Biology subjects

Graber, J. H.

Publications and source records attributed to Graber, J. H..

13 recordsLinked to original sources

bioRxiv2026

Mesenchymal-derived neural progenitors underlie local insulin production and neuronal transdifferentiation during retina regeneration

In humans, retinal-neuron death, optic-nerve injuries, and associated neurodegenerative diseases, such as glaucoma and age-related macular degeneration, often lead to permanent vision loss. While the capacity for regeneration is low in the human nervous system, including the retina, some non-mammalian vertebrate species, including zebrafish, are capable of endogenous neuronal regeneration after injury. Unlike mammals, zebrafish do not form a scar that inhibits axonal and neuronal regeneration after injury. Rather, they harbor neural progenitor and stem-cell populations allowing regeneration of entire parts of the nervous system and restoration of tissue integrity and function. In the zebrafish retina, cycling neural progenitor cells of the ciliary marginal zone and quiescent resident neural stem cells (the latter of which are also called Muller glial cells) participate in neuronal regeneration following different types of injury. In this study, we report the identification of a novel, additional cellular source participating in neuronal regeneration of neurons in the zebrafish retina after genetic ablation of retinal ganglion cells. Before injury, these progenitor cells express molecular markers of neural-crest-cell and/or fibroblast identity, such as sox10, pdgfrb, and eya2, while after neuronal ablation they also express proneural factors including the ascl1a and olig2 genes. Combining genetic ablation of neurons with photoconversion or Cre/Lox-dependent genetic lineage tracing of sox10-expressing cells, we demonstrated that these cells can differentiate into post-mitotic retinal neurons in the ganglion cell layer (GCL) in the absence of cell proliferation. We also showed, surprisingly, that this progenitor population locally produces insulin mRNA, and that insulin signaling is involved in the accumulation of mesenchymal-derived neural progenitors in the GCL and in their subsequent transdifferentiation into RGCs. This work reveals an unexpected and novel cellular mechanism of transdifferentiation, dependent on a neural-crest-derived mesenchymal cell population, participating in neuronal regeneration in the zebrafish retina. The discovery of this plastic cell population could potentially lead to new strategies to promote the formation of new neurons in the mammalian retina.

developmental biology↗

bioRxiv2026

Two axolotl-adapted cell-ablation platforms reveal macrophage-dependent processes essential for spinal-cord and skeletal regeneration

The axolotl (Ambystoma mexicanum) has emerged as the premier model organism for studying scarless repair and adult tissue regeneration, supported by an expanding collection of tissue-specific transgenic lines and translucent skin that enables high-quality live imaging and cell tracking. However, functional characterization of specific cell types during regeneration has been limited by the absence of validated cell-specific ablation systems. Here, we developed and rigorously compared two independent inducible genetic cell-ablation platforms -- bacterial nitroreductase (NTR 2.0) and mammalian inducible caspase-9 (iCasp9), across developmental stages, animal sizes, and administration routes using various transgenic lines and grafting approaches. The NTR 2.0 platform showed limited applicability due to drug toxicity and solubility constraints, restricting its use primarily to larval stages via immersion. In contrast, the iCasp9 system demonstrated superior efficacy across all life stages, including large adults, with multiple viable administration routes. We further validated these platforms by systematically ablating CD68+ macrophages and examined functional consequences during tail regeneration. Sustained depletion revealed essential macrophage-dependent processes despite continuous macrophage repopulation from hematopoietic reservoirs: skeletal-element regeneration was completely abolished, spinal-cord axons degenerated without recovery, and neural crest-derived cells exhibited severe disorganization. These findings establish macrophages as critical orchestrators of central and peripheral nervous-system regeneration and skeletogenesis in axolotls, while providing validated tools for cell-type-specific functional studies across the axolotl lifespan.

developmental biology↗

bioRxiv2025

Integration of a neuronal RNAseq dataset with the draft Gryllus bimaculatus transcriptome refines gene predictions and highlights potential systematic response to injury

The cricket Gryllus bimaculatus presents a compelling model for investigating neuroplasticity due to its unusual capability of adult structural reorganization. The molecular pathways underlying these changes are entirely unknown. Here, we reanalyzed RNAseq data, drawn from deafferented neuronal tissue one, three, and seven days post-injury, that was previously used to assemble a de novo transcriptome. In this current analysis, we aligned our original RNAseq data to the publicly available G. bimaculatus draft genome, and used the resulting alignments to refine and update the existing annotations. We identified over 10,000 missing genes and reported a measurable improvement in BUSCO scores. These updated annotations were then used as the basis for a DESeq2 differential expression analysis and subsequent functional enrichment analysis to further explore the potential molecular basis of this compensatory anatomical plasticity. Days one and three showed the largest post-deafferentation expression differences. Overall, more transcripts were upregulated rather than downregulated. Protein-protein interactions enriched for G-protein-related signaling, hormone metabolism, and membrane dynamics were evident. Changes in expression of factors related to small GTPases and nervous system development were particularly intriguing. We also identified a surprising enrichment of GO terms related to muscle contraction in this neuronal-specific transcriptome. Identifying these and other differentially regulated transcripts can be used to design hypotheses around well-conserved molecular mechanisms that may be involved in this unique example of adult structural plasticity in the cricket.

