Adult Mutant Research Articles

EPCO-10. PROTEIN GENERATOR ENABLES PROTEOMICS-BASED SUBTYPING OF IDH MUTANT ASTROCYTOMA

Abstract The understanding of the proteomic landscape of IDH mutant astrocytoma has been limited in previous studies. To address this knowledge gap, we developed an innovative approach using artificial intelligence (AI) to generate proteomic profiles of tumors based on their transcriptome data. Particularly, we employed a combination of Variational Auto-Encoder and Contrastive Language Image Pretraining techniques to develop an AI model. The model was trained on publicly available databases containing matched RNA and protein data. Leveraging this extensive dataset, we applied the trained model to infer proteomic profiles for over one thousand adult IDH mutant astrocytoma specimens using their transcriptome sequencing data. This novel approach allowed us to explore and identify previously undiscovered subtypes based on the predicted proteomic profiles. As a result, we identified seven distinct clusters: IDHmut-noncodel-Proliferative, IDHmut-noncodel-Mesenchymal, IDHmut-noncodel-Mitochondrial, IDHmut-NFkB-signal, IDHmut-codel-Proliferative, IDHmut-Metabolism, and IDHmut-Neuronal. Of particular clinical significance, the IDHmut-noncodel-Proliferative and IDHmut-noncodel-Immune-Mesenchymal subtypes displayed the worst prognoses, while the IDHmut-Neuronal, IDHmut-NFkB-signal, and IDHmut-Metabolism subtypes exhibited relatively better survival outcomes. Remarkably, within the IDHmut-noncodel-Proliferative cluster, we observed a high prevalence of CDKN2A homozygous deletion, providing a potential explanation for its poor survival prognosis. Conversely, the IDHmut-noncodel-Mesenchymal cluster did not demonstrate any significant genetic events but exhibited elevated expression of mesenchymal marker genes. Furthermore, this cluster displayed notable characteristics, including the presence of gemistocytic differentiated tumor cells and infiltration of activated CD8 T lymphocytes. Moreover, we encompassed an evolutionary analysis of 189 initial-recurrent IDH mutant astrocytoma pairs, and discovered that gemistocytic differentiation represents an early event in tumor progression. In summary, we introduced a novel method utilizing an AI model to generate proteomic profiles, thus enabling the discovery of previously unknown subtypes of IDH mutant glioma with clinical relevance. These findings contribute to a deeper understanding of the molecular landscape of IDH mutant astrocytoma, potentially leading to improved prognostication and personalized treatment strategies.

The functional roles of zebrafish HoxA- and HoxD-related clusters in the pectoral fin development

The paralogs 9–13 Hox genes in mouse HoxA and HoxD clusters are critical for limb development. When both HoxA and HoxD clusters are deleted in mice, significant limb truncation is observed compared to the phenotypes of single and compound mutants of Hox9-13 genes in these clusters. In zebrafish, mutations in hox13 genes in HoxA- and HoxD-related clusters result in abnormal morphology of pectoral fins, homologous to forelimbs. However, the effect of the simultaneous deletions of entire HoxA- and HoxD-related clusters on pectoral fin development remains unknown. Here, we generated mutants with several combinations of hoxaa, hoxab, and hoxda cluster deletions and analyzed the pectoral fin development. In hoxaa−/−;hoxab−/−;hoxda−/− larvae, the endoskeletal disc and the fin-fold are significantly shortened in developing pectoral fins. In addition, we show that this anomaly is due to defects in the pectoral fin growth after the fin bud formation. Furthermore, in the surviving adult mutants, micro-CT scanning reveals defects in the posterior portion of the pectoral fin which is thought to represent latent regions of the limb. Our results further support that the functional role of HoxA and HoxD clusters is conserved in the paired appendage formation in bony fishes.

Foxe1 mutant zebrafish show indications of a hypothyroid phenotype and increased sensitivity to ethanol for craniofacial malformations.

