Wild-type Siblings Research Articles

Identification of the Role of SEL1L in Platelet Function with Implications for Atypical Equine Thrombasthenia in Thoroughbred Horses

Atypical Equine Thrombasthenia (AET) is a recessive heritable platelet disorder in horses due to aberrant platelet signaling after thrombin stimulation, preventing platelets from fully activating or efficiently binding to fibrinogen, leading to prolonged bleeding. To date, AET has only been identified in the Thoroughbred horse breed. A prevalence study performed at one breeding farm found that one in every 150 Thoroughbreds was affected. Despite the negative effects on horse health and racing performance, the underlying etiology of this unique platelet disorder is unknown. Here we show that a variant in SEL1L, a gene which encodes a protein known to play a pivotal role in endoplasmic reticulum-associated protein degradation (ERAD) and not previously known to be involved in platelet function, leads to AET. In order to identify the underlying genetic mechanism, we performed whole genome sequencing on five affected, one equivocal, and eleven control Thoroughbred horses. A whole-genome association study was conducted, and associated variants were filtered by determining if they were in or near genes known to be expressed in platelets. This evaluation identified three putative loci: a missense variant in SEL1L (p.Ile604Val), a 1.6kb deletion in a long non-coding RNA ( AL355838.1) and a 73bp intronic deletion in VIPAS39. The long non-coding RNA AL355838.1 did not show expression in horse platelets, and there was no differential expression of SEL1L or VIPAS39 mRNA identified between affected and unaffected individuals. However, a significant difference in SEL1L protein expression was observed in AET-affected horses' platelets. Flow cytometry and immunofluorescence studies demonstrated that SEL1L is located intracellularly in equine platelets, but not in the α-granule, and moves to the surface of the platelet upon activation with thrombin. Although the template bleeding time was normal, platelets from horses that were homozygous for the SEL1L variant showed defective spreading on collagen. To support SEL1L as the causative gene and dissect the underlying mechanisms, we undertook studies in three additional models. Differentiation of human megakaryocytes revealed the presence of two SEL1L protein isoforms, p100 and p38, and both showed increased expression during megakaryopoiesis, although only p100 was delivered to mature platelets. We perfused whole blood from Mx1-Cre +; Sel1l fl/fl conditional knockouts and controls through a collagen-coated microfluidic chamber. We found that significantly fewer platelets from knockout mice bound to the collagen as compared to control siblings. Finally, a knockout zebrafish line was developed by deleting exons 3-20 using CRISPR/Cas9 mediated genome editing. Similar to the mouse, laser-mediated arterial endothelial injury of 5-day old mutant larvae resulted in significantly fewer thrombocytes (the fish platelet equivalent) adhering to the injured vessel wall as compared to wild-type siblings. Overall, these data support the conclusion that the SEL1L missense variant leads to AET, and that SEL1L has a previously undescribed and conserved role in platelet function, particularly the ability of platelets/thrombocytes to adhere to exposed collagen at sites of endothelial injury. These results will allow the Thoroughbred industry to easily diagnose AET-affected horses and make appropriate decisions for their racing and breeding careers. These data also provide an excellent introduction to further investigation into the understudied and potential role of ERAD proteins in human platelet disorders.

Deletion of prolyl hydroxylase domain-containing enzyme 3 (phd3) in zebrafish facilitates hypoxia tolerance

Prolyl hydroxylase domain (PHD)-containing enzyme 3 (PHD3) belongs to the Caenorhabditis elegans gene egl-9 family of prolyl hydroxylases. PHD3 catalyzes proline hydroxylation of hypoxia-inducible factor α (HIF-α) and promotes HIF-α proteasomal degradation through coordination with the pVHL complex under normoxic conditions. However, the relationship between PHD3 and the hypoxic response is not well understood. In this study, we used quantitative real-time PCR assay and O-dianisidine staining to characterize the hypoxic response in zebrafish deficient in phd3. We found that the hypoxia-responsive genes are upregulated and the number of erythrocytes was increased in phd3-null zebrafish compared with their wildtype siblings. On the other hand, we show overexpression of phd3 suppresses HIF-transcriptional activation. In addition, we demonstrate phd3 promotes polyubiquitination of zebrafish hif-1/2α proteins, leading to their proteasomal degradation. Finally, we found that compared with wildtype zebrafish, phd3-null zebrafish are more resistant to hypoxia treatment. Therefore, we conclude phd3 has a role in hypoxia tolerance. These results highlight the importance of modulation of the hypoxia signaling pathway by phd3 in hypoxia adaptation.

