GJD2 locus has been consistently associated with myopia in multiple independent genome-wide association studies. However, despite the strong genetic evidence, little is known about the functional role of GJD2 in refractive error development. Here, we find that depletion of gjd2a (Cx35.5) or gjd2b (Cx35.1) orthologs in zebrafish, cause changes in the biometry and refractive status of the eye. Our immunohistological and scRNA sequencing studies show that Cx35.5 (gjd2a) is a retinal connexin and its depletion leads to hyperopia and electrophysiological changes in the retina. These findings support a role for Cx35.5 (gjd2a) in the regulation of ocular biometry. Cx35.1 (gjd2b) has previously been identified in the retina, however, we found an additional lenticular role. Lack of Cx35.1 (gjd2b) led to a nuclear cataract that triggered axial elongation. Our results provide functional evidence of a link between gjd2 and refractive error.Myopia is the most common developmental disorder of juvenile eyes, and it has become an increasing cause of severe visual impairment. The  GJD2) gene, which is associated with myopia by genome-wide association studies. They link gjd2 with refractive error and report evidence to suggest that gjd2a plays a role in ocular biometry whilst gjd2b, previously found in the retina, possesses an additional lenticular role.Quint et al. use zebrafish lines deficient in one of two orthologs of the Gap Junction Delta-2 ( Given the current trends, 50% of the world population is expected to have myopia by the year 2050, and 10% will have high myopia with REs of −6 diopter (D) or higher1. The rising global prevalence drives a serious burden as myopia is linked to an increased risk of common and often sight-threatening eye diseases such as myopic macular degeneration, glaucoma, retinal detachment, and cataract3.Refractive errors (REs) arise when the focal length of the optics of the eye are not matched to the length of the eye, causing a defocused image on the retina. Myopia has become the most common type of RE after a significant increase in prevalence over the last decadesGJD2) gene, harboring regulatory elements that could potentially influence the transcription of GJD24. After this initial discovery, the association of this locus to myopia has been replicated by multiple independent studies in numerous ethnicities15. Despite the strong genetic evidence of an RE locus in the vicinity of GJD2, little is known about the functional role of GJD2 in RE.Myopia is a complex trait influenced by an interplay of both genetic and environmental factors. Genome-wide association studies (GWAS) have been successfully used to understand the genetic background of RE. In 2010, one of the first GWAS for RE in European populations identified a locus near the gap junction protein delta-2 (GJD2 encodes the 36-kDa protein connexin 36 (Cx36), a member of the connexin (Cx) protein family, and a key element of neuronal gap junctions. Of note, throughout the manuscript, we will use the official gene name  when referring to the gene and protein name  when referring to the protein. Within the nervous system, gap junctions function as electrical synapses regulating the bidirectional flow of ions and small metabolites between various neural cell types17. Although Cx36 is known to play an essential role in electrical gap junction signaling19 in the majority of retinal cell types31, the molecular mechanism underlying the potential function of Cx36 gap junctions in regulating emmetropization remains unknown.Danio rerio) to gain insight into potential mechanisms for GJD2 in RE. We examined the ocular consequences of the loss of function of two GJD2 (Cx36) zebrafish homologs: gjd2a (Cx35.5) and gjd2b (Cx35.1). Both Cx35.5 (gjd2a) and Cx35.1 (gjd2b) are known to assemble at the electrical synapse across the zebrafish central nervous system (CNS)34. The generally held view is that Cx35.1 (gjd2b) acts as the main retinal connexin, while Cx35.5 (gjd2a) functions mainly in the CNS35. Our results suggest that both gjd2a (Cx35.5) and gjd2b (Cx35.1) are expressed in the retina, and that loss of function leads to alterations in ocular biometry and development of RE.In this study, we used zebrafish (gjd2a) or Cx35.1 (gjd2b)32. To prevent biases induced by potential differences in the mean body sizes of gjd2a (Cx35.5) and gjd2b (Cx35.1) mutants, we compared these fish to wild type (WT) controls that matched the mean body length of the mutant group within a 10% range ) into adulthood (3 mpf) and studied two independent lines that were deficient for Cx35.5 (gjd2a (Cx35.5) mutants, we observed a significant reduction in axial length  mutants  and gjd2b (Cx35.1) mutants showed alterations in corneal thickness, anterior chamber depth, and RPE thickness . Figure 40. We visualized the relative differences in RE between mutant and control fish  mutants showed a progressive positive (hyperopic) RE  mutants was reduced and that thinner or flattened lenses may also lead to hyperopic defocus41, we isolated the lenses of gjd2a (Cx35.5) fish (n = 10) but did not observe any macroscopic abnormalities in lens shape and curvature compared with lenses of WT fish.To determine whether the observed alterations in axial length led to RE, we assessed the refractive status of all lines using eccentric photorefraction. Eccentric photorefraction has served as a sensitive method for refractive measurements in many speciesish Fig. . Consist RE Fig. . Given tgjd2a (Cx35.5), in the gjd2b (Cx35.1) mutant eyes, we found a progressive negative (myopic) RE  mutants showed a relatively large variation in RE measurements  of gjd2b (Cx35.1) mutant eyes showed no visible intensity gradient, a phenomenon we observed in fish with severely enlarged axial lengths  mutant43). We measured a group of lrp2 mutants with moderate axial myopia and found a typical myopic intensity gradient, whereas the gjd2b (Cx35.1) fish displayed a seemingly bifocal signature  mutants, we examined the lenticular appearance of the 1.5, 2, and 3 mpf fish on SD-OCT. We were able to observe the nuclear fiber structures in the gjd2b (Cx35.1) mutant lenses  mutant fish  mutants, relative to WT fish  mutants showed opaque ring-like fiber structures, whereas this was not visible in WT lenses  mutants  mutants.To study the origin of the non-axial form of myopia and bifocal intensity gradient found in the ses Fig. , which wses Fig.  and gjd2ish Fig. . To explish Fig. . At thisses Fig. . Ex vivonts Fig. , g, h, wnts Fig. , f, h. Fgjd2b (Cx35.1) mutants also had a significant increase (7%) in total axial length  mutant lenses for a variety of wavelengths   mutant. The light path of the WT control lenses (n = 3) showed a finer distinction of individual light rays and more homogenous dispersion  Fig. . To furtnm) Fig. . We obsenm) Fig. , f and pnm) Fig.  of the gion Fig. . This obgjd2a) and Cx35.1 (gjd2b) on retinal light processing, we measured the electrical potential of the retina using electroretinograms (ERG) focusing on the B-wave. In total, 2.5 mpf gjd2a (Cx35.5) eyes showed a significant decrease in B-wave amplitude compared to WT control  mutants was not different from WT fish  and gjd2b (Cx35.1) mutants  mutants also showed a significant reduction in nasally directed eye tracking movements per 10-s interval  protein in retinal tissue, we performed immunostaining using a specific anti-Cx35.5 (gjd2a) monoclonal antibody32 in 2.5 mpf fish. In WT fish, we observed a punctate signal throughout the neural retina  plaques were visible in the somata of retinal ganglion cells and more abundantly in the inner plexiform layer (IPL) and outer plexiform layer (OPL). In addition, in the outer retina, a modest distribution of small gap junction plaques was found between adjacent photoreceptors. The signal was absent in the gjd2a (Cx35.5) mutants, confirming the specificity of the antibody  in the zebrafish CNS32. As expected, we found a typical staining pattern of gap junction plaques in the WT retina    and Cx34.7 (gjd1b)51, we examined the staining profile in a mutant for both gjd2 paralogues . A complete loss of immunostaining in the gjd2a; gjd2b double mutant  and Cx35.1 (gjd2b). Hence, we refer to this antibody as the “anti-pan-Cx35”.We also used a commercially available antibody MAB3045 designed against recombinant perch Cx35 proteinina Fig.  and furt.5) Fig.  and gjd2.1) Fig.  mutants.gjd2a/Cx35.5 in both mutants (Supplementary Table gjd2a) and Cx35.1 (gjd2b) gap junctions. In the IPL, the typical punctate staining pattern marked gap junction plaques containing both homologs. In the outer plexiform layer, we found a relatively dense population of Cx35.5 (gjd2a) and Cx35.1 (gjd2b) plaques. Furthermore, we observed modest Cx35.5 (gjd2a) and Cx35.1 (gjd2b) gap junction plaques in the photoreceptor layer between the outer segments as well as across the outer nuclear layer  mutant fish, we investigated the distribution of Cx35.1 (gjd2b) in the lens. Immunostaining of adult WT lenses with anti-pan-Cx35 resulted in modest lateral immunostaining around the outer cortical region; this was lacking in the gjd2b (Cx35.1) mutant. The immunostaining for anti-pan-Cx35 in lenses of the gjd2a (Cx35.5) mutant was similar to the WT lenses  and gjd2b (Cx35.1), we examined single-cell RNA sequencing (scRNAseq) from a whole-embryo dataset52. Relevant retinal cells were captured from the larger dataset using the expression of established tissue and cell markers for retinal cell types (Supplementary Data gjd2a (Cx35.5) and gjd2b (Cx35.1) throughout a variety of retinal cell types , whereas we did not detect gjd2b (Cx35.1) expression in ganglion cells, while putative horizontal cells expressed gjd2b (Cx35.1) but not gjd2a (Cx35.5)  in the scRNAseq data. In short, the histological and scRNAseq findings showed a corresponding expression pattern for both gjd2a (Cx35.5) and gjd2b (Cx35.1).To further investigate the expression of retinal pes Fig. . Only a pes Fig. , c, howepes Fig. . At thispes Fig. . In addi.5) Fig. , c. We nGJD2 in human RE. We performed an in-depth ocular characterization of two GJD2 (Cx36) zebrafish orthologs: gjd2a (Cx35.5) and gjd2b (Cx35.1). We compared the biometry, optics, and electrophysiology of mutant zebrafish eyes with WT eyes, and characterized protein localization by histological and gene expression analyses. Our results suggest that depletion of the orthologs leads to various ocular phenotypes, including biometrical, optical, structural, and electrophysiological changes.In this study, we aimed to increase insight into the potential role of gjd2a (Cx35.5) mutant, we found a hyperopic phenotype mostly determined by a reduction in axial length. The main ocular components that underlined the axial reduction were decreased vitreous chamber depth and lens diameter. Furthermore, our ERG studies showed a reduction in the B-wave amplitude of these mutant fish. This is the first time that biometrical changes and RE have been characterized in a connexin depleted animal model; however, the diminished B-wave potentials have been reported earlier in Gjd2 (Cx36) null mice and have been attributed to defects in the ON-bipolar signaling57. It remains unknown why depletion of gjd2a (Cx35.5) was sufficient to provoke an electrophysiological effect on ERG while not significantly altering the spatial acuity measured by the optokinetic response, as ON-bipolar cell signaling is also highly involved in spatial vision58. Besides, the gjd2a (Cx35.5) mutant fish showed a hyperopic phenotype, but no other eye abnormalities were observed. This finding is in line with what is expected from a gene involved in regulating axial eye growth. It is worth mentioning that in humans, GJD2 (Cx36) is associated with RE in the general population and not with syndromic forms of