neuroscience↗

bioRxiv2025

SGLT2 Inhibition Ameliorates Age-Dependent Renovascular Rarefaction

IntroductionAging is associated with progressive loss of renal function and vascular structure, with and without chronic kidney disease. However, the mechanisms driving renal vascular aging and potential therapeutic interventions remain poorly understood. MethodsTo model this state-of-affairs, we used African turquoise killifish (Nothobranchius furzeri), a naturally short-lived vertebrate. We then inhibited the sodium-glucose co-transporter 2 using dapagliflozin (SGLT2i) to test a potential therapeutic intervention. Histological, immunofluorescent, and 3D vascular imaging were used to evaluate glomerular, tubular, vascular and functional changes. Single-nuclei transcriptomic profiling was performed on whole kidneys to identify age- and treatment-associated molecular signatures. ResultsAged killifish kidneys exhibited hallmark features of renal aging, including glomerulosclerosis, tubular fibrosis, and vascular rarefaction. Functional changes included increased proteinuria and altered tubular transporter function. Transcriptomic profiling revealed a metabolic shift from oxidative phosphorylation to glycolysis and upregulation of pro-inflammatory pathways. Aged vasculature also displayed a marked reduction in tight junctions and cell-cell contacts. SGLT2i attenuated age-related vascular rarefaction, preserved functional capillary networks, reduced albuminuria, restored a youthful transcriptional profile and enhanced intercellular signaling. However, killifish lifespan was not extended. ConclusionThis study establishes the killifish as a translational model for investigating renal vascular aging. We show that SGLT2i preserves renal microvascular structure and function, reduces proteinuria, and reprograms the aged transcriptome. These results support a vascular-protective role of SGLT2i in mitigating age-related renal deterioration. Translational StatementThis study establishes the African turquoise killifish as a model for investigating renal and vascular aging. We found that SGLT2 inhibition preserves microvascular integrity and reduces proteinuria. These results mirror established benefits observed in mammalian models and patients with chronic kidney disease, reinforcing the kidney-protective role of SGLT2 inhibitors. However, the killifish offers a unique opportunity for rapid, translational aging research. By using a naturally short-lived vertebrate with mammalian-like renal aging, our model enables a rapid, preclinical, assessment of vascular outcomes and identifies microvascular preservation as a potential mechanistic target for renoprotection.

systems biology↗

bioRxiv2025

The essential role of connective-tissue cells during axolotl limb regeneration

Axolotls (Ambystoma mexicanum) are known for their remarkable limb-regeneration abilities, which involve the formation of the blastema, a specialized structure consisting of progenitor cells contributed by all major tissues of the limb. Lateral plate mesoderm (LPM)-derived connective tissue (CT) cells dedifferentiate and play a critical role in blastema formation and subsequent limb regeneration. However, the complexity of the blastemas cellular composition and the extent of CT participation and necessity have not been rigorously explored. To address this gap, we conducted spatial transcriptomics using a select array of probes, revealing that CT cells constitute up to 75% of the blastema cells at their peak. Genetic ablation of CT cells significantly delays or truncates limb regeneration, underscoring their necessity during this process. Finally, we analyzed the molecular profile of CT cells throughout the stages of blastema formation and made it accessible through an interactive web platform. Our work reaffirms the central role of CT cells in axolotl limb regeneration and lays the foundation for identifying molecular mechanisms that govern blastema formation during the initial phases of limb regeneration.

developmental biology↗

bioRxiv2025

Zebrafish genetic model of neuromuscular degeneration associated with Atrogin-1 expression