A genetic mosaic mouse model illuminates the pre-malignant progression of basal-like breast cancer.

Basal-like breast cancer (BLBC) is highly aggressive, and often characterized by BRCA1 and p53 deficiency. Although conventional mouse models enabled the investigation of BLBC at malignant stages, its initiation and pre-malignant progression remain understudied. Here, we leveraged a mouse genetic system known as mosaic analysis with double markers (MADM) to study BLBC initiation by generating rare GFP+Brca1, p53-deficient mammary cells alongside RFP+ wild-type sibling cells. After confirming the close resemblance of mammary tumors arising in this model to human BLBC at both transcriptomic and genomic levels, we focused our studies on the pre-malignant progression of BLBC. Initiated GFP+ mutant cells showed a stepwise pre-malignant progression trajectory from focal expansion to hyper-alveolarization and then to micro-invasion. Furthermore, despite morphological similarities to alveoli, hyper-alveolarized structures actually originate from ductal cells based on twin-spot analysis of GFP-RFP sibling cells. Finally, luminal-to-basal transition occurred exclusively in cells that have progressed to micro-invasive lesions. Our MADM model provides excellent spatiotemporal resolution to illuminate the pre-malignant progression of BLBC, and should enable future studies on early detection and prevention for this cancer.

Loss of one copy of vhl in zebrafish facilitates hypoxia tolerance

Insufficient oxygen in the water (hypoxia) is one of the limiting factors for high-density culture in modern aquaculture. Hypoxia tolerance in fish has been reported to be related to the hypoxia-inducible factor (HIF)-α-mediated hypoxia signaling pathway. The well-defined function of the von Hippel-Lindau (VHL) tumor suppressor gene VHL is to mediate proteasomal degradation of hydroxylated HIF-α proteins, resulting in the downregulation of hypoxia signaling pathway. However, whether VHL has an impact on hypoxia tolerance is still elusive. In this study, we find that vhl+/- zebrafish have no gross defects in development, growth, reproduction, and movement. In addition, vhl+/- zebrafish also have normal vasculature. However, under hypoxia, vhl+/- zebrafish are more resistant to hypoxia treatment compared with their wildtype siblings. Further assays indicate that the number of erythrocytes is increased and the expression of hypoxia-responsive genes is enhanced in vhl+/- zebrafish compared with their wildtype siblings. These data indicate that loss of one copy of vhl in zebrafish facilitates hypoxia tolerance, revealing a function of vhl in hypoxia tolerance.

Comparative Biophysical Study of Meibomian Lipids of Wild Type and Soat1-Null Mice: Implications to Meibomian Gland Dysfunction and Dry Eye Disease.

The biophysical roles of Meibomian lipids (MLs) played in health and meibomian gland dysfunction (MGD) are still unclear. The purpose of this research is to establish the composition-structure-functional correlations of the ML film (MLF) using Soat1-null mice and comprehensive in vitro biophysical simulations. MLs were extracted from tarsal plates of wild type (WT) and Soat1 knockout (KO) mice. The chemical composition of ML samples was characterized using liquid chromatography - mass spectrometry. Comprehensive biophysical studies of the MLFs, including their dynamic surface activity, interfacial rheology, evaporation resistance, and ultrastructure and topography, were performed with a novel experimental methodology called the constrained drop surfactometry. Soat1 inactivation caused multiple alternations in the ML profile. Compared to their WT siblings, the MLs of KO mice were completely devoid of cholesteryl esters (CEs) longer than C18 to C20, but contained 7 times more free cholesterol (Chl). Biophysical assays consistently suggested that the KO-MLF became stiffer than that of WT mice, revealed by reduced film compressibility, increased elastic modulus, and decreased loss tangent, thus causing more energy loss per blinking cycle of the MLF. Moreover, the KO mice showed thinning of their MLF, and reduced evaporation resistance. These findings delineated the composition-structure-functional correlations of the MLF and suggested a potential biophysical function of long-chain CEs in optimizing the surface activity, interfacial rheology, and evaporation resistance of the MLF. This study may provide novel implications to pathophysiological and translational understanding of MGD and dry eye disease.

The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish.