myopia. Therefore, alterations in axial length without other gross abnormalities resemble the human situation.In the gjd2a (Cx35.5) mutant fish and the reported literature, we hypothesize that the uncoupling of retinal gap junctions inhibits ocular growth. Evidence supporting this notion includes the observation that pharmacological uncoupling of gap junctions using meclofenamic acid protects against form-deprivation myopia (FDM), an experimental form of myopia, in chicks60. Furthermore, it has been shown that high-intensity light has a protective effect against childhood64 and experimental67 myopia, whereas chicks and monkeys exposed to continuous high-intensity light developed severe hyperopia68. It is known that lighting conditions can modulate retinal gap junction coupling through various pathways and neuromodulators by modifying its phosphorylation state69. For example, it has been described that Cx35/Cx36 remains in a dephosphorylated (uncoupled) state under bright lighting conditions73. Therefore, we speculate that high-illuminant light inhibits ocular growth through the uncoupling of retinal gap junctions. Two important modulators are dopamine and adenosine. Light-directed Cx35/36 (de)phosphorylation has been described to be regulated by dopamine signaling (effect: dephosphorylation/uncoupling)82 and adenosine signaling (effect: phosphorylation/coupling)82. Furthermore, studies showed that adenosine antagonists (uncoupling effect) appeared effective against childhood83 and experimental86 myopia, whereas stimulating dopamine signaling (uncoupling effect) in the eye appeared protective against experimental myopia88. In short, the previous findings suggest that the uncoupling of retinal gap junctions through either meclofenamic acid, high-intensity light, or adenosine inhibition/dopamine stimulation all have inhibitory effects on axial growth  mutants may have resulted from the permanent ablation of Cx35.5 (gjd2a) gap junctions in the retina, leading to a lower degree of cellular coupling in the retina.The precise molecular mechanism explaining the relation between retinal gap junction coupling and eye growth remains unknown. However, based on our results in the 89. Other studies exploring the intricate relation between the phosphorylation state of Cx36 (GJD2) in the retina and RE are warranted.A limited number of studies have explored the phosphorylation state of Cx36 in animal models of myopia. A recent study describes an FDM murine model in which eyelids were sutured for 40 days. After removing the lid suture, mice showing a negative RE were selected for immunohistochemistry studies in which the phosphorylation state of Cx36 was assessed. The authors report increased phosphorylation and coupling of Cx36 between AII amacrine cells in the studied eyes and attribute their finding to a compensation mechanism aiming to improve the signal-to-noise ratio caused by the defocused state during lid suturegjd2b (Cx35.1) mutant, we observed a normal axial length; however, unexpectedly, a negative (myopic) RE by photorefraction. In our photorefraction studies, we discovered that the intensity gradient of the gjd2b (Cx35.1) mutants showed a bifocal signature when compared to the myopic lrp2 mutants. By SD-OCT and DIC, we found that the RE in the gjd2b (Cx35.1) mutants had a lenticular origin due to a progressive, cataract-like, opacification of the nuclear layers. In the mammalian lens, gap junctions include connexin 43 (Cx43), 46 (Cx46), and 50 (Cx50)93. Consistent with our results in the gjd2b (Cx35.1) mutant, the loss of mammalian Cx46 and Cx50 also leads to cataract development93, and mutations in human Cx46 and Cx50 have been associated with autosomal dominant congenital cataract97. Although to date, Cx35.1 (gjd2b) has not been described as a lenticular connexin, four other main connexins, Cx43, Cx44.1, Cx48.599, and the larger Cx79.829, have previously been found in the lens. Whereas Cx48.5 zebrafish morphants showed modest nuclear opacities at 9.5 dpf99, the gjd2b (Cx35.1) mutant showed a cataract phenotype in a much later developmental stage (60% at 3 mpf). Similar to mammalian Cx46 and Cx5091, the immunostaining for Cx35.1 (gjd2b) appeared around the equatorial region of the lens. We presume that Cx35.1 (gjd2b), like other vertebrate lenticular connexins100, is essential in zebrafish for lens circulation by facilitating an outward intracellular current of water, ions, and small metabolites. Disruption of this microcirculation system may have led to the accumulation of metabolic waste102 inducing the observed scattering and diffraction. As the lenticular immunoreactivity was only modest, further investigation is needed to pinpoint the exact topographic distribution of Cx35.1 (gjd2b) throughout the zebrafish lens. Translation of our findings into human cataracts is challenging given that expression of GJD2 (Cx36) in human lenses has not been reported. This may suggest that gjd2b (Cx35.1) has evolved and diverged in function in zebrafish.In the gjd2b (Cx35.1) mutant fish, in humans, patients with nuclear cataract experience lenticular myopia due to an increased refractive index105. The severity of the nuclear cataract in those patients correlates strongly with the severity of the myopic shift103, which is consistent with the progression of both cataract and RE in the gjd2b (Cx35.1) mutants. Given that no biometric changes were observed during the first 3 months, we hypothesize that the initial myopic shift observed with photorefraction may have been induced by the converging properties of the opacifying lens nucleus. During adulthood, the cataract progressed substantially, leading to a degradation of retinal image quality, which may have triggered the late-onset axial elongation observed at 6 mpf. In the juvenile fish (1.5 and 2 mpf), we did not observe changes in axial length due to the fact that only 25% of the fish at this developmental stage showed a significant cataractous phenotype, reducing the sample size and power at these two particular time points. We further demonstrated light distortion by the cataractous lenses of the gjd2b (Cx35.1) mutant, indicative of a level of image degradation compared to the translucent diffusers used to trigger FDM110. The susceptibility for FDM in adult teleost fish has also been confirmed in tilapia111. In conclusion, we postulate that the cataract, negative RE, and late-onset axial elongation were more an effect of the lenticular and not intraretinal loss of Cx35.1 (gjd2b) gap junctions and that the function of gjd2b (Cx35.1) in zebrafish diverges from the function of human GJD2. Also, the reduction of the number of eye-tracking movements after the loss of Cx35.1 (gjd2b) may have been the result of the emerging cataract rather than an intraretinal signaling defect, as the B-wave amplitude was not altered.Consistent with the negative RE in the gjd2a), Cx35.1 (gjd2b) has previously been reported as a retinal connexin in zebrafish. A large part of the described functional and topographic characteristics of Cx35.1 (gjd2b) in databases was based on studies into “Cx35” before discovering Cx35.5 (gjd2a)112. These studies generally used antibodies such as the cross-reacting anti-pan-Cx35 (MAB3045)50 and anti-Cx36 (Ab298)50 that binds an epitope conserved between Cx35.5 (gjd2a) and Cx35.1 (gjd2b). Further, functional studies into light-dependent phosphorylation and coupling of “Cx35” used phosphospecific anti-Cx35 antibodies113 that showed cross-reactivity with homologs of other vertebrates70. In this study, we showed specific expression and functionality of gjd2a (Cx35.5) in the zebrafish retina. Therefore, we presume that the reported topography, and to some extent, functional characteristics of the former “Cx35” was composed of a shared contribution of Cx35.5 (gjd2a) and Cx35.1 (gjd2b).In contrast to Cx35.5 (gjd2a) and Cx35.1 (gjd2b) was similar to each other, and as expected, to mammalian Cx36118.We found that the intraretinal distribution of Cx35.5  and Cx35.1 (gjd2b) in the photoreceptor layer and modest expression of gjd2a (Cx35.5) and gjd2b (Cx35.1) in the rods and cones of the 2–5 dpf larvae. In the OPL of adult zebrafish, we found a dense population of Cx35.5 (gjd2a) and Cx35.1 (gjd2b) gap junction plaques resembling the distribution in mammalian species with cone rich retinas such as humans and guinea pigs70. The scRNA-seq study showed that gjd2a (Cx35.5) and gjd2b (Cx35.1), similar to mammalian Cx36 (GJD2)119, were both expressed in bipolar and amacrine cells. Interestingly, gjd2b (Cx35.1) expression was also detected in a number of horizontal cells, similar to Cx35 in carp120, whereas expression of mammalian Cx36 (GJD2) has not been reported in horizontal cells24. In our scRNA-seq, we detected gjd2a (Cx35.5), but not gjd2b (Cx35.1), expression in larval ganglion cells. As the somatas in the GCL showed a positive signal for the anti-pan-Cx35 in the gjd2a (Cx35.5) mutant, the absence of gjd2b (Cx35.1) in ganglion cells in the scRNA-seq study may have been a consequence of the early stage in development or the limited total retinal cell count in the data set. Similarly, Cx36 (GJD2) has been found in dendrodendritic gap junctions between alpha-type ganglion cells114 and ganglion-to-amacrine cells119. It is worth noting that given the limited total retinal cell count of the scRNA-seq data, caution is required when making broad interpretations of the data.Cx36 containing gap junctions in the photoreceptor layer have previously been described in a cone–cone and rod–cone configurationGJD2 (Cx36) have consistently been associated with RE (rs634990 and rs524952)15. In the reported studies, the risk allele has been associated with a myopic RE. However, as observed in other complex traits, the examination of the coding region of GJD2 (Cx36) has not revealed coding variants explaining the GWAS signal, and it has been suggested that the identified variants might play a role through gene regulation4. Here, we found that the absence of gjd2a (Cx35.5) expression in zebrafish leads to a hyperopic phenotype, and we hypothesized that the observed phenotype is the result of the uncoupling of retinal cells. Therefore, one could speculate that in humans, the identified risk variants might up-regulate the expression of GJD2, facilitating the coupling of Cx36 (GJD2) gap junctions in the eye. This needs further exploration in studies of human ocular tissues, particularly those exploring regulatory elements such as expression (eQTL) or methylation (meQTL) quantitative trait loci121.In humans, two common genetic variants downstream of gjd2a (Cx35.5) and gjd2b (Cx35.1). Given the phenotypic changes and the gene expression pattern observed in both the gjd2a (Cx35.5) and gjd2b (Cx35.1) mutants, we conclude that the hyperopic phenotype observed in the gjd2a (Cx35.5) mutant is related to the retinal role of Cx35.5 (gjd2a). In contrast, the phenotype observed in the gjd2b (Cx35.1) mutant is caused by a cataract, suggesting that the function of Cx35.1 (gjd2b) in zebrafish diverges from its human counterpart. Our results suggest that the cellular function of Cx35.5 (gjd2a) is most similar to mammalian Cx36 (GJD2) in terms of retinal localization, functionality , and the potential regulation of the axial length. However, the exact molecular mechanism underlying the reduced axial length after gjd2a (Cx35.5) depletion is yet to be determined. Studies examining, for example, the retinal cell-specific transcriptome of the gjd2a (Cx35.5) mutant may help understand the retina-to-sclera signaling pathway that follows the lower degree of cellular coupling, and as a result, inhibition of axial growth. Our study demonstrates that zebrafish is a suitable model to study REs. Furthermore, the gjd2a (Cx35) mutant provides unique opportunities for exploring the