The degenerative loss of muscle associated with aging leading to muscular atrophy is called sarcopenia. Currently, practicing regular physical exercise is the only efficient way to delay sarcopenia onset. Identification of therapeutic targets to alleviate the symptoms of aging requires in vivo model organisms of accelerated muscle degeneration and atrophy. The zebrafish undergoes aging, with hallmarks including mitochondrial dysfunction, telomere shortening, and accumulation of senescent cells. However, zebrafish age slowly, and no specific zebrafish models of accelerated muscle atrophy associated with molecular events of aging are currently available. We have developed a new genetic tool to efficiently accelerate muscle-fiber degeneration and muscle-tissue atrophy in zebrafish larvae and adults. We used a gain-of-function strategy with a molecule that has been shown to be necessary and sufficient to induce muscle atrophy and a sarcopenia phenotype in mammals: Atrogin-1 (also named Fbxo32). We report the generation, validation, and characterization of a zebrafish genetic model of accelerated neuromuscular atrophy, the atrofish. We demonstrated that Atrogin-1 expression specifically in skeletal muscle tissue induces a muscle atrophic phenotype associated with locomotion dysfunction in both larvae and adult fish. We identified degradation of the myosin light chain as an event occurring prior to muscle-fiber degeneration. Biological processes associated with muscle aging such as proteolysis, inflammation, stress response, extracellular matrix (ECM) remodeling, and apoptosis are upregulated in the atrofish. Surprisingly, we observed a strong correlation between muscle-fiber degeneration and reduced numbers of neuromuscular junctions in the peripheral nervous system, as well as neuronal cell bodies in the spinal cord, suggesting that muscle atrophy could underly a neurodegenerative phenotype in the central nervous system. Finally, while atrofish larvae can recover locomotive functions, adult atrofish have impaired regenerative capacities, as is observed in mammals during muscle aging. In the future, the atrofish could serve as a platform for testing molecules aimed at treating or alleviating the symptoms of muscle aging, thereby opening new therapeutic avenues in the fight against sarcopenia.

developmental biology↗

bioRxiv2025

The small molecule ML233 is a direct inhibitor of tyrosinase function

Melanogenesis is the biological process regulating the synthesis of melanin pigments in melanocytes. Defective melanogenesis is associated with numerous human skin diseases, including, but not limited to, albinism, vitiligo, melasma, and hypo- and hyperpigmentation disorders. Tyrosinase is the rate-limiting enzyme controlling melanogenesis, and hence tremendous efforts have been made to identify potent and safe inhibitors of tyrosinase function. However, despite decades of research, currently there is no effective treatment that inhibits melanogenesis or tyrosinase activity with no adverse side effects. In this study, we report characterization of the ML233 chemical as a potent inhibitor of tyrosinase activity in vivo and in vitro. We demonstrate that ML233 reduces melanin production in the zebrafish model with no observable significant toxic side effects, and in murine melanoma cells. We also predict that these effects are mediated through direct tyrosinase-ML233 interaction, i.e., binding of the ML233 molecule to the active site of the protein to inhibit its function. Together, our results reveal that ML233 plays roles in both healthy and pathological skin cells via inhibition of melanin production. ML233-mediated tyrosinase inhibition is a potentially safe and effective approach to alleviate the symptoms of melanocyte-associated diseases and thereby substantially improve human health.

pharmacology and toxicology↗

bioRxiv2022

Macrophage differentiation is marked by increased abundance of the mRNA 3' end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3 UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.

molecular biology↗

bioRxiv2022

A Genomically and Clinically Annotated Patient Derived Xenograft (PDX) Resource for Preclinical Research in Non-Small Cell Lung Cancer

Patient-derived xenograft models (PDXs) are an effective preclinical in vivo platform for testing the efficacy of novel drug and drug combinations for cancer therapeutics. Here we describe a repository of 79 genomically and clinically annotated lung cancer PDXs available from The Jackson Laboratory that have been extensively characterized for histopathological features, mutational profiles, gene expression, and copy number aberrations. Most of the PDXs are models of non-small cell lung cancer (NSCLC), including 37 lung adenocarcinoma (LUAD) and 33 lung squamous cell carcinoma (LUSC) models. Other lung cancer models in the repository include four small cell carcinomas, two large cell neuroendocrine carcinomas, two adenosquamous carcinomas, and one pleomorphic carcinoma. Models with both de novo and acquired resistance to targeted therapies with tyrosine kinase inhibitors are available in the collection. The genomic profiles of the LUAD and LUSC PDX models are consistent with those observed in patient tumors of the same tumor type from The Cancer Genome Atlas (TCGA) and to previously characterized gene expression-based molecular subtypes. Clinically relevant mutations identified in the original patient tumors were confirmed in engrafted tumors. Treatment studies performed for a subset of the models recapitulated the responses expected based on the observed genomic profiles. SignificanceThe collection of lung cancer Patient Derived Xenograft (PDX) models maintained at The Jackson Laboratory retain both the histologic features and treatment-relevant genomic alterations observed in the originating patient tumors and show expected responses to treatment with standard-of-care agents. The models serve as a valuable preclinical platform for translational cancer research. Information and data for the models are freely available from the Mouse Models of Human Cancer database (MMHCdb, http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).

cancer biology↗

bioRxiv2020

Differential Effects of RASA3 Mutations on Hematopoiesis are Profoundly Influenced by Genetic Background and Molecular Variant