The double stranded RNA binding protein Adad1 (adenosine deaminase domain containing 1) is a member of the adenosine deaminase acting on RNAs (Adar) protein family with germ cell-specific expression. In mice, Adad1 is necessary for sperm differentiation, however its function outside of mammals has not been investigated. Here, through an N-ethyl-N-nitrosourea (ENU) based forward genetic screen, we identified an adad1 mutant zebrafish line that develops as sterile males. Further histological examination revealed complete lack of germ cells in adult mutant fish, however germ cells populated the gonad, proliferated, and entered meiosis in larval and juvenile fish. Although meiosis was initiated in adad1 mutant testes, the spermatocytes failed to progress beyond the zygotene stage. Thus, Adad1 is essential for meiosis and germline maintenance in zebrafish. We tested if spermatogonial stem cells were affected using nanos2 RNA FISH and a label retaining cell (LRC) assay, and found that the mutant testes had fewer LRCs and nanos2-expressing cells compared to wild-type siblings, suggesting that failure to maintain the spermatogonial stem cells resulted in germ cell loss by adulthood. To identify potential molecular processes regulated by Adad1, we sequenced bulk mRNA from mutants and wild-type testes and found mis-regulation of genes involved in RNA stability and modification, pointing to a potential broader role in post-transcriptional regulation. Our findings suggest that the RNA regulatory protein Adad1 is required for fertility through regulation of spermatogonial stem cell maintenance in zebrafish.

Disruption of sirtuin 7 in zebrafish facilitates hypoxia tolerance

SIRT7 is a member of the sirtuin family proteins with nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase activity, which can inhibit the activity of hypoxia-inducible factors independently of its enzymatic activity. However, the role of SIRT7 in affecting hypoxia signaling invivo is still elusive. Here, we find that sirt7-null zebrafish are more resistant to hypoxic conditions, along with an increase of hypoxia-responsive gene expression and erythrocyte numbers, compared with their wildtype siblings. Overexpression of sirt7 suppresses the expression of hypoxia-responsive genes. Further assays indicate that sirt7 interacts with zebrafish hif-1αa, hif-1αb, hif-2αa, and hif-2αb to inhibit their transcriptional activity and mediate their protein degradation. In addition, sirt7 not only binds to the hypoxia responsive element of hypoxia-inducible gene promoters but also causes a reduction of H3K18Ac on these promoters. Sirt7 may regulate hypoxia-responsive gene expression through its enzymatic and nonenzymatic activities. This study provides novel insights into sirt7 function and sheds new light on the regulation of hypoxia signaling by sirt7.

Integration of alligator cathelicidin gene via two CRISPR/Cas9-assisted systems enhances bacterial resistance in blue catfish, Ictalurus furcatus

CRISPR/Cas9-mediated genome editing has paved new avenues for improving production-valued traits in aquaculture by knocking out or disrupting functional genes. However, utilizing CRISPR/Cas9-based knock-in (KI) of exogenous genes can expedite genetic improvement of traits such as disease resistance, which remains problematic in farmed fish. In this study, we successfully generated transgenic blue catfish (Ictalurus furcatus) of primarily Rio Grande strain ancestry with site-specific KI of the alligator (Alligator sinensis) cathelicidin (As-Cath) gene into the luteinizing hormone (lh) locus via two CRISPR/Cas9-mediated KI systems, assisted by the linear double-stranded DNA (dsDNA) and double-cut plasmid, respectively. High integration rates were observed with linear dsDNA (16.67%, [13/78]) and double-cut plasmid strategies (24.53%, [26/106]). In addition, the on-target KI efficiency of the double-cut plasmid strategy (16.04%, [17/106]) was 1.67 times higher than that of the linear dsDNA strategy (10.26%, [8/78]) based on the odds ratio. The relative expression of the As-Cath transgene of P1 founders was detected in nine tissues, dominated by the kidney, skin, and muscle (14.30-, 7.71- and 6.92-fold change, P < 0.05). Moreover, the As-Cath transgenic blue catfish showed a higher cumulative survival rate than that of wild-type controls (80% vs. 30%, P < 0.05) following Flavobacterium covae infection. Survival during culture supports the challenge data as survival of As-Cath transgenic individuals was 97.1% while that of pooled non-transgenic individuals was observed to be less 87.0% (P = 0.15). The growth rates and external morphology of the transgenic and wild-type siblings were not different (P > 0.05), indicating no pleiotropic effects of the As-Cath transgene integration at the lh locus in the P1 founders for this trait. Taken together, our findings demonstrate that CRISPR/Cas9-assisted KI of an antimicrobial peptide gene can be achieved in blue catfish with high integration efficiency, and As-Cath transgenic blue catfish have improved disease resistance, which is a promising strategy for disease reduction in aquaculture.