FOXE1 mutations in humans are associated with cleft palate and hypothyroidism. We previously developed a foxe1 mutant zebrafish demonstrating mineralization defects in larvae. In the present study, we investigate the thyroid status and skeletal phenotype of adult foxe1 mutants. Mutant fish have increased expression of tshβ in the pituitary, and of hepatic dio1 and dio2. In plasma, we found higher Mg levels. Together these findings are indicative of hypothyroidism. We further observed mineralization defects in scales due to enhanced osteoclast activity as measured by increased expression levels of tracp, ctsk, and rankl. Gene-environment interactions in the etiology of FOXE1-related craniofacial abnormalities remain elusive, which prompts the need for models to investigate genotype-phenotype associations. We here investigated whether ethanol exposure increases the risk of developing craniofacial malformations in foxe1 mutant larvae that we compared to wild types. We found in ethanol-exposed mutants an increased incidence of developmental malformations and marked changes in gene expression patterns of cartilage markers (sox9a), apoptotic markers (casp3b), retinoic acid metabolism (cyp26c1), and tissue hypoxia markers (hifaa, hifab). Taken together, this study shows that the foxe1 mutant zebrafish recapitulates phenotypes associated with FOXE1 mutations in human patients and a clear foxe1-ethanol interaction.

Photoreceptor regeneration occurs normally in microglia-deficient irf8 mutant zebrafish following acute retinal damage

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers.

Maternal dnd1 is essential for migration and maintenance of PGCs in Nile tilapia at larval stage

As a germ-cell-specific maternal mRNA and RNA binding protein, dead end (dnd1) plays an important role in migration and maintenance of primordial germ cells (PGCs), and therefore, disruption of dnd1 results in no germ cells. However, the effect and duration of maternal dnd1 on PGCs in zygote mutants have not been reported yet. In the present study, CRISPR/Cas9 was used to mutate dnd1 in tilapia. Supported by the maternal dnd1 mRNA, germ cells of the dnd1 homozygous mutants proliferated and migrated normally before 7 dpf (days post fertilization). After the degradation of maternal dnd1 mRNA, significant downregulation of piwil1, piwil2, nanos2, nanos3 and vasa mRNA, and blocked migration of germ cells were observed at 14 dpf. In dnd1 zygote mutants, the number of germ cells was decreased significantly compared with wild type (WT) at 25 dpf, and no germ cell was observed at 30 dpf. Consistently, no expression of vasa and nanos3 was detected in the adult dnd1 mutants by qPCR. Interestingly, Cyp19a1a/cyp19a1a and foxl2 were expressed in dnd1-deficient XX gonads, while Cyp11c1/cyp11c1 and dmrt1 were expressed in dnd1-deficient XY gonads as in WT gonads. Correspondingly, both serum E2 level in XX and 11-KT level in XY fish did not differ significantly between dnd1 mutant and WT fish. Taken together, the present study demonstrates that disruption of dnd1 lead to progressive loss of germ cells, while the somatic cells are not affected in tilapia. The sexual fate of gonads is determined by the genetic sex instead of germ cell number, which makes tilapia a potential surrogate model for germ cell transplantation.

Primer on fibroblast growth factor 7 (FGF 7)

Fibroblast growth factor 7 (FGF7), also known as keratinocyte growth factor (KGF), is an important member of the FGF family that is mainly expressed by cells of mesenchymal origin while affecting specifically epithelial cells. Thus, FGF7 is widely expressed in diverse tissues, especially in urinary system, gastrointestinal tract (GI-tract), respiratory system, skin, and reproductive system. By interacting specifically with FGFR2-IIIb, FGF7 activates several downstream signal pathways, including Ras, PI3K-Akt, and PLCs. Previous studies of FGF7 mutants also have implicated its roles in various biological processes including development of essential organs and tissue homeostasis in adults. Moreover, more publications have reported that FGF7 and/or FGF7/FGFR2-IIIb-associated signaling pathway are involved in the progression of various heritable or acquired human diseases: heritable conditions like autosomal dominant polycystic kidney disease (ADPKD) and non-syndromic cleft lip and palate (NS CLP), where it promotes cyst formation and affects craniofacial development, respectively; acquired non-malignant diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), mucositis, osteoarticular disorders, and metabolic diseases, where it influences inflammation, repair, and metabolic control; and tumorigenesis and malignant diseases, including benign prostatic hyperplasia (BPH), prostate cancer, gastric cancer, and ovarian cancer, where it enhances cell proliferation, invasion, and chemotherapy resistance. Targeting FGF7 pathways holds therapeutic potential for managing these conditions, underscoring the need for further research to explore its clinical applications. Having more insights into the function and underlying molecular mechanisms of FGF7 is warranted to facilitate the development of effective treatments in the future.Here, we discuss FGF7 genomic structure, signal pathway, expression pattern during embryonic development and in adult organs and mutants along with phenotypes, as well as associated diseases.

Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein

Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal β –hexosaminidase A (HexA). We report that 1) recombinant HEXA alone increased HexA activity and reduced GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein comprised of HEXA linked to two blood brain barrier (BBB) entry elements, a transferrin receptor binding sequence and G-CSF, associates with HEXB in vitro; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, in vivo; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by ∼40% within 6 weeks , when injected intravenously to adult TS mutant mice mimicking the slow course of late onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, reduce the accumulation of GM2 and improve muscle strength, thereby providing potential treatment for late-onset TS.

Characterisation of an Adult Zebrafish Model for SDHB-Associated Phaeochromocytomas and Paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours arising from chromaffin cells. Pathogenic variants in the gene succinate dehydrogenase subunit B (SDHB) are associated with malignancy and poor prognosis. When metastases arise, limited treatment options are available. The pathomechanism of SDHB-associated PPGL remains largely unknown, and the lack of suitable models hinders therapy development. Germline heterozygous SDHB pathogenic variants predispose to developing PPGLs with a life-long penetrance of around 50%. To mimic the human disease phenotype, we characterised adult heterozygous sdhb mutant zebrafish as a potential model to study SDHB-related PPGLs. Adult sdhb mutant zebrafish did not develop an obvious tumour phenotype and were anatomically and histologically like their wild-type siblings. However, sdhb mutants showed significantly increased succinate levels, a major hallmark of SDHB-related PPGLs. While basal activity was increased during day periods in mutants, mitochondrial complex activity and catecholamine metabolite levels were not significantly different. In conclusion, we characterised an adult in vivo zebrafish model, genetically resembling human carriers. Adult heterozygous sdhb mutants mimicked their human counterparts, showing systemic elevation of succinate levels despite the absence of a tumour phenotype. This model forms a promising basis for developing a full tumour phenotype and gaining knowledge of the pathomechanism behind SDHB-related PPGLs.

Characterization of zebrafish coagulation cofactors Fviii and Fv mutants and modeling hemophilia A and factor V deficiency.

The aim of this study is to characterize zebrafish coagulation cofactors fviii and fv mutant fish and assess if they phenocopy classical hemophilia A and factor V deficiency in humans. The embryos from fviii and fv zebrafish heterozygote mutants generated by ENU mutagenesis were purchased from the ZIRC repository. They were reared to adulthood and genotyped. The heterozygote male and female were crossed to get homozygote, heterozygote, and wild-type fish. Functional kinetic coagulation assays and bleeding assays were performed on normal and mutant adult fish, and venous laser injury assays were performed on the larvae. The DNA from fviii and fv mutants were sequenced to confirm if they have a premature stop codon in exon 19, and in exon 2, respectively, and in both mutants, the amino acid glutamine is replaced with a stop codon. Homozygous and heterozygous 5 days post fertilization (dpf) larvae for fviii and fv deficient mutants exhibited prolonged time to occlusion after venous laser injury compared to wild-type controls. The homozygous and heterozygous fviii adult mutants showed modest bleeding and delayed fibrin formation in the kinetic partial thromboplastin time (kPTT) assay with their plasma. fv homozygous larvae had poor survival beyond 12 dpf. However, heterozygous fv mutants exhibited heavy bleeding and prolonged fibrin formation in the kPTT and kPT assay compared with wild-type siblings. Our characterization showed fviii and fv mutants from ZIRC phenocopied to a considerable extent classical hemophilia A and factor V deficiency in humans, respectively. These models should be useful in studying and developing novel drugs that reverse the phenotype and in generating suppressor mutations to identify novel factors that compensate for these deficiencies.

