in the retina.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe previous data showed that HSF4\u003csup\u003edel\u003c/sup\u003e mutation upregulated P21\u003csup\u003ecip1\u003c/sup\u003e expression, which triggered lens epithelial cells to undergo senescence[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Hsf4\u003csup\u003edel\u003c/sup\u003e retina expressed a high level of the inflammatory interleukins at the postnatal age, implying that HSF4\u003csup\u003edel\u003c/sup\u003e might cause cells to undergo senescence or apoptosis in the retina. To prove this, we immunoblotted the expression of P21\u003csup\u003ecip1\u003c/sup\u003e and P16\u003csup\u003eINK4a\u003c/sup\u003e in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e vs. wild-types. P16\u003csup\u003eINK4a\u003c/sup\u003e was upregulated at both protein and mRNA in Hsf4\u003csup\u003edel\u003c/sup\u003e retina of P10, 7 and 13 months old mice but not in P1 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B and D). P21\u003csup\u003ecip1\u003c/sup\u003e is slightly upregulate in P1 Hsf4\u003csup\u003edel\u003c/sup\u003e retina as compared to that in wild-type, and aging underpinned the upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C and E). Reconstitution of AAV2-HSF4b-Flag to one-month HSF4\u003csup\u003edel\u003c/sup\u003e retina partially downregulated P21\u003csup\u003ecip1\u003c/sup\u003emRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In addition, the upregulation of P16\u003csup\u003eINK4a\u003c/sup\u003e and P21\u003csup\u003ecip1\u003c/sup\u003e mRNA was also observed in zHsf4\u003csup\u003enull\u003c/sup\u003e retina at 4dpf, 10dpf, 7mpf and 13mpf as compared to that in wild-type counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H). These results indicated that dysfunction of Hsf4 upregulated CKIs expression in retinal tissues. To determine whether Hsf4\u003csup\u003edel\u003c/sup\u003e retina undergo apoptosis, we then performed TUNEL assay. The TUNEL-positive cells were much more in 7 -and 13- months retina than that in P10 Hsf4\u003csup\u003edel\u003c/sup\u003e retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Few TUNEL- positive cells were observed in the wild-type retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Those results indicated that dysfunction of HSF4 elevated the cells\u0026rsquo; senescence or apoptosis in ONL and RPE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eHSF4\u003c/em\u003e \u003csup\u003e \u003cem\u003edel\u003c/em\u003e \u003c/sup\u003e \u003cem\u003emice deregulate the expression of heat shock proteins in the retina\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe fundamental function of Hsf4 is to regulate the expression of heat shock proteins. In lens tissue, HSF4 exerts a dual regulation on heat shock protein expression. It upregulates the expression of small heat shock proteins, such as Hsp25, Cryab and γ-crystallins, and simultaneously downregulates the expression of Hsp70, FGF4/7[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, its regulatory effects on the expression of heat shock proteins in the retina remain unclear. The immunoblot results showed that HSF4\u003csup\u003edel\u003c/sup\u003e mutation downregulated the expression of Hsp90 and Hsp25, but upregulated CRYAB expression at both protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D) and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G) in the retina of P1, P10, 7 M and 13 M mice as compared to that in wild-type counterparts. The results of immunofluorescent and RPE whole mount staining assays indicated that CRYAB expression, was significantly upregulated in Hsf4\u003csup\u003edel\u003c/sup\u003e retina as compared to wild-type (Fig. S4, A and B). The recapitulation of AAV2-Hsf4b-Flag into one-month Hsf4\u003csup\u003edel\u003c/sup\u003e retina restored the changed expression of heat shock proteins at both protein and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-J). Since Hsf4b-Flag is predominantly expressed in RPE cells (Fig S3B), we further tested the regulation of Hsf4 on those heat shock proteins\u0026rsquo; expression in the primary cultured RPE cells in vitro. As the results indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, the expression of Hsp90 and Hsp25 was downregulated and CRYAB expression was upregulated in RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e cells as compared to that in RPE/wt cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, lanes 1\u0026ndash;6 and L). Overexpression of Hsf4b-Flag partially restored the expression change of Hsp90, Hsp25 and CRYAB in the RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, lanes 4\u0026ndash;9 and L). These results confirmed that Hsf4b exhibits a distinctive regulation on different heat shock proteins\u0026rsquo; expression in RPE cells. In addition, we also tested the expression of heat shock proteins in the retina of Hsf4\u003csup\u003enull\u003c/sup\u003e vs. wild-type zebrafish. Deficiency of Hsf4 upregulated CRYAB and downregulated Hsp25 in the retina of 4dpf, 10dpf, 7mpf, and 13mpf zebrafish compared to wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM-O). In contrast, Hsf4\u003csup\u003enull\u003c/sup\u003e downregulated Hsp90 only in 7 and 13mpf zebrafish retina, but not in embryonic zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). These results suggested that dysfunction of HSF4 deregulated the expression pattern of heat shock proteins during retinal development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMice lacking Hsf4 develop microphthalmia. One of the pathological defects is lens cataracts. However, the regulatory effect of Hsf4 on other ocular tissues’ homeostasis, such as the retina, remains unclear. Using Hsf4\u003csup\u003edel\u003c/sup\u003e mice and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish, we find that in addition to the lens, Hsf4 participates in the regulation of retinal homeostasis. The inframe-deleted mutation of Hsf4 in mice (Hsf4\u003csup\u003edel\u003c/sup\u003e) or removing Hsf4 expression in zebrafish (zHsf4\u003csup\u003enull\u003c/sup\u003e) results in the downregulation of the expression of visual cycle enzymes (RPE65, RDH5 and RLBP1) in RPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. S3), and the activation of gliosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig. S2) and cellular senescence (upregulation of P21\u003csup\u003ecip1\u003c/sup\u003e and P16\u003csup\u003eINK4a\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), without changing retinal structural (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) at early postnatal age. With age increase, the retinal structure of Hsf4\u003csup\u003edel\u003c/sup\u003e mice and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish undergoes atrophy, characterized by disorganization of INL and IPL, ONL atrophy, RPE disconnection, lipofuscin accumulation, the increased neovascularization and apoptotic cells. Our results demonstrate that Hsf4 is an indispensable transcription factor for retinal homeostasis during retinal development.\u003c/p\u003e \u003cp\u003eThe atrophic retina is mainly visible in 7 and 13-month-old HSF4 mutant mice and 13 mpf zebrafish but not in postnatal age. However, Hsf4\u003csup\u003edel\u003c/sup\u003e mice and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish exhibited downregulation of visual cycle enzymes and upregulation of gliosis at postnatal ages. Impairment of visual cycle enzymes are associated with congenital pigmental retinitis and aged-related retinal degeneration. Abnormal activation of Gliosis is the predominantly pathological causative for retinal atrophy. Therefore, we thought that the retinal atrophy of aged Hsf4\u003csup\u003edel\u003c/sup\u003e mice and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish is the consequence of the activation of gliosis and the downregulation of visual cycle enzymes’ expression. The time line of retinal atrophy is overlapped with severe cataracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, those deteriorations in the retina have not been addressed in the previously reported Hsf4-deleted cataractous mice and zebrafish[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The possible reason is that most of previous studies regarding Hsf4 knockout mice focus on lens development at postnatal or rejuvenate ages, in which Hsf4 knockout mice or zebrafish have not developed retinal atrophy based on our data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although the structure of the retina does not change in the postnatal age, Hsf4\u003csup\u003edel\u003c/sup\u003e mice exhibit functional damage in the retina. For example, Hsf4\u003csup\u003edel\u003c/sup\u003e mice at postnatal age decrease the dark-adaptive response in ERG assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We postulate that this downregulation of ERG is associated with the downregulation of visual cycle enzymes, such as RPE65, RDH5 and RLBP1 in Hsf4\u003csup\u003edel\u003c/sup\u003e retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig. S3 and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) rather than lens opacification, because the downregulation of those visual cycle-enzymes occurred in the retina as early as P1 mice and 4 dpf zebrafish whereas lens looks normal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. S3 and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Another supporting evidence is that recapitulation of Hsf4-Flag restores the expression of visual cycle enzymes in Hsf4\u003csup\u003edel\u003c/sup\u003e mice in vivo and in REP/Hsf4\u003csup\u003edel\u003c/sup\u003e cells in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-N and Fig. S3). These results suggest that Hsf4 is an essential transcriptional factor for visual cycle enzymes expression during early retinal development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e–\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Defects of visual cycle turn out the accumulated vitamin A intermediates (e.g., A2E), the inducer of the oxidative stress and retinal degeneration [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore,we proposed that those early changes in Hsf4\u003csup\u003edel\u003c/sup\u003e mice and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish might exert a causative for the retinal degeneration.