Poly (A)-specific ribonuclease deficiency impacts oogenesis in zebrafish

Poly (A)-specific ribonuclease (PARN) is the most important 3′–5′exonuclease involved in the process of deadenylation, the removal of poly (A) tails of mRNAs. Although PARN is primarily known for its role in mRNA stability, recent studies suggest several other functions of PARN including a role in telomere biology, non-coding RNA maturation, trimming of miRNAs, ribosome biogenesis and TP53 function. Moreover, PARN expression is de-regulated in many cancers, including solid tumours and hematopoietic malignancies. To better understand the in vivo role of PARN, we used a zebrafish model to study the physiological consequences of Parn loss-of-function. Exon 19 of the gene, which partially codes for the RNA binding domain of the protein, was targeted for CRISPR-Cas9-directed genome editing. Contrary to the expectations, no developmental defects were observed in the zebrafish with a parn nonsense mutation. Intriguingly, the parn null mutants were viable and fertile, but turned out to only develop into males. Histological analysis of the gonads in the mutants and their wild type siblings revealed a defective maturation of gonadal cells in the parn null mutants. The results of this study highlight yet another emerging function of Parn, i.e., its role in oogenesis.

SLC35E1 promotes keratinocyte proliferation in psoriasis by regulating zinc homeostasis

Keratinocyte hyperproliferation is a key pathogenic factor in psoriasis. However, the mechanisms that regulate keratinocyte hyperproliferation in this condition remain unclear. Here, we found that SLC35E1 was highly expressed in keratinocytes of patients with psoriasis and that Slc35e1−/− mice displayed a less severe imiquimod (IMQ)-induced psoriasis-like phenotype than their wild-type siblings. In addition, SLC35E1 deficiency inhibited keratinocyte proliferation in both mice and cultured cells. On a molecular level, SLC35E1 was found to regulate zinc ion concentrations and subcellular localization, while zinc ion chelation reversed the IMQ-induced psoriatic phenotype in Slc35e1−/− mice. Meanwhile, epidermal zinc ion levels were decreased in patients with psoriasis and zinc ion supplementation alleviated the psoriatic phenotype in an IMQ-induced mouse model of psoriasis. Our results indicated that SLC35E1 can promote keratinocyte proliferation by regulating zinc ion homeostasis and zinc ion supplementation has potential as a therapy for psoriasis.

Comparative analysis of transcriptional changes in zebrafish cep290 and bbs2 mutants by RNA-seq reveals upregulation of inflammatory and stress-related pathways.

Acute injury to the adult zebrafish retina triggers the release of pro-inflammatory cytokines and growth factors that stimulate multiple gene regulatory networks, which ultimately stimulate Müller glia to proliferate and regenerate neurons. In contrast, zebrafish carrying mutations in cep290 or bbs2 undergo progressive loss of cone photoreceptors and exhibit signs of microglia activation and inflammation, but the mutants fail to stimulate a regeneration response. To identify transcriptional changes that occur in zebrafish mutants undergoing progressive photoreceptor degeneration, RNA-seq transcriptional profiling was performed on cep290-/- and bbs2-/- retinas. The PANTHER Classification System was used to identify biological processes and signaling pathways that were differentially expressed between mutants and wild-type siblings during degeneration. As expected, genes associated with phototransduction were downregulated in cep290 and bbs2 mutants compared to wild-type siblings. Although both cep290 and bbs2 mutants undergo proliferation of rod precursors in response to retinal degeneration, the process of negatively regulating proliferation is enriched for upregulated genes, and this negative regulation may restrict proliferation of Müller glia and inhibit regeneration. A total of 815 differentially expressed genes (DEGs) were shared by cep290 and bbs2 retinas. Genes in pathways associated with inflammation, apoptosis, stress response, and PDGF signaling were overrepresented. Identifying the genes and biological pathways that are common in zebrafish models of inherited retinal degeneration provides a foundation for future studies on the mechanisms that regulate cell death as well as processes that prohibit Müller cell reprogramming or proliferation in a model capable of retinal regeneration. The pathways will provide targets for future interventions that may promote successful regeneration of lost photoreceptors.

Leukocyte Tyrosine Kinase (Ltk) Is the Mendelian Determinant of the Axolotl Melanoid Color Variant.