Phosphorylation of cardiac sodium channel at Ser571 anticipates manifestations of the aging myopathy.

Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction.NEW & NOTEWORTHY We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

Zebrafish dspb-/- mutant as model for arrhythmogenic cardiomyopathy - impact of physical activity

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background Zebrafish has been reported as a model to study the Arrythmogenic cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. Purpose Our aim was the characterization of dspb-/- mutants, studying the impact of physical activity in development of the disease. Methods The characterization of phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) and adult (8 months old) individuals. The impact of physical activity was determined through moderated training periods (1 month) and endurance assays using a swimming tunnel. Results Viability assays showed that dspb mutants suffer a premature death, with significant higher mortality rates than the wildtype group (survival WT 89.7% vs dspb-/- 61.3%, p<0.01). Larvae cardiac function showed dspb mutants had a higher heart rate (WT 106.8 ± 26.8 vs dspb-/- 141.0 ± 7.2, p<0.01) while echocardiography showed differences in heart rate (WT 127.2 ± 49,8 vs dspb-/- 219.2 ± 26.8, p<0.05), systolic (WT 26.9 ± 5.3 vs dspb-/- 18.5 ± 5.2, p<0.05) and diastolic (WT 37.6 ± 8.4 vs dspb-/- 23.7 ± 5.4, p<0.05) areas. Gene expression analysis in adult heart showed dspb expression drastically decreased (WT 0.9 ± 0.1 vs dspb-/- 0.4 ± 0.01, p<0.01), whilst pkp2 expression increased (WT 0.9 ± 0.1 vs dspb-/- 2.59 ±0.22, p<0.001) as well as dspa gene expression (wt 0.9 ± 0.1 vs dspb-/- 4.41 ± 0.18, p< 0.001) as a compensation mechanism. Moderated long term physical activity had a positive impact in heart rate (dspb-/-pre 219.2 ± 26.8 vs dspb-/-post 138 ± 7.5, p<0.01) and diastolic area (dspb-/-pre 24 ± 5 vs dspb-/-post 32 ± 2.1, p<0.05). Adult mutants showed a better endurance showing improvement in immobile cumulative duration (dspb-/-pre 849.3 ± 29.3 vs dspb-/-post 179.8 ± 29.7, p<0.01) and immobile frequency (dspb-/-pre 5399.5 ± 1127.8 vs dspb-/-post 1310 ± 481.9, p<0.01). Conclusion The dspb-/- zebrafish presented pathological cardiac phenotype at larvae and adult stages, and less endurance in intensity exercises. The moderate exercise has turned out to be a beneficial modulator of the phenotype.

Genetic Disruption of cyp21a2 Leads to Systemic Glucocorticoid Deficiency and Tissues Hyperplasia in the Teleost Fish Medaka (Oryzias latipes).

cytochrome P-450, 21-hydroxylase (cyp21a2), encodes an enzyme required for cortisol biosynthesis, and its mutations are the major genetic cause of congenital adrenal hyperplasia (CAH) in humans. Here, we have generated a null allele for the medaka cyp21a2 with a nine base-pair insertion which led to a truncated protein. We have observed a delay in hatching and a low survival rate in homozygous mutants. The interrenal gland (adrenal counterpart in teleosts) exhibits hyperplasia and the number of pomca-expressing cells in the pituitary increases in the homozygous mutant. A mass spectrometry-based analysis of whole larvae confirmed a lack of cortisol biosynthesis, while its corresponding precursors were significantly increased, indicating a systemic glucocorticoid deficiency in our mutant model. Furthermore, these phenotypes at the larval stage are rescued by cortisol. In addition, females showed complete sterility with accumulated follicles in the ovary while male homozygous mutants were fully fertile in the adult mutants. These results demonstrate that the mutant medaka recapitulates several aspects of cyp21a2-deficiency observed in humans, making it a valuable model for studying steroidogenesis in CAH.