relationship between gap junction uncoupling and eye growth and promises to generate insights into the biological mechanism underlying human REs.In summary, we have provided an in-depth ocular characterization of zebrafish gjd2a (gjd2afh437) and gjd2b (gjd2bfh454) mutant lines were provided by Dr. Adam Miller from the Institute of Neuroscience at the University of Oregon. The lines were generated using TALENs by targeting the first exon32. Furthermore, to minimize potential differences due to genetic background, we used (for both gjd2a (Cx35.5) and gjd2b (Cx35.1)) the offspring of a heterozygous incross , to generate the WT control and mutant lines studied here. The lrp2/−− (lrp2mw1) mutant line43 was kindly provided by Prof. Brian A. Link . Stable zebrafish lines were genetically validated by Sanger sequencing, and raised in tanks with a matched population size and constant feeding pattern to minimize the environmental influence of body size in ocular development. During OCT, photorefraction, OKR, and ERG, zebrafish were anesthetized using a 0.016% tricaine methanesulfonate solution , buffered to pH  =  7.The All animals were raised and treated in accordance with the Dutch animal welfare legislation and the guidelines from the experimental animal health care center (EDC: Experimenteel Dier Centrum) of the Erasmus Medical Center Rotterdam, The Netherlands. All experiments were conducted in accordance with the European Commission Council Directive 2010/63/EU . All fish were kept on a 14-h light: 10-h dark cycle at a constant temperature of 28.5 °C.gjd2a (Cx35.5) and gjd2b (Cx35.1)) the offspring of a heterozygous incross , to generate the WT control and mutant lines studied here. 3D compositions of the eye were generated by a Thorlabs SD-OCT 900 nm Ganymade system. The fish were positioned on a rotatable platform and kept under constant anesthesia. The measured eye was positioned unilaterally above the tricaine solution. The total XYZ field of view was 1.7 × 1.7 × 2.2 mm with a pixel depth of 2 µm in the Z-direction. Custom MATLAB software was used to separate the XYZ planes, borders were marked manually for each ocular component. The dimensions of the ocular components were calculated by the script based on the refractive index of the corresponding tissue type. The corneal refractive index was set to 1.33124. The refractive index of the zebrafish lens was calculated from the gradient refractive index as reported by multiple independent studies and set to 1.4127. The refractive index for the anterior chamber and vitreous chamber was set to 1.34 and the retinal refractive index was set to 1.38129.To rule out variability in eye metrics as a result of alterations in total body length, body sizes were measured at each time point. To isolate the intraocular biometrical changes from extraocular and globally altered body dimensions, only WT control fish within a 10% range from the average mutant body size were included as a control  was aligned perpendicular to the ocular surface with a slight angle relative to the water surface. This prevented irregularities in the brightness profiles as the result of surface reflections. Custom C++ software was used to measure the slope of the gradient brightness profile in real-time. Hundred independent measurements were averaged for each eye. The slope of the brightness gradient was converted into RE by calibrating the system with ophthalmic lenses (−5 to +10 D range). The resulting plot showed a high coefficient of determination (r2 = 0.971) and a conversion factor of 1.924. The conversion factor was used to convert the slope of the brightness profile into the RE (in Diopter). This RE may have been subject to a small eye retinoscopic artifact40 i.e., the infrared light reflects back due to the myelin-rich layer of the retinal nerve fiber layer, while during normal conditions the light is optimally focused on the light-sensitive part of the photoreceptors. Considering the visual acuity of zebrafish135 we assumed that WT fish have a refractive status close to emmetropia. The relative RE was calculated by subtracting mutant REs by the baseline RE of the WT group.The refractive state of the fish was measured by a custom eccentric infrared photorefractorish Fig. . The fisThe presence and proportion of cataract lenses were quantified in coronal  sections at the center of the anterior-posterior axis of 1.5–3 mpf fish. An additional 6 mpf group was analyzed by OCT for the presence of cataracts and relative changes in axial length. For this 6 mpf time-point, the extent of the opacities in the lens was quantified in ImageJ. Binary images of coronal lens sections were created and by automated thresholding, the ratio of opaque pixels was measured. For the ex vivo quantification of the proportion of cataract lenses in the 6 mpf group, a differential interference contrast microscope (Nikon WideField Ti-Eclipse inverted microscope with Coolsnap CCD camera) was used. Lenses were classified as cataractous when the nuclear ring structure was clearly visible.To evaluate image distortion by cataract, changes in optical capacities were assessed in isolated lenses by transmission measurements and light path visualization. The customized setup for transmission measurements consisted of a transmission meter , emitting light at three wavelengths; IR light (940 nm), UV light (365 nm), and broad-spectrum visible light (380–760 nm). A light sensor detected the relative loss of intensity as a result of optical changes of the lens.136.To visualize the light path a customized setup Fig.  consisti137. Two 100 ms139 light stimuli with a light intensity of ~6000 lux were given with an 8000 ms interval. The response was amplified 10.000 times with a band-pass of 700–0.1 Hz and recorded with the Signal6.03 software (Cambridge Electronic Design Limited). The recordings were baseline corrected, with the baseline signal determined as the average signal before the stimulus was given, during a 50 ms timespan. The average response to the two light stimuli was plotted and the maximal B wave amplitude was calculated and additionally normalized for body length.Before the ERG recordings, the fish were dark-adapted for at least 30 min and handled under dim, red light illumination. In total, 2.5 mpf fish were subsequently anesthetized using 0.016% tricaine methanesulfonate solution and were placed in a petri dish filled with agarose gel, with the reference electrode placed into the gel and the right eye facing the light source. The spinal cord was severed to stop the heartbeat. Next, a small incision was made with a 25-gauge syringe needle at the edge of the cornea of the right eye, in which the recording electrode filled with E2 medium was placedVisual acuity was measured in 3 mpf fish with a custom recording device , starting at 0.15 cpd, until an OKR could no longer be elicited. The highest spatial frequency was repeated to verify the loss of the OKR pattern. The response was considered positive when three or more consecutive optokinetic nystagmus patterns were detected in both the temporal to nasal and nasal to temporal direction. The visual acuity was defined as the maximum spatial frequency in cpd that was reached. As a second analysis, the slope of the slow phase of the OKR pattern was quantified and the eye velocity was calculated in d/s. This was done for three different spatial frequencies; 0.15, 0.20, and 0.25 cpd. Calculated values were quantified for the temporally and nasally directed movements. The OKR graphs were analyzed in Python (version 3.8. 0) and the optokinetic gain was determined as the ratio between the eye velocity and drum velocity, in which a gain of 1 indicates a perfect tracking of the moving stimulus. In addition, the number of eye-tracking movements per 10 s interval was quantified for each spatial frequency.gjd2a/Cx35.5  an anti-rabbit-cy5 (Jackson ImmunoResearch), both 1:200. As a cytoplasmic marker Phalloidin546 (1:200) (ThermoFisher) was used. After 1 h incubation at RT, slides were washed and mounted with ProLong Gold Antifade Mountant with DAPI (ThermoFisher). Imaging was done using a Confocal (Zeiss LSM700) microscope.For immunostaining of retinal sections, the eyes of 2.5 mpf fish were dissected and embedded in OCT medium . The eyes were frozen in nitrogen/penta-butane, and stored at −20 °C. Cryosections of 8 µm were cut laterally and placed on adhesion slides . Slides were dried with pressurized air and fixated in 4% PFA for 8 min. The slides were washed in PBS and subsequently blocked for 1 h at room temperature in a 5% sheep serum (Sigma-Aldrich) diluted in PBS solution. A wash step of 3 times 5 min in PBS was performed, and the primary antibody (1:100) was applied. After overnight incubation at 4 °C, slides were washed and secondary antibodies applied. For MAB3045 (Millipore) an anti-mouse-cy5 (Sigma-Aldrich) was used and for anti-For whole-mount imaging of the lens, 6 mpf zebrafish lenses were dissected in PBS and fixed in 1.5% PFA in PBS for 24 h at room temperature. Lenses were washed 3× times 10 min in PBS and permeabilized in a PBS-0,1% Tween-20 solution, overnight at 4 °C, and subsequently incubated with primary antibodies diluted 1:100 in PBS-0,1% Tween-20 and, again, incubated overnight at 4 °C. Lenses were washed three times and secondary antibodies were diluted 1:200 in PBS-0,1% Tween-20 and incubated overnight at 4 °C. For each genotype, the immunostaining was performed twice. For imaging, lenses were fixated in 1.8% low melting point agarose (Invitrogen) covered with PBS. A confocal microscope (Leica SP5 AOBS) with a water immersion objective (HCX PL APO 20× NA 1.0) and 561 nm laser DIC were used to record z-stacks of the lenses. Subsequently, coronal optical slices were evaluated over the anterior-posterior axis.52 and modified slightly as noted below. Briefly, cells were dissociated from the Tg(olig2:GFP)vu12 and Tg(elavl3:GCaMP6s) larvae sampled at 1, 2, and 5 days post-fertilization. Dissociated cells were then run on a 10X Chromium platform using 10× v.2 chemistry140. To ensure that the full transcript of the Connexin-encoding genes was represented in the dataset, we updated the Ensembl release 97 General Transfer Format (GTF) gene model file by using pooled, short-read RNA-seq data. Using this updated GTF file, we aligned reads to the zebrafish GRCz11_93 genome using the 10X Cellranger pipeline (version 3.1)140. We used the Seurat142 V3 software package for R, v3.6.2 using standard quality control, normalization, and analysis steps to cluster and we used 140 principle components to analyze the final 68,766 cells. UMAP analysis was performed on the whole dataset with a resolution of 13.0, which produced 226 clusters and one singleton. Retinal analysis was performed on a selection of seven clusters based on canonical markers of cell types, capturing a total of 2218 cells. We restricted the retinal analysis to cells originating from the 2 and 5 dpf fish.Details as described in Farnsworth et al.143.Statistical analyses were performed using R (version 4.0.2). Given that measurements from the two eyes of the fish were available, we used linear mixed models (LMM) to analyze the SD-OCT, photorefraction, DIC, lens intensity, and lens opacity data. The LMM accounts for the correlations between paired eyes of a fish by adding a random intercept. LMM has been described to appropriately account for inter-eye correlation while maximizing power and precisionIn all the performed mixed models we controlled for genotype (WT or mutant) as a fixed factor, and for fish (subject) as a random factor. The following outcomes were investigated: axial length, anterior chamber depth, lens diameter, vitreous chamber depth, retinal diameter, and RPE thickness in µm (SD-OCT), RE in Diopters (photorefraction), the number of opaque pixels (lens opacity by OCT) and the percentual loss of transmitted light through the lenses.Even though LMM also allows the analyses of longitudinal data, individual labeling of fish is challenging. Therefore, we analyzed the studied time points  separately  and not as longitudinal data.For the ERG, Optical properties of the lens, OKR, and ex vivo light propagation experiments we used Welch’s ANOVA (Prism version 8.4.1).Further information on research design is available in the Transparent Peer Review FileSupplementary InformationDescription of Supplementary FilesSupplementary Data S1Supplementary Data S2Supplementary Data S3Supplementary Data S4Reporting Summary