The great diversity of color patterns observed among amphibians is largely explained by the differentiation of relatively few pigment cell types during development. Mexican axolotls present a variety of color phenotypes that span the continuum from leucistic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, proportionally fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single-origin hypothesis of pigment cell development, wherein it has been proposed that all three pigment cell types derive from a common progenitor cell, with pigment metabolites playing potential roles in directing the development of organelles that define different pigment cell types. Specifically, these studies identified xanthine dehydrogenase (XDH) activity as a mechanism for the permissive differentiation of melanophores at the expense of xanthophores and iridophores. We used bulked segregant RNA-Seq to screen the axolotl genome for melanoid candidate genes and identify the associated locus. Dissimilar frequencies of single-nucleotide polymorphisms were identified between pooled RNA samples of wild-type and melanoid siblings for a region on chromosome 14q. This region contains gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and leukocyte tyrosine kinase (Ltk), a cell surface signaling receptor that is required for iridophore differentiation in zebrafish. Wild-type Ltk crispants present similar pigment phenotypes to melanoid, strongly implicating Ltk as the melanoid locus. In concert with recent findings in zebrafish, our results support the idea of direct fate specification of pigment cells and, more generally, the single-origin hypothesis of pigment cell development.

Abstract 3636: Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS

Abstract Background: 25% of patients diagnosed with colorectal cancer (CRC) have liver metastasis at presentation, and ~80% of all metastatic CRC are developed in the liver. We previously reported that liver endothelial cells (ECs), a key component of the liver microenvironment, secrete LRG1 to promote CRC growth via activating human epidermal growth factor receptor (ERbB3, also known as HER3). However, we found that LRG1-induced HER3 activation is distinct from the canonical neuregulin 1 (NRG1)-induced HER3 pathway. The present study further validated LRG1 as a new HER3 ligand for promoting mCRC growth and elucidated the novel downstream signaling pathway induced by LRG1-HER3. Methods: We first measured the binding affinity between HER3 and LRG1 by Biolayer interferometry (BLI). We then used in vitro and in vivo xenograft approaches to determine the effect of LRG1 monoclonal antibody (15C4) on HER3 activation and CRC growth. To further determine the role of LRG1 in promoting CRC growth in the liver, we used murine CRC cells in a syngeneic orthotropic liver injection model to establish CRC allografts in the liver of LRG1−/− mice with systemic LRG1 knockout and wild-type siblings (LRG1+/+). We also performed unbiased phospho-MS analysis and subsequent validations to determine the downstream signaling pathway activated by LRG1-HER3. Results: We identified that LRG1 binds to HER3 with the affinity at ~100nM. The LRG1 antibody 15C4 completely attenuated LRG1-induced HER3 activation and in vitro and xenograft growth in vivo. Moreover, LRG1−/− mice with CRC allografts in the liver had 2 times longer overall survival than tumor-bearing LRG1+/+ mice. Lastly, unbiased -omics analysis identified eIF4-protein synthesis is significantly activated by LRG1. With target-specific inhibitors, we further determined that LRG1-HER3 activates the PI3K-PDK1-RSK1/3-eIF4 axis independent of AKT. Conclusions: We identified LRG1 as a novel HER3 ligand and demonstrated that the liver microenvironment-derived LRG1 plays a key oncogenic role in mCRC, by activating a novel RSK-eIF4 survival pathway. Our findings highlighted the potential of blocking LRG1-HER3 and involved downstream pathways for treating patients with mCRC. Citation Format: Moeez Ghani Rathore, Michelle Wright, Wei Huang, Derek Taylor, Yamu Li, Jordan Winter, Zhenghe Wang, John Greenwood, Stephen Moss, Rui Wang. Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3636.

Cellular activity of autophagy and multivesicular bodies in lens fiber cells during early lens development in rbm24a mutant of zebrafish: Ultrastructure analysis