Allelic heterogeneity of TTNtv dilated cardiomyopathy can be modeled in adult zebrafish.

Allelic heterogeneity (AH) has been noted in truncational TTN-associated (TTNtv-associated) dilated cardiomyopathy (DCM); i.e., mutations affecting A-band-encoding exons are pathogenic, but those affecting Z-disc-encoding exons are likely benign. The lack of an in vivo animal model that recapitulates AH hinders the deciphering of the underlying mechanism. Here, we explored zebrafish as a candidate vertebrate model by phenotyping a collection of zebrafish ttntv alleles. We noted that cardiac function and sarcomere structure were more severely disrupted in ttntv-A than in ttntv-Z homozygous embryos. Consistently, cardiomyopathy-like phenotypes were present in ttntv-A but not ttntv-Z adult heterozygous mutants. The phenotypes observed in ttntv-A alleles were recapitulated in null mutants with the full titin-encoding sequences removed. Defective autophagic flux, largely due to impaired autophagosome-lysosome fusion, was also noted only in ttntv-A but not in ttntv-Z models. Moreover, we found that genetic manipulation of ulk1a restored autophagy flux and rescued cardiac dysfunction in ttntv-A animals. Together, our findings presented adult zebrafish as an in vivo animal model for studying AH in TTNtv DCM, demonstrated TTN loss of function is sufficient to trigger ttntv DCM in zebrafish, and uncovered ulk1a as a potential therapeutic target gene for TTNtv DCM.

Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function.

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it "bigheart." Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

Mutagenesis of Odorant Receptor Coreceptor Orco Reveals the Odorant-Detected Behavior of the Predator Eupeodes corollae.

The successful mating of the hoverfly and the search for prey aphids are of great significance for biological control and are usually mediated by chemical cues. The odorant receptor co-receptor (Orco) genes play a crucial role in the process of insect odor perception. However, the function of Orco in the mating and prey-seeking behaviors of the hoverfly remains relatively unexplored. In this study, we characterized the Orco gene from the hoverfly, Eupeodes corollae, a natural enemy insect. We used the CRISPR/Cas9 technique to knock out the Orco gene of E. corollae, and the EcorOrco-/- homozygous mutant was verified by the genotype analysis. Fluorescence in situ hybridization showed that the antennal ORN of EcorOrco-/- mutant lack Orco staining. Electroantennogram (EAG) results showed that the adult mutant almost lost the electrophysiological response to 15 odorants from three types. The two-way choice assay and the glass Y-tube olfactometer indicated that both the larvae and adults of hoverflies lost their behavioral preference to the aphid alarm pheromone (E)-β-farnesene (EBF). In addition, the mating assay results showed a significant decrease in the mating rate of males following the knock out of the EcorOrco gene. Although the mating of females was not affected, the amount of eggs being laid and the hatching rate of the eggs were significantly reduced. These results indicated that the EcorOrco gene was not only involved in the detection of semiochemicals in hoverflies but also plays a pivotal role in the development of eggs. In conclusion, our results expand the comprehension of the chemoreceptive mechanisms in the hoverflies and offers valuable insights for the advancement of more sophisticated pest management strategies.

Stag2a or Stag2b Knockout Zebrafish Are Viable, Yet Loss of Stag2a Is Associated with Abnormal Expression of Early and Late Myeloid and Lymphoid Markers