In NSCLC, CDCA3 protein levels are regulated by the ubiquitin ligase APC/C and cofactor Cdh1. Here, we identified that the degradation of CDCA3 is modulated by activity of casein kinase 2 (CK2) which promotes an interaction between CDCA3 and Cdh1. Supporting this, pharmacological inhibition of CK2 with CX-4945 disrupts CDCA3 degradation, elevating CDCA3 levels and increasing sensitivity to platinum agents. We propose that combining CK2 inhibitors with platinum-based chemotherapy could enhance platinum efficacy in CDCA3low NSCLC tumours and benefit patients.Platinum-based chemotherapy remains the cornerstone of treatment for most non-small cell lung cancer (NSCLC) cases either as maintenance therapy or in combination with immunotherapy. However, resistance remains a primary issue. Our findings point to the possibility of exploiting levels of cell division cycle associated protein-3 (CDCA3) to improve response of NSCLC tumours to therapy. We demonstrate that in patients and in vitro analyses, CDCA3 levels correlate with measures of genome instability and platinum sensitivity, whereby CDCA3 Kildey et al find that high levels of mitotic regulator CDCA3 correlates with sensitivity to platinum agents in non-small cell lung cancer patients and cell lines. They show that interfering with CDCA3 degradation through CK2 inhibition enhances CDCA3 levels and increases sensitivity to platinum agents suggesting a therapeutic route. The most commonly diagnosed form of lung cancer is non-small-cell lung cancer (NSCLC) which is broadly subdivided into the adenocarcinoma (ADC) and squamous cell carcinoma (SqCC) histologies. Patients with NSCLC may undergo adjuvant chemotherapy for resectable disease, chemoradiotherapy, and maintenance immunotherapy for locally advanced disease or systemic treatment  for advanced disease and palliative care. However, despite improvements in these strategies and the introduction of targeted therapies (including immunotherapy), the global 5-year survival rate remains poor at 19%3.Lung cancer is the leading cause of cancer-related mortality accounting for 18.4% of all cancer-related deaths worldwidecis-Diamineplatinum (II) dichloride, is a common platinum agent used in the adjuvant setting, and in combination with radiation or immunotherapy for advanced NSCLC. The mechanism of action for cisplatin is the induction of DNA damage via the formation of inter- or intrastrand crosslinks (ICLs). These crosslinks impede DNA strand separation which occur during the cellular processes of replication fork progression and transcription4. While most NSCLC patients receive platinum-based chemotherapy, intrinsic or acquired resistance remains a primary issue with <50% of patients having an objective partial response7. Identifying and providing alternative treatment options for those patients who will not benefit from platinum-based chemotherapy might improve NSCLC patient health outcomes. Hence, implementing biomarkers or additional combinational therapies is key to improving the effectiveness of platinum-based chemotherapy.Chemotherapy employing platinum-based doublet combinations remains the cornerstone of treatment for non-oncogene driven NSCLC. Cisplatin, 9. While other molecular functions are yet to be determined, CDCA3 is associated with an emerging role in solid malignancies with upregulated expression noted in oral squamous cell carcinoma tissues10, liver cancer11, gastric cancer13, colon cancer15 and breast cancer17. We have previously identified a role for CDCA3 in NSCLC18. CDCA3 is commonly upregulated in NSCLC versus non-malignant tissue where elevated malignant expression of CDCA3 strongly correlates with poor patient outcomes. We also identified that CDCA3 is functionally important in mediating proficient G2/M cell cycle progression and tumour cell proliferation where depletion of this protein induces tumour cell senescence.In the search for novel biomarkers or therapeutic targets, we have studied the protein cell division cycle associated protein-3 (CDCA3), also known as trigger of mitosis entry 1 (TOME-1). This protein primarily modulates cell cycle progression from the G2 phase to mitosis. CDCA3 reportedly forms part of a SKP1-Cullin-RING-F-box (SCF) ubiquitin ligase (E3) protein complex to promote the timely degradation of the inhibitory tyrosine kinase WEE1In the present study, we examined the potential of CDCA3 as a biomarker for tumour cell response to platinum-based chemotherapy. Our data demonstrate that CDCA3 levels, in patients and in vitro, correlate strongly with sensitivity to platinum agents. We also demonstrate that while CDCA3 protein levels are regulated by the anaphase-promoting complex/cyclosome (APC/C) in NSCLC, pharmacological strategies to block this protein–protein interaction enhance sensitivity to platinum agents.19 and other solid malignancies20. To this end, we undertook bioinformatics analyses of TCGA datasets to correlate relative CDCA3 transcript levels with a homologous recombination deficiency (HRD) score in the ADC and SqCC NSCLC histologies. As shown in Fig. CDCA3 expression significantly correlated with a HRD score in ADC and SqCC disease determined firstly by a multigene signature representative of HR deficiencies21 23. While first applied in breast cancers to predict pathologic complete response (pCR) to chemotherapy23, this analysis has been used to assess NSCLC cases24 and shows utility for DNA-damaging therapeutics. In ADC  in a panel of NSCLC cell lines. Both cisplatin and carboplatin induced a dose-dependent reduction in cell viability across all cell lines tested  in a smaller panel of NSCLC cell lines treated with cisplatin. As shown in Fig. CDCA3 transcripts in this small selection of cell lines.To explore mechanisms regulating CDCA3 levels, cisplatin treated NSCLC cell lines were examined for CDCA3 by western blot analysis. These data revealed that except for CRL-5889 cells, CDCA3 levels were upregulated ~1.5–2.5-fold in the majority of cell lines following 12 h of cisplatin treatment Fig. . CDCA3 tWe next asked whether platinum-induced stress could affect CDCA3 protein stability, thus yielding the increased levels of the protein. To do so, we chose the SKMES-1 and H460 cells, as model squamous and non-squamous NSCLC cell lines respectively, and also given that these cells have abundantly detectable CDCA3 protein levels suitable for protein half-life experiments. These cells were treated with cycloheximide to block protein synthesis in the presence or absence of cisplatin over the course of 4 h. Western blot analysis revealed that in the absence of cisplatin, CDCA3 levels were moderately reduced by ~30–50% in both H460 Fig.  and SKME8 and a D-box R-X-X-L motif commencing at Arg198. Each of these motifs are necessary for substrate recognition by the co-activator proteins Cdh1 or Cdc20, which serve to recruit substrates for poly-ubiquitination by the large multi-subunit E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) for degradation by the 26S proteasome. To confirm which of the APC/C co-activators is required to promote CDCA3 degradation in NSCLC, we ectopically expressed escalating levels of either HA-tagged Cdh1 or Cdc20 in H460 cells. For these experiments, H460 cells were used as a model NSCLC cell line suitable for transfections and as a cell line with readily detectable endogenous CDCA3 by western blot analysis. As shown in Fig. 8, is the predominant co-activator required to regulate levels of CDCA3 in NSCLC. Noticeably, CDCA3 lacks a classical ABBA motif, which is reported to be required for Cdc20 binding25, perhaps defining the specificity for CDCA3 recognition by Cdh1 versus Cdc20.To explore the possibility that CDCA3 levels are regulated by protein degradation, we searched the primary protein sequence of CDCA3 for known motifs recognised by ubiquitin ligases. As shown in Fig. 26 and PSIPRED27 protein sequence analyses suggested that CDCA3 is an intrinsically disordered protein which forms transient helical structures, particularly over sequences containing conserved motifs, including the KEN and D-box motifs  simulations to evaluate an interaction between CDCA3 and Cdh1. Firstly, PrDOS29, in the presence and absence of cisplatin induced the upregulation of CDCA3 protein , we next assessed whether the CK2 phosphorylation status of immunoprecipitated CDCA3 varied across cell cycle phases. Phospho-CK2 immunoreactivity was detected in H460 cells arrested at the G1/S boundary by double thymidine treatment but not in mitotic arrested nocodazole treated cells  in a panel of eight cell lines. CX-4945 reduced cell viability in a dose-dependent manner across all cell lines tested , whereby CDCA3low cell lines showed greater sensitivity . Although not statistically significant, carboplatin sensitivity was also inversely proportional to CX-4945 sensitivity  and n + 4 for both serines (Glu256 and Glu231) which are also observed in other substrates48. Moreover, immunoprecipitated CDCA3 is immunoreactive with a CK2 phosphorylation specific antibody that is sensitive to CX-4945 . All methods were also performed in accordance with relevant guidelines and regulations and approved by Queensland University of Technology .All human data was obtained from public resources with non-identifiable patients providing informed consent according to TCGA Ethical Board regulations (51) and the CDCA3-FLAG expression construct was generated by subcloning the full-length CDCA3 sequence in pcDNA3.1+ using the BamHI and EcoRI restriction enzyme sites. N-terminal consensus kozak sequence and C-terminal triple FLAG sequence were introduced using PCR.The CDCA3 antibody (HPA026587), monoclonal FLAG M2 antibody (F1804) and α-Tubulin antibody (T9026) were purchased from Sigma Aldrich. Antibodies against the HA tag (#3724) and phospho-CK2 substrate motif (#8738) were purchased from Cell Signaling Technology . The Cdh1 (ab77885) and γH2AX (ab26350) antibodies were from Abcam. The FANCI antibody (A301-254A) was from Bethyl Laboratories, Inc. 4′-6-diamidino-2-phenylindole (DAPI) was from Life Technologies and Complete EDTA-free protease inhibitor mixture was from Roche Applied Sciences. Donkey anti-rabbit and anti-mouse Alexa Fluor 488 antibodies were purchased from Life Technologies. CX-4945 was purchased from Selleck Chemicals Llc and proTAME was purchased from BostonBiochem. All other reagents were from Sigma-Aldrich except where noted. Mammalian expression construct for HA-Cdh1 was a gift from Marc Kirschner (Addgene plasmid #11596l-glutamine (Life Technologies), supplemented with 10% foetal bovine serum . All cell lines were cultured at 37 °C in a humidified 5% CO2 atmosphere.All NSCLC cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI-1640-medium + FZR1 (Cdh1 protein) and CSNK2A1 (Life Technologies). siCdh1-1 (ID: s27991); siCdh1-2 (ID: s27993); and siCK2 (DI: s3637). For expression construct transfection, FuGene HD transfection reagent was used (Promega Corporation).For transfections of the customised siRNA library, Lipofectamine RNAimax was used (Life technologies). The customised Silencer Select human kinase siRNA library  contained 