Use of zebrafish as animal model for various diseases during early developmental stages has been exponentially increased with the aim to achieve the best representative results in this transparent fish. Recent studies documented that Rbm24a mutant causes cataract formation and resulted in blindness using the zebrafish model. Therefore, correct interpretation of studies that aimed for molecular approaches, a description of comparative and in-depth analysis of development of lens in wildtype and mutant is crucial to obtain the correct conclusion. In this study, we use a gold standard method the Transmission Electron Microscopy (TEM) to analysis the lens development in rbm24a mutant zebrafish. Firstly, we compare the cellular structures at 16–20 h post fertilization (hpf), the lens placode in ectoderm indicated delay lens development in rbm24a mutant than wildtype (siblings) zebrafish. At 33 hpf, loosely appeared lens fiber cells showed heterogenous electron density with numbers of mitochondria in lens of rbm24a mutant, revealed the influence of gene mutation in lens development. A detail ultrastructure of lens of rbm24a mutant also presented at 33 hpf. Comparatively in wildtype (siblings) at 33 hpf, lens exhibited homogenous electron density in tightly packed lens fiber cells with few mitochondria. Furthermore, to characterize the lens in rbm24a mutant we obtained data of cellular structures on 25 hpf and 1.5 days’ post fertilization (dpf). At 25 hpf in mutant zebrafish, the detached solid sphere lens mass from ectoderm showed karyorrhexis, mitophagy and vesicles (also multivesicular bodies), these cellular structures supposed to hamper the development of future fiber cells. Moreover, at 1.5 dpf in mutant, nuclear excisosome, multilamellar bodies and irregular shaped mitochondria in heterogenous electron dense cytoplasm of lens fiber cells, collectively shown affected lens transparency. In summary the ultrastructure results of lens of rbm24a mutant zebrafish expand our knowledge and give reflection of different cellular activities like autophagy, apoptosis, vesicles (multivesicular bodies) and nuclear excisosomes which play their role in transparency achievement.

Transcriptomic and Metabolomic Studies Reveal That Toll-like Receptor 2 Has a Role in Glucose-Related Metabolism in Unchallenged Zebrafish Larvae (Danio rerio).

Toll-like receptors (TLRs) have been implicated in the regulation of various metabolism pathways, in addition to their function in innate immunity. Here, we investigate the metabolic function of TLR2 in a larval zebrafish system. We studied larvae from a tlr2 mutant and the wild type sibling controls in an unchallenged normal developmental condition using transcriptomic and metabolomic analyses methods. RNAseq was used to evaluate transcriptomic differences between the tlr2 mutant and wild-type control zebrafish larvae and found a signature set of 149 genes to be significantly altered in gene expression. The expression level of several genes was confirmed by qPCR analyses. Gene set enrichment analysis (GSEA) revealed differential enrichment of genes between the two genotypes related to valine, leucine, and isoleucine degradation and glycolysis and gluconeogenesis. Using 1H nuclear magnetic resonance (NMR) metabolomics, we found that glucose and various metabolites related with glucose metabolism were present at higher levels in the tlr2 mutant. Furthermore, we confirmed that the glucose level is higher in tlr2 mutants by using a fluorometric assay. Therefore, we have shown that TLR2, in addition to its function in immunity, has a function in controlling metabolism during vertebrate development. The functions are associated with transcriptional regulation of various enzymes involved in glucose metabolism that could explain the different levels of glucose, lactate, succinate, and malate in larvae of a tlr2 mutant.

Sex-specific meiosis responses to Gsdf in medaka (Oryzias latipes).

The meiotic entry of undifferentiated germ cells is sexually specific and strictly regulated by the testicular or ovarian environment. Germline stem cells with a set of abnormal sex chromosomes and associated autosomes undergo defective meiotic processes and are eventually eliminated by yet to be defined post-transcriptional modifications. Herein, we report the role of gsdf, a member of BMP/TGFβ family uniquely found in teleost, in the regulation of meiotic entry in medaka (Oryzias latipes) via analyses of gametogenesis in gsdf-deficient XX and XY gonads in comparison with their wild-type siblings. Several differentially expressed genes, including the FKB506-binding protein 7 (fkbp7), were significantly upregulated in pubertal gsdf-deficient gonads. The increase in alternative pre-mRNA isoforms of meiotic synaptonemal complex gene sycp3 was visualized using Integrative Genomics Viewer and confirmed by real-time qPCR. Nevertheless, immunofluorescence analysis showed that Sycp3 protein products reduced significantly in gsdf-deficient XY oocytes. Transmission electron microscope observations showed that normal synchronous cysts were replaced by asynchronous cysts in gsdf-deficient testis. Breeding experiments showed that the sex ratio deviation of gsdf-/- XY gametes in a non-Mendelian manner might be due to the non-segregation of XY chromosomes. Taken together, our results suggest that gsdf plays a role in the proper execution of cytoplasmic and nuclear events through receptor Smad phosphorylation and Sycp3 dephosphorylation to coordinate medaka gametogenesis, including sex-specific mitotic divisions and meiotic recombination.