Studies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5-13.5 due to severe hemorrhage and decreased fetal liver erythropoiesis. Conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-Cre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in mouse models scat (G125V) and hlb381 (H794L) show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. Global transcriptomic studies in scat suggest potential targets to ameliorate disease progression. Author SummaryHematopoiesis is the process by which blood cells are formed. The individual must have a normal complement of red blood cells to prevent anemia, platelets to control bleeding, and white blood cells to maintain immune functions. All blood cells are derived from hematopoietic stem cells that differentiate into progenitor cells that then develop into mature circulating cells. We studied several mouse strains carrying different mutations in RASA3. We show that RASA3 is required at the earliest stages of blood formation, the stem and progenitor cells, and that the complement of genes other than RASA3, or the genetic background of the mutant strain, profoundly alters the overall effect on blood formation. Further, the molecular nature of the mutation in RASA3 also has a profound and independent effect on overall blood formation. One strain, designated scat, suffers cyclic anemia characterized by severe anemic crisis episodes interspersed with remissions where the anemia significantly improves. Comparison of scat crisis and remission hematopoietic stem and progenitor cells reveals striking differences in gene expression. Analyses of these expression differences provide clues to processes that potentially drive improvement of anemia in scat and provide new avenues to pursue in future studies to identify novel therapeutics for anemia.

genetics↗

bioRxiv2019

Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity

The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.

developmental biology↗

bioRxiv2018

Reference trait analysis reveals correlations between gene expression and quantitative traits in disjoint samples

Systems genetic analysis of complex traits involves the integrated analysis of genetic, genomic, and disease related measures. However, these data are often collected separately across multiple study populations, rendering direct correlation of molecular features to complex traits impossible. Recent transcriptome-wide association studies (TWAS) have harnessed gene expression quantitative trait loci (eQTL) to associate unmeasured gene expression with a complex trait in genotyped individuals, but this approach relies primarily on strong eQTLs. We propose a simple and powerful alternative strategy for correlating independently obtained sets of complex traits and molecular features. In contrast to TWAS, our approach gains precision by correlating complex traits through a common set of continuous phenotypes instead of genetic predictors, and can identify transcript-trait correlations for which the regulation is not genetic. In our approach, a set of multiple quantitative \"reference\" traits is measured across all individuals, while measures of the complex trait of interest and transcriptional profiles are obtained in disjoint sub-samples. A conventional multivariate statistical method, canonical correlation analysis, is used to relate the reference traits and traits of interest in order to identify gene expression correlates. We evaluate power and sample size requirements of this methodology, as well as performance relative to other methods, via extensive simulation and analysis of a behavioral genetics experiment in 258 Diversity Outbred mice involving two independent sets of anxiety-related behaviors and hippocampal gene expression. After splitting the dataset and hiding one set of anxiety-related traits in half the samples, we identified transcripts correlated with the hidden traits using the other set of anxiety-related traits and exploiting the highest canonical correlation (R = 0.69) between the trait datasets. We demonstrate that this approach outperforms TWAS in identifying associated transcripts. Together, these results demonstrate the validity, reliability, and power of the reference trait method for identifying relations between complex traits and their molecular substrates.\n\nAUTHOR SUMMARYSystems genetics exploits natural genetic variation and high-throughput measurements of molecular intermediates to dissect genetic contributions to complex traits. An important goal of this strategy is to correlate molecular features, such as transcript or protein abundance, with complex traits. For practical, technical, or financial reasons, it may be impossible to measure complex traits and molecular intermediates on the same individuals. Instead, in some cases these two sets of traits may be measured on independent cohorts. We outline a method, reference trait analysis, for identifying molecular correlates of complex traits in this scenario. We show that our method powerfully identifies complex trait correlates across a wide range of parameters that are biologically plausible and experimentally practical. Furthermore, we show that reference trait analysis can identify transcripts correlated to a complex trait more accurately than approaches such as TWAS that use genetic variation to predict gene expression. Reference trait analysis will contribute to furthering our understanding of variation in complex traits by identifying molecular correlates of complex traits that are measured in different individuals.

genomics↗

bioRxiv2018

Bioinformatics workflows for genomic analysis of tumors from Patient Derived Xenografts (PDX): challenges and guidelines

Bioinformatics workflows for analyzing genomic data obtained from xenografted tumor (e.g., human tumors engrafted in a mouse host) must address several challenges, including separating mouse and human sequence reads and accurate identification of somatic mutations and copy number aberrations when paired normal DNA from the patient is not available. We report here data analysis workflows that address these challenges and result in reliable identification of somatic mutations, copy number alterations, and transcriptomic profiles of tumors from patient derived xenograft models. We validated our analytical approaches using simulated data and by assessing concordance of the genomic properties of xenograft tumors with data from primary human tumors in The Cancer Genome Atlas (TCGA). The commands and parameters for the workflows are available at https://github.com/TheJacksonLaboratory/PDX-Analysis-Workflows.

bioinformatics↗