62 lyophilised siRNAs targeting 38 different human kinases  or CX-4945 (5 µM) for 12 h or proTAME (10 µM) for 6 h. For protein half-life experiments, cells were treated with cycloheximide (70 µM) for 4 h in the presence or absence of cisplatin (5 µM). To block cells in prometaphase, cells were treated with either nocodazole (0.1 µg/mL) for 16 h. To block cells at the G1/S cell cycle boundary, cells were treated with thymidine (2 mM) for 16 h, followed by recovery in fresh culture media for 8 h before a second 16-h thymidine treatment.For whole cell lysate collection, cells were washed with phosphate-buffered saline and lysed in lysis buffer (50 mM HEPES (pH 7.5), 150 mM KCl, 5 mM EDTA, 0.05% IGEPAL CA-630 (v/v), 1x protease inhibitor cocktail (Roche) and 1x phosphatase inhibitor cocktail ). Following sonicated and centrifugation, total protein yield was determined by Bicinchoninic Acid (BCA) Protein assay (Sigma Aldrich). Total protein (20 µg) samples were denatured in 1x Laemmli Buffer supplemented with 8% β-mercaptoethanol for 5 min at 80 °C.For immunoprecipitation, protein samples were prepared with 400 µg protein in 400 µl of lysis buffer. Lysates were incubated overnight with 3 µg of FLAG antibody, HA antibody or CDCA3 antibody overnight at 4 °C. Following incubation, lysates were incubated with protein A or G Dynabeads pre-equilibrated with lysis buffer (Invitrogen). The Dynabeads were denatured using 2x Laemmli sample buffer supplemented with 8% β-mercaptoethanol for 5 min at 80 °C.Samples were separated on Bolt 4–12% Bis-Tris Plus pre-cast gels (Life Technologies) and transferred onto nitrocellulose membrane  using the semi-dry transfer Novex system (Life Technologies). Membranes first blocked using Odyssey blocking buffer (Li-Cor) were incubated with primary antibody overnight at 4 °C in a 1:1 solution of Odyssey blocking buffer and PBS-T. All primary antibodies were used at a dilution of 1:1000 except for antibodies targeting CDCA3 (1:800) and tubulin (1:5000). Following incubation, membranes were washed with PBS-T and incubated with appropriate secondary antibodies and imaged using the Li-Cor Odyssey system (Li-Cor). Images were acquired and subject to densitometry analysis.CDCA3 (forward primer 5′-GGACCCTGAGACTCCCAGAT-3′ and reverse primer 5′-GCCGCTTACCCTGTCGTAG-3′) and 7SL transcript levels (forward primer 5′-ATCGGGTGTCCGCACTAAGTT-3′ and reverse primer 5′-CAGCACGGGAGTTTTGACCT-3′) were determined using SYBR Green (Thermo Fisher Scientific) on a ViiA7 RT-PCR system (Thermo Fisher Scientific). CDCA3 levels were normalised to 7SL levels and analysed using the comparative CT method.Total RNA was isolated using an Isolate II RNA mini kit (Bioline). Equal amounts of RNA (1 µg), quantified using a Nanodrop Lite spectrophotometer (Thermo Fisher Scientific), were reverse transcribed to generate cDNA using the Tetro cDNA Synthesis kit (Bioline). 4 to 1.2 × 104 cells per well on a glass-base 96-well plate (Corning) at and left to adhere for 12 h overnight before drug treatment. For treatments, cells were exposed to IC50 concentrations of cisplatin for 12 h either alone or in the presence of IC50 concentrations of CX-4945. For recovery experiments, drug treated cells were washed with PBS and cultured in fresh media for 8 h. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature (RT) and permeabilised with 0.1% Triton X-100 in PBS for 5 min at ambient temperature. The cells were subsequently blocked with 2% Donkey serum in PBS for 30 min at ambient temperature. Primary antibodies were diluted in 0.5% Donkey serum in PBS and incubated overnight at 4 °C. Antibodies targeting FANCI, γH2AX and CDCA3 were used at dilutions of 1:100, 1:1000 and 1:300 respectively. Fluorescent secondary antibodies conjugated with Alexa Fluor® dye were diluted in 0.5% Donkey serum in PBS and incubated for 1 h at RT (1:600 dilution). Following this, the cells were stained with Hoechst 33342 stain diluted in PBS  and incubated at RT for 5 min before imaging. Images were collected on an InCell Analyzer 6500 high content microscopy imaging system . Images were analysed using the CellProfiler software v3.1.9. FANCI or γH2AX foci were reported as foci per nuclei per field of view, n = 13–16 fields. A minimum of 800 cells were quantified per condition.Cells were seeded at 1.0 × 1050) calculated using the GraphPad Prism 8 software.CellTiter-Glo 2.0 Luminescent assay (Promega Corporation) was used to determine cell viability for dose response assays. Cells at a density of 500 cells/well in white walled 384-well plates were drug treated 24 h following seeding. Following 48 h of treatment, CellTiter-Glo 2.0 was added to each well according to the manufacturer’s instructions. Luminescence was recorded using a PHERAstar FSX detection system (BMG Labtech). Following background value removal, data was normalised to untreated controls. Dose response curves were generated and drug potency values (ICS. cerevisiae APC/C activator protein Cdh1 bound to the three degrons of APC/C-CDH1 modulator 1 (PDB code: 4BH6)52. We report a molecular model of the WD40 propeller domain of human Cdh1 (Ser172-Ser474) and C-terminal domain of CDCA3 (Ser193-Glu267). The N-terminal domains outside of these sequences were not modelled, due to lack of homology with known 3D structures and the intrinsic disorder of the proteins. The KEN and D-box motifs of APC/C-Cdh1 modulator 1 were aligned with the two homologous degron sequences present in the C-terminal of CDCA3. We also looked for regions of negative charge in CDCA3 that could mimic the ABBA motif of APC/C-CDH1 modulator 1 and found a non-canonical ABBA-like motif from Arg220-Leu231. Firstly, the degrons motifs were modelled using position restraints and the remaining residues were then modelled from the template to CDCA3 using Modeller tool in UCSF Chimera. The S. cerevisiae cdh1 molecule is structurally homologous to human cdh1 53 and was therefore replaced using MatchMaker tool in UCSF Chimera54. The KEN, D box and non-canonical ABBA motifs of CDCA3 were modelled to interact with their respective sites on the Cdh1 molecule similar to the wild-type Cdk2-cyclinA2-Cks2-cdc20 complex (PDB code: 6Q6G)55.A molecular model of CDCA3 was built based on the structure of 56. The Na+ ions were added to neutralise the system charge. The AMBER ff99SB*-ildn force field parameters57 and TIP3P model58 were used for the proteins/peptides and water molecules, respectively. MD simulation was performed using PMEMD.CUDA59 from AMBER 16 suite of programmes on NVIDIA GPU, which were compiled with CUDA 7.5. The electrostatic interactions were calculated using the Particle Mesh Ewald method with 12 Å short-range electrostatic and van der Waals cutoffs. All simulations were performed under periodic boundary conditions in a cubic water box. The equations of motion were solved using a time step of 2 fs. The SHAKE algorithm and Langevin dynamics were applied to constrain bonds to non-heavy atoms and to control the temperature. Each system was minimised using steepest descent for 1000 steps and conjugate gradient for another 500 steps. After minimisation, the system was heated from 0 to 300 K in 50 ps simulation by applying 10 kcal/(mol•Å2) harmonic position restraints to the protein and peptide heavy atoms with a constant particle number, volume and temperature (NVT) ensemble. Each system was further equilibrated in the constant particle number, pressure and temperature (NPT) ensemble at 1 atm and 300 K for 650 ps with same restraints as in the NVT run. Another 20 ns equilibration was performed by applying 2 kcal/(mol•Å2) harmonic position restraints on the KEN, D box and the non-cannonical ABBA motifs under NPT condition. Finally, 400 ns production run was performed to study the stability and the conformational changes in the peptide-protein complex. Snapshots from last 300 ns  were combined for plotting secondary structures and obtaining stastistics on hydrogen bonds using the CPPTRAJ module of AmberTools 16. UCSF Chimera was used for visualisation of trajectory and preparing images of the molecular models.Atomistic molecular dynamics (MD) simulation was applied to refine and explore the conformational space of the modelled CDCA3-cdh1 complex. The complex was solvated in explicit water using tleap from the AmberTools 16 package2-transformed level 3 Illumina HiSeq RNASeq V2 mRNA levels (RSEM) were used. Homologous recombination deficiency (HRD) scores were generated from a 230 multi-gene signature (Peng)21 or unweighted sum of three  genomic scars (sum)22 as previously described. The phamacogenomic predictor of sensitivity to chemotherapy (PPSC) was determined according to a previously described multigene signature23. Somatic copy number variation analyses to assess chromosome arm gains or losses or the ploidy content of tumours were determined from SNP6 array copy number data as previously described60. Whole-genome doubling events were calculated using the ABSOLUTE method as previously described61. Within the two NSCLC histologies, the correlation between relative CDCA3 transcript expression and each parameter was assessed by linear regression analysis with P-values, R-values and 95% confidence intervals reported according to Spearman’s rank correlation. Differences in reported sample numbers between figures are due to availability of appropriate copy number or gene expression data. These analyses were performed in the R statistical environment .Correlations between CDCA3 transcript expression and measures of genomic instability or response to chemotherapeutic drugs were assessed from The Cancer Genome Atlas (TCGA) RNAseq datasets. Specifically, logt tests or Pearson correlation coefficients. P-values below 0.05 were considered significant and indicated using the following abbreviations: *P < 0.05, ****P < 0.0001.For in vitro experiments, data and statistical analyses were performed using GraphPad Prism V8 software. Results are shown as mean ± SD unless otherwise stated. Data were analysed using two-tailed Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplemental Data 1Reporting Summary