\u003c/p\u003e \u003cp\u003eA number of transcription factors have been reported to participate in regulating retinal development through controlling the expression of visual cycle enzymes, such as Pax9, Otx2, Sox9 and Lhx2 [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e–\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Among these transcription factors, Sox9 can regulate the transcriptional expression of multiple visual cycle enzymes RPE65 and RLBP1 by associating with Otx2 binding to these promoters[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Dysfunctional mutations of those transcriptional factors are associated with congenital retinitis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our results in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e indicate that Hsf4 acts as a novel transcriptional factor for the expression of visual cycle enzymes during early retinal development. However, the regulatory mechanism is still under investigation.\u003c/p\u003e \u003cp\u003eInterestingly, the regulation of Hsf4 on the expression of heat shock proteins differs from lens to retina, especially on Cryab protein. During lens development, Hsf4 upregulates small heat shock protein expression, such as Hsp25, Cryab, γ-crystallin, and simultaneously downregulates Hsp70, FGF and vimentin[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, in the retina of Hsf4\u003csup\u003edel\u003c/sup\u003e mice and zHsf4\u003csup\u003enull\u003c/sup\u003e zebrafish, we find that dysfunction of Hsf4 downregulates Hsp25 and Hsp90 but upregulates Cryab in the P10 retina, and these regulatory effects are enlarged with age increase. Hsf4\u003csup\u003edel\u003c/sup\u003e mice exhibit the upregulation of Cryab predominantly in RPE and glial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and S4). The onset of induction of Cryab is in P10 Hsf4\u003csup\u003edel\u003c/sup\u003e retina, but not in P1, and subretinal administration of AAV2-Hsf4b-Flag into Hsf4\u003csup\u003edel\u003c/sup\u003e retina or overexpression of Hsf4-Flag in the primary RPE/Hsf4\u003csup\u003edel\u003c/sup\u003e cells decreases the high expression of Cryab, which implies that Hsf4 inhibits Cryab expression at least in RPE cells. Cryab regulates diverse signal pathways, including anti-apoptosis, cell migration, myofiber remodeling in skeletal and cardiomyocyte and inflammation[\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The upregulation of Cryab in RPE and Glial cells allow us to postulate that Cryab may be involved in regulating gliosis, inflammation or cell survival, and this hypothesis needs further investigating.\u003c/p\u003e \u003cp\u003eOur previous data show that Hsf4\u003csup\u003edel\u003c/sup\u003e mice upregulate the expression of P21\u003csup\u003ecip1\u003c/sup\u003e and senescence-associated inflammatory factors in the lens[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; The MEF cells lacking Hsf4 undergo replicative senescence[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and mice lacking Hsf4 counteract P53-mutation-induced lymphoma and liver cancers[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Those results suggest that the big role of Hsf4 is the anti-cellular senescence. Consistent with its regulatory roles in lens, Hsf4\u003csup\u003edel\u003c/sup\u003e mice upregulate senescent biomarkers, such as, P21\u003csup\u003ecip1\u003c/sup\u003e and P16\u003csup\u003eINK4a\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and inflammatory interleukins, IL-1β, Il-6, IL8 and growth factor VEGFA in early postnatal retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig. S2). In addition, HSF4\u003csup\u003edel\u003c/sup\u003e mutation activate gliosis in the retina of P10 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Recapitulation of Hsf4 restores the expression of those changed proteins. These results suggest that Hsf4 plays a role of anti-senescence or anti-gliosis in the retina in addition to regulating visual cycle. As the consequence, the TUNEL results showed that there are more apoptotic cells in RPE and ONL of 7 and 13-month-old Hsf4\u003csup\u003edel\u003c/sup\u003e retina than in the P10 retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore, the up-regulation of cellular senescence and apoptosis may be the rational for retinal atrophy in the aged Hsf4\u003csup\u003edel\u003c/sup\u003e mice.