Sphingosine Kinase 1 Deficiency in Smooth Muscle Cells Protects against Hypoxia-Mediated Pulmonary Hypertension via YAP1 Signaling.

Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.

SRSF2P95Hand FLT3ITDSynergistically Drives Leukemic Transformation in Zebrafish

Internal Tandem Duplication of human FMS-like tyrosine kinase (FLT3-ITD) is one of the most frequent genetic alterations in de novo acute myeloid leukemia (AML) and is often acquired during transformation of myelodysplastic syndrome (MDS) into AML, predicting an unfavorable prognosis. RNA splicing factor mutations (e.g. hot spot mutations in serine and arginine rich splicing factor 2 - SRSF2) are often found in MDS and chronic myelomonocytic leukemia (CMML). However, the clinicopathologic features of co-mutated FLT3-ITD and SRSF2 in MDS/CMML and transformation to AML are presently unclear. Here we investigated whether FLT3-ITD cooperates with SRSF2-P95H to promote leukemogenesis in zebrafish. Transgenic zebrafish carrying human FLT3-ITD or SRSF2-P95H as driven by Runx1 promoter were generated by Tol2 transgenesis. Tg(Runx1:FLT3ITD) was outcrossed with Tg(Runx1: SRSF2P95H) to generate Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish. The presence of human FLT3-ITD or SRSF2-P95H was confirmed through Sanger sequencing, and the expression of each transcript was detected by qRT-PCR in both embryos and adult kidney marrow. Their clinicopathologic features were examined. At the embryonic stage, the number of leukocytes (l-plastin+) was significantly increased in Tg(Runx1: FLT3ITDSRSF2P95H) embryos compared with their single mutant and wildtype (WT) siblings, whereas the abundance of neutrophils (SBB+) was unaffected. At six months of age, Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish showed monocytosis in peripheral blood (PB) (1 out of 4), but not in the single mutant and WT siblings. In kidney marrow (KM), there was expansion of both immature and mature monocytes, and dysplasia of granulopoiesis and erythropoiesis. At 9 months of age, all four Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed neutropenia and substantial increase in monocytosis. At 1 year of age, one out of the nine Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed a significant increase in blasts in KM, reminiscent of human AML, and the other eight Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish demonstrated phenotypes consistent with human CMML-like diseases. Increased monocytic compartment of KM was confirmed by α-naphthyl acetate esterase sensitive to fluoride inhibition. In addition, both the spleen area and cellularity were expanded in Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish compared with Tg(Runx1: SRSF2P95H) single mutant and WT siblings. Taken together, our findings demonstrated that FLT3-ITD cooperates with SRSF2-P95H to drive the initiation and progression of CMML and the subsequent transformation to AML. Understanding the molecular underpinnings of these events will shed light to the diverse clinicopathologic features in CMML patients.

Pathogenetic Role of RNA Splicing and NRAS Mutation Combination in Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by monocytosis and recurrent gene mutations including those of SRSF2, ASXL1, TET2, RUNX1 and NRAS. Different mutation combinations may explain the diverse clinicopathologic features in this disease, ranging from those of myelodysplastic syndrome (MDS) to myeloproliferative neoplasm (MPN) phenotype and even transformation to acute myeloid leukemia (AML). A zebrafish program was initiated to understand the pathogenetic roles of mutation combinations in CMML and generate animal models for high throughput drug screening. In this study, we reported our findings based on SRSF2 and NRAS mutation combination. Transgenic zebrafish lines expressing human SRSF2P95H and NRASG12Dmutations (F0), as driven by Runx1 promoter, were generated using Tol2 transgenesis. These lines were out-crossed with wildtype (WT) to generate F1. F1 SRSF2P95Hmutant were then crossed with F1 NRASG12D mutant to generate WT, Tg(Runx1:SRSF2P95H), Tg(Runx1: NRASG12D) and Tg(Runx1:SRSF2P95HNRASG12D). Expression of mutations was confirmed in embryos and adult kidney marrow (KM) by quantitative RT-PCR and DNA sequencing. At 3 months, there was no morphologic changes in peripheral blood (PB) or KM in double or single mutant fish. From 6-12 months, there was progressive weight loss in Tg (Runx1: SRSF2P95H NRASG12D) fish but not single mutant and WT siblings. In the double mutant fish, the PB showed monocytosis and presence of blasts. KM showed increase in cellularity, immature monocytes and blast population but decrease in erythropoiesis. Neutrophils were dysplastic as evident by hypogranularity, abnormal nuclear configuration and features resembling pseudo Pelgar-Huet anomaly as in human MDS. Increased monocytic compartment in KM was confirmed by non-specific esterase (NSE) which was sensitive to fluoride inhibition. Survival of the Tg(Runx1:SRSF2P95HNRASG12D) was significantly shorter than those of single mutant and WT siblings. Both the double mutant and Tg(Runx1:NRASG12D) showed splenomegaly. Of the 49 Tg(Runx1:SRSF2P95HNRASG12D) adult fish examined, 9 developed MDS/AML with 10-19% KM blasts and 18 developed AML with ≥ KM 20% blasts. Zebrafish model of CMML based on co-expression of SRSF2 and NRAS mutations recapitulated the diverse clinical repertoire of this disease, ranging from MDS to MPN and leukemic transformation into AML occurred in some animals. The molecular basis underlying the difference in penetrance might account for the diverse clinicopathologic characteristics in patients and it warrants further investigations.