N-acylethanolamine.The diversification of land plants largely relies on their ability to cope with constant environmental fluctuations, which negatively impact their reproductive fitness and trigger adaptive responses to biotic and abiotic stresses. In this limiting landscape, cumulative research attention has centred on deepening the roles of major phytohormones, mostly auxins, together with brassinosteroids, jasmonates, and abscisic acid, despite the signaling networks orchestrating the crosstalk among them are so far only poorly understood. Accordingly, this review focuses on the Arabidopsis Amidase Signature (AS) superfamily members, with the aim of highlighting the hitherto relatively underappreciated functions of AMIDASE1 (AMI1) and FATTY ACID AMIDE HYDROLASE (FAAH), as comparable coordinators of the growth-defense trade-off, by balancing auxin and ABA homeostasis through the conversion of their likely bioactive substrates, indole-3-acetamide and  Beneath the apparent simplicity of the sessile lifestyle of plants, an intricate hormonebased machinery becomes crucial to face an often hostile environment. Infectious pathogens, herbivorous predators, soil salinity, drought, or temperature fluctuations, are among the diverse biotic and abiotic stresses challenging their survival and optimal reproduction ,2. In thde novo biosynthesis to inactivation through conjugation, sequestration and degradation [Under favorable circumstances, a vast miscellany of plant growth and developmental aspects, such as promotion of cell elongation, expansion, and differentiation, have been so far majorly ascribed to those signaling molecules of the auxin class ,7,8. In radation ,23, the radation ,25.N-acylethanolamines (NAEs) by a fatty acid amide hydrolase (FAAH), based on the strong evidence to bridge their growth-inhibiting shared roles to ABA signaling pathway. Thereby, our review may fuel future synergistic research approaches, integrating the converging roles of their preferred substrates in early plant growth, thus leading future biotechnological innovations to sustainably increase crop yields and meet the worldwide growing demand.Hence, this work provides updated information on the Arabidopsis AS superfamily members, focusing our main attention not only on the Amidase 1 (AMI1)-dependent IAA biosynthesis, but also on the hydrolysis of cisSer-Lys catalytic triad, instead of the typical Ser-His-Asp triad found in the active sites of serine proteases [The ubiquitous AS members are a numerous group of amidohydrolases widely distributed throughout prokaryotes and eukaryotes . AS members are highly divergent both in terms of substrate preferences and function ,27,28. Troteases ,30.This group of enzymes include: the plant AMI1, a specific indole-3-acetamide (IAM) amidohydrolase that synthesizes IAA from IAM ; FAAH, aOnly two proteins, AMI1 and FAAH, out of the seven different coding genes constituting this small enzyme family were characterized for their enzymatic activity ,36,37,38cisSer-Lys triad remains conserved (PS00571 in the PROSITE dictionary) [cisSer-Lys catalytic center described in Rhodococcus rhodochrous strain J1 or Sulfolobus solfataricus [The apparent molecular mass of AMI1 is around 45 kDa, its subcellular localization is in the cytoplasm, and the canonical residue composition of the Ser-tionary) ,44. Howeataricus , Ser137 ataricus  assumes ive site .Agrobacterium tumefaciens IaaH gene product acts against IAM and NAM [Aside from the expected common features to other AS members, AMI1 exhibits some striking differences, being the only member with indole-3-acetamide hydrolase activity, with IAM and phenylacetamide as its preferred substrate . This am and NAM . On the  and NAM , yeast t and NAM . Further and NAM ,35,45,46 and NAM .L-tryptophan (L-Trp) into IAA via the intermediate IPyA [Prunus jamasakura, Citrus unshiu, Cucurbita maxima and A. thaliana [Triticum aestivum and Pisum sativum tissues [Oryza sativa [Poncirus trifoliata [General auxin biosynthesis is mainly derived from the indole-3-pyruvate (IPyA) anabolic pathway, wherein, the fine-tuning of tryptophan aminotransferases (TAA1/TAR2) and flavin containing monooxygenases (YUC1-11), becomes crucial to converting ate IPyA ,53. Eithate IPyA ,55,56,57thaliana ,59,60,61 tissues , Oryza sa sativa ,64 and Pifoliata . Lastly,ifoliata ,37,66,67A. thaliana, up to 95% of IAM has been proven to originate from the precursor IAOx by a hitherto unidentified enzyme [L-Trp-derived production of key glucosinolates [Within the Brassicaceae family and concretely in d enzyme . Moreoved enzyme ,70. Therinolates , such asinolates , thus ininolates ,74. Striinolates ,44, the inolates . In lineinolates ,76.cyp79b2 cyp79b3 plants [YUC8 and ILL5/IAR3, no other significant differentially expressed auxin homeostasis-related genes were identified in ami1-2 [YUC8, which takes part in auxin biosynthesis [UGT75D1 and GH3.17, together with a number of auxin transport- and signaling-related genes, including LAX2, PIN4, PIN5, IAA1, IAA12, IAA14, ARF6, ARF7, and ARF16, most likely in response to IAA overproduction. Further, no other alternative auxin biosynthesis routes, such as IAMH1 and IAMH2, were transcriptionally induced in cyp79b2 cyp79b3 to balance the loss of IAOx source [However, despite the severely reduced IAM contents of 3 plants , both IA3 plants . Herein,n ami1-2 . Both thynthesis , along wynthesis ,78, mighx source ,69,79 orx source , but demx source . Taken tx source , all thex source , nourishx source ,82, are in planta, most probably impacting cellular auxin homeostasis by means of balancing the IAM pool [HAK/KT12 and KUP4 [ami1 mutants displayed a slight growth reduction of aerial parts, together with a significant reduction of root branching, as well as total root length and area [AMI1 expression is majorly located in proliferating tissues, such as young seedlings and developing flowers, but is repressed during early stages of germination ,44,51,83IAM pool ,84. In tIAM pool . These pand KUP4 . Besidesand area . On the and area ,37,86,87A.thaliana were recently reported [ami1 rty parentals, set a reduced number of siliques producing non-viable seeds. The offspring aborted germination just after radicle extrusion [rty ancestors, thus explaining the parentals sterility.Therefore, in the line of assessing the described plant growth repressing role of IAM, it was highly relevant to characterize the impact of endogenous IAM accumulation, aiming at deepening on the molecular and physiological mechanisms for IAM signal perception and integration. As a starting point to tackle this question, the consequences derived from the simultaneous genetic interruption of the indole glucosinolate and IAM pathways in reported . Strikinxtrusion . Consistami1 rty embryos and seeds, additional IAM and IAA mass spectrometric analysis of these homozygous seeds were performed, finding higher IAM:IAA ratios in ami1 rty relative to those of wt [rty allelic sur1-1 mutant [WRKY33, a transcriptional repressor of this camalexin anabolic route [GRF3 and GRF5 on IAM treated wt Arabidopsis seedlings [TCP10 and TCP23 [To further dissect the observed IAM dwarfish effect on se of wt  and the 1 mutant . Alternaic route . So, theeedlings . Furthernd TCP23 , underpind TCP23 ,89.Of spnd TCP23 .ami1-2 mutants belong to different transcription factor classes [YUC8 and ILL5/IAR3 in auxin homeostasis, these ami1-2 misregulated genes have been associated with biotic stress responses [ami1 seedlings in response to osmotic stress conditions, the repression of AMI1 activity has been proposed as a first line adaptation mechanism. Additionally, the remarkable number of identified differentially expressed small heat shock proteins by whole-genome transcript sequencing of ami1 rty seeds, points towards the misregulation of desiccation tolerance processes, involved in drought stress adaptive responses [Altogether, IAM or other putative by-products, might act as a signaling molecule with prominent impact on gene expression regulatory processes. More than 12% of the differentially expressed IAM-responsive genes in  classes . As repr classes . In this classes ,37. For esponses ,91. On aesponses .GNC and CGA1 transcription factors, both DELLAs downstream effectors [NCED3, either by means of IAM exogenous application, or as a result of ABA accumulation in the ami1 alleles [The above-mentioned osmotic stress responses involve ABA-dependent and ABA-independent pathways ,93. In tffectors . Furtherffectors , as wellffectors ,97, haveffectors . In the  alleles . Hence, To date, FAAH is the sole known integral membrane protein within the AS enzymes, which catalyzes the hydrolytic central step in NAEs metabolism, converting these lipid signaling molecules into their corresponding free fatty acid and ethanolamine products, thus terminating their regulatory actions . There aAmborella trichopoda [A. thaliana, Brassica napus, Camelina sativa) and castor (Ricinus communis), as those having FAAHs uniquely belonging to the group I. So, it has been suggested, in the basis of the joint presence of both FAAH groups in the A. trichopoda genome, an ancestral plant FAAH evolutionary bifurcation, predating angiosperms emergence, and later differential taxa-depending loss of orthologs. Consequently, throughout evolution, A. thaliana and its relatives had entirely lost the group II FAAH orthologs, whereas, e.g., group I is preferentially represented in Solanaceous species, while Gossypium or the leguminous plants have mostly group II instead of group I FAAHs [Thus, the elucidation of the FAAH crystal structure , jointlyichopoda , the phy I FAAHs .Glicine max (soybean) group I and group II FAAHs were performed on the basis of the AtFAAH 3D structure template (PDB: 6DHV [cisSer-Lys) of the AS superfamily, this exhaustive inspection uncovered significant contrasts between the novel group II of FAAHs and the AtFAAH. In brief, conversely to the group I polar residues conforming the surroundings of the substrate-binding pocket, those of the group II are predominantly nonpolar and more reminiscent of mammalian FAAH, including some bulkier aromatic residues in the ligand-binding site. In the same manner, this lower group II hydrophilicity has been also predicted for its cytosolic access channel building residues, most probably to fit more hydrophobic head group substrates. Therefore, this structural diversity expands the so far underestimated plant signaling communication system, as evidenced by the putative vast collection of naturally occurring potential substrates for group II FAAHs, beyond NAEs themselves. So, according to emerging literature, these unnoticed signaling molecules may range from plant fatty acid amides, such as the alkamides [L-homoserine lactones (AHLs), which are essential in N-acyl amide-mediated plant-microbe interactions [Herein, homology modeling between DB: 6DHV ). Thus, lkamides , to micrractions .N-acylphosphatidylethanolamine (NAPE)-derived origin of NAEs, and further identified the mammalian phospholipase D (PLD) as the specific enzyme catalyzing the conversion of NAPE to NAE in vivo [Although there are marked structural differences between NAEs, the inherent FAAH promiscuity to hydrolyze these ubiquitous signaling bioactive acylamides, appear to be conserved throughout eukaryotic organisms ,103, alt in vivo . In plannverters , likely nverters  or phospnverters  mediatedA. thaliana FAAH is equally efficient hydrolyzing either the hydroxylated or the unsubstituted NAE 18:2 [However, the orchestration of numerous behavioral and physiological processes in vertebrates, not only depends on the anandamide binding to the membrane G protein-coupled cannabinoid receptors, CB1 and CB2 ,109, butNAE 18:2 , althougNAE 18:2 ,116, is NAE 18:2 ,98.As in the case of animals, mounting experimental evidence has so far underpinned the crucial role of NAE signaling in regulating multiple plant physiological processes . In the AtFAAH overexpressing seedlings compared to wt, with concordant reduction of endogenous NAE levels, and in contrast to loss-of-function AtFAAH lines, which exhibited enhanced sensitivity to NAE [AtFAAH knockouts did not display any other phenotype than the increased sensitivity to exogenous NAE [AtFAAH overexpressors was the jeopardized innate immunity to usually non-pathogenic organisms [AtFAAH overexpressing mutants displayed an associated induction of the flowering master regulator FLOWERING LOCUS (FT), under both inductive long day (LD) and non-inductive short day (SD) conditions [AtFAAH overexpressors under 14 SD conditions growth, comprising a 9% less total NAE than wt. Moreover, wt Arabidopsis plants exogenously treated with NAE 12:0 showed a significant delayed flowering [Specifically, micromolar concentrations of exogenously applied NAE 12:0 and NAE 18:2, not only exert a dose-dependent reduction in seedling growth, but also provoke root cell and cytoskeletal organization alterations ,120,121.y to NAE ,123,124.y to NAE ,119,124,rganisms . On the nditions ,128,129.Arabidopsis genetic research with ABA biosynthesis and ABA-insensitive (ABI) mutants, the negative regulating role of the ABA signaling cascade, inhibiting seed germination and arresting seedling growth, have been robustly established [AtFAAH overexpressors, these lines display an enhanced sensitivity to ABA, likely due to elevated PA levels [Over the past years, primarily based on ablished ,135,136.ablished ,137,138 ablished ,133,139,ablished ,141, as ablished . Within ablished ,145,146,ablished ,148,149 ablished . Indeed,A levels ,123. Nothibitors .abi3-1 seedlings, displayed arrested seedling growth in response to higher NAE 12:0 concentrations, but not to supplied ABA, it has been postulated that alternative ABA-independent mechanisms operate in regulating seedling development [RD29 drought response gene in absence of its major activator, the ABI3 transcription factor, within a narrow developmental window. Remarkably, even without growth inhibition effects, and outside this sensitivity time frame, there was only an ABA-responsive upregulation of RD29B, but not to NAE treatment, thus reinforcing the divergences between both metabolites in the regulation of growth [Furthermore, even more compelling experimental results point towards the crosstalk between NAE and ABA signaling pathways to negatively regulate early seedling development ,122,123.In the basis of the recapitulated literature, it becomes clear that, both NAE and IAM signaling metabolites, can negatively impact plant growth processes, such as germination or early seedling development, either through the regulation of ABA-responsive or -non responsive genes. Thus, their common growth inhibiting roles, derived from elevated contents of either NAE or IAM, drive proper adaptive responses by means of modulating the growth-defense trade-off, as schematized in 4+ . Thereby