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eHsf4 exerts multiple functions during retinal postnatal development, such as upregulating the expression of the visual cycle enzymes, tuning the expression of heat shock proteins, and downregulating glia-mediated inflammation and the expression of CKIs. Deregulation of the vision cycle and glia-mediated inflammation are early events for Hsf4\u003csup\u003edel\u003c/sup\u003e-induced age-related retinal atrophy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest with respect to the research, authorship, and publication of this article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData available\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the finding of this study are available within the paper and its supplementary information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by grants from the National Nature Science Foundation of China (No.81970785,\u0026nbsp;81570825, U1604171and 31802314),\u0026nbsp;the Joint Construction Project of Henan Medical Science and Technology Research Plan (Grand number: SBGJ202102157 and SBGJ202103068), and the Key Science and Technology Program of Henan Province (Grant No. 222102310467).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBaixue Liu and Youfei Lang\u003csup\u003e\u0026nbsp;\u003c/sup\u003eperformed most of experiments and data collection. Mengjiao Xue performed the tissue section and H\u0026amp;E staining assays. Mingjun Jiang assisted in animal breeding and isolation of Hsf4\u003csup\u003edel\u003c/sup\u003e RPE cells. Xiaolin Jia and Guiling Zhou assisted in Hsf4\u003csup\u003enull\u003c/sup\u003ezebrafish maintaining, breeding and genotyping. Dan Dan Chen assisted in ERG analysis. Fengyan Zhang and \u0026nbsp;Xuyan Peng contributed in project design and data analysis. Yanzhong Hu: support the whole project including material supplies, experiment design, data collection and manuscript draft. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaunier V, Merle BMJ, Delyfer MN, Cougnard-Gregoire A, Rougier MB, Amouyel P, Lambert JC, Dartigues JF, Korobelnik JF, Delcourt C (2018) Incidence of and Risk Factors Associated With Age-Related Macular Degeneration: Four-Year Follow-up From the ALIENOR Study. 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Exp Cell Res 382:111459. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.yexcr.2019.06.004\u003c/span\u003e\u003cspan address=\"10.1016/j.yexcr.2019.06.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heat shock factor 4, Visual cycle, Gliosis, Retinal degeneration, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4220460/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4220460/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eLoss of function of heat shock factor 4(HSF4) causes microphthalmia with lens opacification. The objective of this study is to uncover the regulation of HSF4 on retinal homeostasis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHsf4\u003csup\u003edel\u003c/sup\u003e mutant mice and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish models were recruited in this study. H\u0026amp;E was used to determine retinal structure. The immunoblot, qRT-PCR and immunofluorescence staining were used to measure the expression of mRNA and protein. AAV2-Hsf4-Flag virus were used to the reconstitution assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe retinal structure of Hsf4\u003csup\u003edel\u003c/sup\u003e mice and Hsf4\u003csup\u003enull\u003c/sup\u003e zebrafish, which is comparable to wild-type at P10 days old, undergoes atrophy at 7 and 13 months old. Dysfunction of Hsf4 downregulates the expression of visual cycle enzymes (e.g., RPE65, RLBP1 and RDH5 ) and heat shock proteins (e.g., HSP90 and HSP25), and simultaneously activates retinal gliosis (e.g., upregulating the expression of GFAP, GS, CRYAB, inflammatory interleukins, and VEGFA) and the expression of senescent P16\u003csup\u003eINK4a\u003c/sup\u003e and P21\u003csup\u003ecip1\u003c/sup\u003e in the retina of postnatal P1- P10 mice and embryonic zebrafish, and those changes are enhanced in 7 and 13 months old mice and zebrafish. Subretinal administration of AAV2-Hsf4b to the retina of one-month Hsf4\u003csup\u003edel\u003c/sup\u003e mice partially rescued the expression of changed proteins. ERG results showed that the downregulation of amplitude of a- and b- waves at scotopic response was detected at P15. Overexpression of Flag-Hsf4b in the in vitro cultured primary Hsf4\u003csup\u003edel\u003c/sup\u003e RPE cells restores the expression of visual cycle enzymes and heat shock proteins. TUNEL assay shows that there are more apoptotic cells in the ONL and the RPE of 7-and 13-month-Hsf4\u003csup\u003edel\u003c/sup\u003e retina than in P10 retina.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn addition to causing cataracts, the loss of function of HSF4 impairs the visual cycles and activates the gliosis in early postnatal age, which are associated with the retinal atrophy.\u003c/p\u003e","manuscriptTitle":"Dysfunction of heat shock factor 4 impairs retinal structure and visual function in mice and zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-15 04:38:50","doi":"10.21203/rs.3.rs-4220460/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3bd3a251-a824-4265-ada1-b268938bdaf6","owner":[],"postedDate":"April 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T18:58:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-15 04:38:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4220460","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4220460","identity":"rs-4220460","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}