Deletion of eif6 Partially Rescues Survival, but Not Neutropenia, of Zebrafish Model of Shwachman-Diamond Syndrome: The Roles of Sbds and Eif6 in Perturbing Lipid Metabolism

Introduction: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency, skeletal defects, neutropenia, and an increased risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Biallelic mutations in SBDS account for ~90% of patients. SBDS interacts physically with EFL1 to release EIF6 from the pre-60S ribosomal subunit and promote the assembly of the mature 80S ribosome in the cytoplasm. A subset of SDS patients acquire del(20q), resulting in loss of EIF6. Additionally, somatic heterozygous EIF6 mutations have been found in SDS patients. SDS patients harboring del(20q) and EIF6 mutations are linked to a lower risk of developing MDS/AML when compared to other SDS patients. Together these factors point to a potential mechanism whereby a reduction in EIF6 levels may lead to a suppression in ribosome biogenesis defects. Methods and Results: We have created sbds knockout (KO) zebrafish strains that phenocopy the human syndrome with stunted growth, neutropenia and atrophy in the pancreas, liver, and intestine. Unlike the Sbds-null mice which die in early embryogenesis, sbds-null fish survive to early juvenile stages. The sbds KO fish display an accumulation of Eif6 in liver and intestine. Interestingly, we also found an accumulation of EIF6 in SDS patient-derived lymphoblasts. EIF6 has been described as a key regulator of metabolism, specifically stimulating glycolytic and fatty acid synthesis. Lipid metabolomics of sbds KO fish show a decrease in free fatty acids and phosphatidylcholine. Expression of several genes critical in lipid metabolism (srebp1, fasn and pparg) is upregulated in sbds KO fish. To study the role of Eif6 in the pathophysiology of SDS, we created an eif6 KO zebrafish with an insertion of one base pair resulting in a premature stop codon. By immunoblotting we demonstrated that mutants did not display Eif6 by 6 days post fertilization (dpf), and heterozygous fish displayed less protein than wildtype siblings. This new strain showed early mortality (~10 dpf), neutropenia, tp53 pathway activation, with a pronounced upregulation of cdkn1a as early as 5 dpf. RNA-Seq data of eif6 KO fish at 5 dpf showed a total of 2225 differentially expressed genes (DEGs): 668 downregulated and 1557 upregulated. These DEGs are involved in two main gene expression pathways: ribosome biogenesis and oxidative phosphorylation. We also observed a dysregulation of lipid metabolism markers in the eif6 KO fish. Furthermore, we bred the sbds KO with the eif6 KO, we observed a partial rescue on survival only in sbds-/- with one copy of eif6(eif6+/-) but failed to rescue their neutropenia. Conclusions: Our organismal models of sbds or eif6 deletion provide new insights into the pathophysiology of human SDS: 1) SBDS affects lipid metabolism possibly due to an accumulation of EIF6, 2) Loss of eif6 affects development/survival at an earlier stage than loss of sbds, and 3) Loss of either sbds or eif6 markedly upregulates cdkn1a, which is downstream of tp53. Interestingly, Eif6 partially rescues survival of sbds-null organisms, but only in the haploinsufficient state. EIF6 may offer a promising target for a novel therapeutic strategy in SDS.