To assess the knowledge of S and F in the above-mentioned skills and their continuation in the 2nd level of CTR training, surveys were administered: pre-test, at the beginning, post-test, sometime during the FLTCs, and satisfaction at the end of the FLTCs. RESULTS/ANTICIPATED RESULTS: Fifty eight (58) S/F from UPRMSC, UCC and 7 other institutions participated. Forty two  answered a pre-test and 31/42 (74%) completed the post-test. Results showed that S/F: who correctly defined CTR increased from 7% to 77 %; their ability to identify a CT researcher increased from 10% to 83%. Fifty five percent (55 %) (21/38) S/F that were certified in the FLTCs, answered the satisfaction survey. One hundred percent (100%) indicated that the materials offered contributed in the identification of a CT researcher and a topic in CTR; 100% answered that the FLTCs contributed higher knowledge in and provided new skills in CTR. Moreover, 31/38 (82%) S/F started the 2nd level of training. DISCUSSION/SIGNIFICANCE OF IMPACT: The FLTCs were successful in increasing S/F knowledge of CTR and to further engage in 2nd level of trainings. Title V impact extended island wide, increasing the diversity of represented health professions and science fields among participants. The interventions were deemed to be of high quality.OBJECTIVES/GOALS: To assess the impact of FLTCs on CTR on S and F from health professions and basic science academic programs island wide in Puerto Rico. Cycles supported by the Title V Cooperative Project at University of Puerto Rico-Medical Sciences Campus (UPRMSC) and Universidad Central del Caribe (UCC)(Title V). METHODS/STUDY POPULATION: After offering FLTCs in CTR to S and F from UPRMSC and UCC, Title V expanded it to S and F from other institutions island wide in PR. These FLTCs were offered the 2


Bone development is a tightly regulated process. Several integrated signaling pathways including HH, PTHrP, WNT, NOTCH, TGF-β, BMP, FGF and the transcription factors SOX9, RUNX2 and OSX are essential for proper skeletal development. Misregulation of these signaling pathways can cause a large spectrum of congenital conditions categorized as skeletal dysplasia. Since the signaling pathways involved in skeletal dysplasia interact at multiple levels and have a different role depending on the time of action (early or late in chondrogenesis and osteoblastogenesis), it is still difficult to precisely explain the physiopathological mechanisms of skeletal disorders. However, in recent years, significant progress has been made in elucidating the mechanisms of these signaling pathways and genotype–phenotype correlations have helped to elucidate their role in skeletogenesis. Here, we review the principal signaling pathways involved in bone development and their associated skeletal dysplasia. Skeletal development in mammals occurs through two distinct mechanisms: intramembranous and endochondral ossification. In the intramembranous process, osteoblast cells differentiate directly from mesenchymal cells and form flat bones like cranial vault, parts of the jaw and lateral clavicles. At the end of the bone formation period, osteoblasts die by apoptosis or become embedded in the matrix as osteocytes, which then eventually undergo apoptosis . In the Both ossification processes are strictly regulated by several signaling pathways grouped by their response to the following signaling ligand families: Hedgehog (HH), Parathyroid Hormone-related Protein (PTHrP), Wingless and int-1 (WNT), NOTCH, Transforming Growth Factor-beta (TGF-β), Bone Morphogenic Protein (BMP) and Fibroblast Growth Factor (FGF). These pathways converge on specific transcription factors including SRY-related HMG-box 9 (SOX9), Runt-related transcription factor 2 (RUNX2) and Osterix (OSX). Dysregulation of these signaling pathways cause a large spectrum of skeletal diseases. Skeletal dysplasia (SD), also named osteochondrodysplasia, is a group of rare genetic disorders mainly characterized by cartilage and bone growth anomalies. Today, thanks to next-generation DNA sequencing, 461 different SD have been recognized and classified in 42 groups depending on their clinical, radiographic and/or molecular bases [Hedgehog (HH) signaling is involved in many developmental processes, such as growth, patterning and morphogenesis of many tissues. In mammals, three HH ligands are produced: Sonic (SHH), Indian (IHH), and Desert Hedgehog (DHH). HH ligands interact with the 12-pass transmembrane Patched receptors (PTCH 1 and 2) causing the activation of the smoothened (SMO) protein, a 7- pass transmembrane protein with intrinsic intracellular activity. Its translocation to the base of primary cilium activates Gli transcriptor factors  ,6,7. In In the first case, IHH upregulates the expression of PTHrP, which maintains growth plate chondrocytes in a proliferative state and inhibits their hypertrophic maturation . Indeed,HH is also involved in osteoblast development during ossification. In endochondral ossification, IHH produced by pre-hypertrophic and hypertrophic chondrocytes acts on osteoblast progenitors to induce the specification in Runx2-positive osteoblast precursors in the perichondrium and the primary spongiosa ,20. In aIHH cause brachydactyly type A1 [IHH have also been associated with Syndactyly with craniosynostosis [SHH mutations cause congenital hand deformities and severe craniofacial and neurological syndromes like Werner syndrome, Acheiropodia [As demonstrated by all these studies, HH signaling is critical for bone development and its misregulation results in several human skeletal diseases associated with a wide spectrum of limb and facial skeletal anomalies. In particular, as mentioned before, HH ligands diffuse across the developing limb bud and produce a concentration gradient that acts as a morphogen to specify digit development. HH signaling activating mutations, like those inactivating GLI3 repressor, cause polydactyly in Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome and Preaxial polydactyly type 4 ,24,25,26 type A1 . Mutationostosis , Syndactnostosis  and acronostosis . This lairopodia , differeiropodia , and Lauiropodia .s and Gαq). Through Gαs, PTHrP receptor activates adenylate cyclase (AC) and the production of 3’, 5’-adenosine monophosphate (cAMP), which in turn activates protein kinase A (PKA) [q activates phospholipase Cβ (PLCβ) and the formation of diacylglycerol (DAG) and 1,4,5-inositol triphosphate (IP3). DAG activates in turn proteinase kinase C (PKC), while the production of IP3 leads to an increase of intracellular Ca2+ [PTHrP is a paracrine factor expressed by cartilage and bone. In particular, PTHrP is expressed by periarticular resting cells of the perichondrium and at a lower level by proliferating chondrocytes ,34. PTHr A (PKA) ,36. On tlar Ca2+ ,38.These two signaling pathways have opposite actions on chondrocyte differentiation B. PTHrP PTHR1 mutations in Blomstrand dysplasia cause prenatal lethality, shortened limbs, advanced bone mineralization and increased bone density due to reduction of proliferating and resting chondrocytes within the growth plate [PTHR1 has been identified in Eiken dysplasia, a disorder with opposite phenotype compared to Blomstrand dysplasia. Patients with Eiken dysplasia show severely delayed skeletal maturation, as well as mild growth retardation [PRKAR1A, which is the type 1 regulatory subunit of PKA or mutations in phosphodiesterase 4 (PDE4D), both causing an impaired PKA activation [GNAS gene, coding for the binding protein Gαs, cause Pseudohypoparathyroidism type IA, also known as Albright hereditary osteodystrophy (AHO) [s-mediated hypertrophy inhibition could result in promotion of the Gαq-mediated chondrocytes’ hypertrophy, leading to premature growth plate closure and favoring short stature.The critical role of PTHrP receptor in bone development is highlighted by the discovery of two severe chondrodysplasias: Blomstrand lethal chondrodysplasia and metaphyseal chondrodysplasia Jansen type. Inactivating recessive th plate ,48. Metath plate . Interesardation . These fardation . A more tivation ,53. Furthy (AHO) ,55. Thishy (AHO) . It has GNAS gene compared to AHO [HDAC4 gene, cause brachydactyly–mental retardation syndrome characterized by brachydactyly type E, craniofacial abnormalities and intellectual disabilities [On the other hand, McCune-Albright syndrome or isolated fibrous dysplasia (FD) of bone is caused by gain-of-function mutations in distinct sites of the d to AHO ,58. Thesd to AHO . These ibilities .2+. All these signaling pathways are activated by the binding of extracellular WNT ligands to one of the seven-pass transmembrane Frizzled (FZD) receptors. Depending on the pathway, the activation occurs with or without specific co-receptors like lipoprotein-related protein (LRP) or receptor tyrosine kinase-like orphan receptors (ROR) [2+ pathway, WNT binds to FZD receptors and ROR causing the intracellular Ca2+ release and the activation of some calcium-sensitive enzymes. More downstream, this event activates the nuclear factor of activated T cells (NF-AT), which translocates to the nucleus and induces specific gene expressions. WNT signaling pathways play a central role in bone development regulating chondrocyte and osteoblast differentiation  and the WNT/Cars (ROR) ,62. In tntiation A.SOX9 promoter [WNTs secreted by the ectoderm act via a β-catenin-dependent pathway. It blocks the cartilage formation in limb bud mesenchymal cells, by inhibiting SOX9 expression through a repressive chromatin mark (H3K27me3) and DNA methylation over the promoter . The inapromoter ,65. WNT promoter . Some mepromoter ,67. Somepromoter ,69,70,71WNT1, involved in the canonical pathway, are responsible for osteogenesis imperfecta (OI) type XV and for early onset osteoporosis [WNT3, playing an essential role in axis formation and limb growth, causes tetra-melia syndrome type 1, consisting on the absence of all four limbs [WNT5A gene, involved in the WNT/PCP non canonical pathway, is linked to the autosomal dominant form of Robinow syndrome (RS) type 1 [WNT6 have been identified in acro-pectoro-vertebral dysplasia (F-syndrome) and mutations in WNT7A gene, involved in limb development and contributing to anteroposterior patterning, have been identified Al-Awadi-Raas-Rothscild syndrome and Fuhrmann syndrome. Finally, mutations in the WNT10B gene, a key regulator of osteogenesis, playing an important role in the development of hands and feet, cause split-hand/foot malformation type 6 [AMER1 (or WTX), an intracellular inhibitor of WNT are responsible for X-linked osteopathia striata with cranial sclerosis [WNT ligands consist of 19 different cysteine-reach secreted glycoproteins. Several of them have been associated with SD. For example, loss-of-function mutations in oporosis . Loss-ofur limbs . The WNT) type 1 . Mutation type 6 ,76,77,78n type 6 ,86,87,88clerosis . FinallyThe discovery and the understanding of all these diseases showed how WNT signaling is essential to regulate bone density in humans, since its hyperactivation causes skeletal diseases characterized by high-bone mass phenotype, while its inhibition causes decreased bone formation. Furthermore, it explains why WNT is used as therapeutic target to improve bone mass in patients affected by skeletal diseases such as osteoporosis or osteogenesis imperfecta.SOX9 enhancer and suppress the SOX9 gene expression, which is essential for chondrocyte differentiation [Similar to WNT/β-catenin signaling, Notch signaling also suppresses chondrogenesis through four NOTCH receptors (NOTCH1-4) and at least five ligands  . In the ntiation ,97.Notch signaling is also critical for axial skeleton segmentation during somitogenesis ,99. DefeNOTCH2, the major receptor involved in the chondrocyte and osteoblast differentiation, are responsible for Hadju–Cheney syndrome, a disorder characterized by focal bone lysis of distal phalanges, progressive bone loss and fractures [NOTCH1, RBPJ and DLL4 are responsible for Adams Oliver syndrome, a congenital condition characterized by aplasia cutis congenita of the scalp and transverse limb defects [In parallel, Notch signaling acts upstream of RUNX and regulates the osteochondro-progenitor cell proliferation and lineage specification . Dependiractures ,112. Los defects ,114.Since the Notch pathway can have different roles depending on the cell differentiation stage, it could be very difficult to target this pathway for therapies.The TGF-β superfamily is a group of more than 40 members including 3 TGF-β proteins , and 14 BMPs, Activins and Growth and Differentiation Factors (GDFs) . It is iThis has been demonstrated by studying several conditional knockout mouse models for TGF-β, its receptors and SMAD proteins. All these models are characterized by an impairment of endochondral and intramembranous bone formation .During chondrogenesis, BMP and TGF-β signaling are required for the expression and maintenance of SOX9 and, in consequence, for the formation of chondrogenic mesenchymal condensations. TGF-β, BMPs and their receptors are also expressed in proliferating chondrocytes and hypertrophic chondrocytes and regulate chondrocyte proliferation and differentiation. In particular, TGF-β promotes the condensation and differentiation of mesenchymal cells, induces chondrocyte proliferation, but inhibits terminal chondrocyte differentiation ,120. On In osteoblastogenesis, TGF-β promotes expansion and proliferation of osteoprogenitors, early differentiation and commitment to the osteoblastic lineage through the MAPKs and SMAD 2/3 pathways. Conversely, it prevents the terminal osteoblast differentiation . ActivatFocusing on BMP signaling, genetic studies have shown that several BMPs, such as BMP-2, 4, 5, 6 and 7 have strong osteogenic capacity . In partGDF5 [SMAD4, the only common SMAD for both TGF-β and BMP signaling, results in Myhre syndrome, characterized by short stature, facial dysmorphism, stiff joints and brachydactyly [In humans, activating mutations in one of the Type I receptors, the Activin receptor IA/Activin-Like kinase 2 (ACVR1/ALK2), cause Fibrodysplasia ossificans progressive (FOP), a rare disease characterized by heterotopic endochondral ossification, through an increased BMP Smad-dependent and non-canonical p38 MAPK signaling activity . MutatioGDF5 . PatientGDF5 . Gain-ofydactyly .BMPER cause diaphanospondylodysostosis characterized by craniofacial anomalies and vertebral segmentation [LEMD3 gene are associated with osteopoikilosis with and without melorheostosis, characterized by multiple round foci of increased bone density and hyperostosis respectively [The TGF-β/BMP signaling is also negatively regulated by several mechanisms: intracellular antagonists, like SMAD 6 and 7; the inner nuclear membrane protein LEMD3 and the SKI protein influencing the SMAD signaling; extracellular ligand antagonists, like NOGGIN and BMPER; intracellular ubiquitin ligases and transcriptional repressors. Over-expression of NOGGIN causes osteopenia, fractures and multiple synostoses syndrome, consisting of facial dysmorphism, craniosynostosis and multiple joint fusion . Heterozentation . Moreoveectively . Finallyectively .As mentioned in this paragraph, numerous human SD are related to TGF-β/BMP signaling pathways confirming their essential role in bone development. In particular, it seems that increased TGF-β signaling can result in either increased bone formation or craniosynostosis. Indeed, the clinical consequences depend on tissue-specificity expression of the mutant allele and on the interaction with other signaling pathways. On the other hand, impaired BMP signaling mostly causes distal and axial skeletal defects, demonstrating a prominent role of BMP in chondrocyte differentiation and establishment of joint boundaries.Fibroblast growth factors (FGFs) are a large family of 18 or more proteins which mostly bind to four cell surface tyrosine kinase FGF receptors (FGFR 1-4). This binding induces the dimerization of the receptor tyrosine kinase domains and their sequential trans-phosphorylation . ConsequFGFs and their receptors are expressed during all stages of the bone development in a time- and space-dependent manner. For example, FGFR1 is expressed during mesenchymal condensation, in perichondrium and periosteum and in hypertrophic chondrocytes of the growth plate . FGFR2 iFgfr3 showed an increase in osteoblast number but a decrease in osteoid mineralization [FGF signaling is fundamental also in intramembranous ossification and in the regulation of all steps of osteoblastogenesis. In particular, FGFR1 is expressed in calvaria mesenchyme and later in osteoblasts. During early development, it promotes osteoblast differentiation without affecting RUNX2 expression, while, in mature osteoblasts, it inhibits their mineralization activity . In contlization .FGFRs cause human skeletal dysplasia with a variable degree of severity. The most common genetic skeletal dysplasia in humans is achondroplasia, a rhizomelic short limb dwarfism characterized by short stature, limited elbow extension and frontal bossing. It is caused by gain-of-function mutations in the FGFR3 gene leading to an overactivation of the FGF signaling [FGFR3 cause skeletal overgrowth, campodactyly, tall stature and hearing loss (CATSHL) syndrome [FGFR2 [FGFR3 mutations in craniosynostosis disorders like Crouzon syndrome with acanthosis nigricans and Muenke syndrome [FGFR1 and FGFR2. Crouzon, Apert, Beare-Stevenson with cutis gyrata, Bent-bone dysplasia, Saethre-Choetzen, Jackson-Weiss and Pfeiffer syndromes are all caused by activating mutation in FGFR2 gene and the consequent accelerated osteoblast maturation in the sutures [FGFR1, like the Hartsfield syndrome and the osteoglophonic dysplasia [Mutations in ignaling ,150,151.ignaling ,153,154.ignaling ,156,157,ignaling ,159. Allsyndrome ,161 and e [FGFR2 . The rolsyndrome ,164. Cra sutures ,166,167.ysplasia ,170,171.ysplasia ,173,174.ysplasia .NPR2, the gene encoding NPR-B, are responsible for short and tall stature phenotype, such as in acromesomelic dysplasia type Maroteaux and tall stature with long halluces type NPR2, respectively [During bone development, the FGF signaling can be modulated by C-type natriuretic peptide (CNP) action. When CNP binds to its NPR-B receptor, the MAPK signaling activated by FGFR3 is inhibited C. Similaectively ,179. TheSOX9 gene have been associated with campomelic dysplasia, an autosomal dominant skeletal dysplasia characterized by hypoplastic scapulae, progressive disc degeneration, small chest, short long bones with bowing of the lower extremities, Pierre Robin sequence and sex reversal in males [SOX9 is a transcription factor of the SRY family that regulates sex determination and developmental events like cartilage development. SOX9 is the earliest nuclear factor required for chondrogenesis. It is initially expressed in mesenchymal condensations of the early skeletal precursors. It is dispensable for the initial mesenchymal condensation, but crucial for the subsequent chondrocyte differentiation. In fact, in its absence, chondrogenesis is blocked. In the growth plate, SOX9 is expressed at highest levels in proliferating and prehypertrophic chondrocytes and less expressed in chondrocytes undergoing hypertrophy ,180. SOXin males ,189,190.RUNX2 causes cleidocranial dysplasia, characterized by short stature, delayed closure of fontanelles, prominent forehead, drooping shoulders and abnormal dental development. The distinctive radiological features are shortened or absent clavicles, delayed ossification of the skull bones, and delayed ossification of pelvic bones [RUNX2 gene are associated with metaphyseal dysplasia with maxillary hypoplasia, characterized by metaphyseal flaring of long bones, enlargement of the medial halves of the clavicles, maxillary hypoplasia, and dystrophic teeth [RUNX2 is a transcription factor belonging to the RUNX family composed by RUNX1, RUNX2 and RUNX3 . RUNX2 iic bones ,204. On ic teeth ,206.OSX gene has been identified in a patient with a moderate form of osteogenesis imperfecta, presenting with bone fractures, mild bone deformities and delayed tooth eruption. This confirms the importance of a proper OSX expression for bone health [OSX, a transcription factor containing three C2H2-type zinc fingers, controls the genetic program of osteoblast differentiation and bone formation. Expression of OSX in osteoblast precursors induces the differentiation of these cells into mature and functional osteoblasts and, then, into osteocytes ,208. OSXe health .In this review, we discussed the main signaling pathways involved in bone development and how mutations in their components have been associated with SD. It is important to highlight that even if the signaling pathways have been discussed independently, there is a complex cross-talk among them at multiple levels. This, in association with the evidence that the mutation consequences depend on the specificity of the mutations and on their temporal and spatial mode of action, makes more difficult the understanding of the physiopathological mechanisms of these diseases. Moreover, these signaling pathways can be secondarily affected by alterations in other cellular processes, such as extracellular matrix regulation or metabolic processing. Indeed, several skeletal dysplasia, that we decided to omit in this review, have been associated with mutations in these processes. Fortunately, in the last decade, the development of new technologies, like whole exome and genome sequencing has accelerated the identification of skeletal dysplasia-causing mutations. On the other hand, the development of CRISPR-Cas9 technology and of several mouse models is helping the deciphering of the physiopathological mechanisms. Advanced genetic testing is also helping the diagnosis of skeletal dysplasia. The diagnosis and management of these pathologies have long been based on clinical feature and skeletal imaging. Today, these key techniques are increasingly combined with the genetic testing in order to obtain a more accurate and early diagnosis of SD. It also aids in prognosis and in counselling families regarding genetic recurrence risk and preconceptional reproductive planning ,213,214.