Introduction:Mutations in Stromal antigen 2 ( STAG2) are frequently found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In mice, loss of Stag2 is embryonic lethal. Conditional knockout of Stag2 in adult mice results in MDS-like hematopoietic anomalies. Zebrafish harbor two paralogues of STAG2 ( stag2a and stag2b). To date, a complete stag2-null zebrafish model organism has not been developed. Recently reported, a stag2b-/- zebrafish demonstrated downregulation in primitive erythropoiesis. However, any hematopoiesis beyond 2 days post fertilization (2 dpf) was not detailed. Furthermore, a stag2a mutant zebrafish has not been reported. Methods and Results: Using CRISPR/Cas9 genome editing, we created a stag2a mutant anda stag2b mutant zebrafish. Both mutations result in a frame shift and premature protein truncations. Unlike the murine Stag2 -/- mutant, zebrafish stag2a -/- mutants and stag2b -/-mutants survive to adulthood, with no obvious morphological phenotypes. Detection of hemoglobin, using O-dianisidine staining, reveals anemia at 31 hours post fertilization (31 hpf) in stag2a +/- mutants (10/29), stag2a -/-mutants (10/15), stag2b +/-mutants (6/11), and stag2b -/-mutants (5/7); wildtype siblings appear unaffected. Intriguingly, at 4 dpf, stag2a maternal-zygotic mutants show increased expression levels of early and late myeloid and lymphoid markers. ( Figure 1) Work is ongoing to analyze blood cell lineages from zebrafish whole kidney marrow, the equivalent of mammalian bone marrow, in adult stag2a and adult stag2b mutants. In addition, we are characterizing the zebrafish stag2a;stag2b double mutants, which we will present. Conclusions: In zebrafish, stag2a and stag2b contribute to proper primitive and definitive hematopoiesis. Our organismal model of Stag2 deletion in the zebrafish will yield new insight into how loss of STAG2 may lead to MDS/AML.

Gene Expression Profiling in Trigeminal Ganglia from Cntnap2-/- and Shank3b-/- Mouse Models of Autism Spectrum Disorder.

Sensory difficulties represent a crucial issue in the life of autistic individuals. The diagnostic and statistical manual of mental disorders describes both hyper- and hypo-responsiveness to sensory stimulation as a criterion for the diagnosis autism spectrum disorders (ASD). Among the sensory domain affected in ASD, altered responses to tactile stimulation represent the most commonly reported sensory deficits. Although tactile abnormalities have been reported in monogenic cohorts of patients and genetic mouse models of ASD, the underlying mechanisms are still unknown. Traditionally, autism research has focused on the central nervous system as the target to infer the neurobiological bases of such tactile abnormalities. Nonetheless, the peripheral nervous system represents the initial site of processing of sensory information and a potential site of dysfunction in the sensory cascade. Here we investigated the gene expression deregulation in the trigeminal ganglion (which directly receives tactile information from whiskers) in two genetic models of syndromic autism (Shank3b and Cntnap2 mutant mice) at both adult and juvenile ages. We found several neuronal and non-neuronal markers involved in inhibitory, excitatory, neuroinflammatory and sensory neurotransmission to be differentially regulated within the trigeminal ganglia of both adult and juvenile Shank3b and Cntnap2 mutant mice. These results may help in disentangling the multifaced complexity of sensory abnormalities in autism and open avenues for the development of peripherally targeted treatments for tactile sensory deficits exhibited in ASD.

Tlx1 regulates acinar and duct development in mouse salivary glands.

Tlx1 encodes a transcription factor expressed in several craniofacial structures of developing mice. The role of Tlx1 in salivary gland development was examined using morphological and immunohistochemical analyses of Tlx1 null mice. Tlx1 is expressed in submandibular and sublingual glands but not parotid glands of neonatal and adult male and female C57Bl/6J (Tlx1+/+ ) mice. TLX1 protein was localized to the nuclei of terminal tubule cells, developing duct cells and mesenchymal cells in neonatal submandibular and sublingual glands, and to nuclei of duct cells and connective tissue cells in adult glands. Occasionally, TLX1 was observed in nuclei of epithelial cells in or adjacent to the acini. Submandibular glands were smaller and sublingual glands were larger in size in mutant mice (Tlx1-/- ) compared to wild-type mice. Differentiation of terminal tubule and proacinar cells of neonatal Tlx1-/- submandibular glands was abnormal; expression of their characteristic products, submandibular gland protein C and parotid secretory protein, respectively, was reduced. At 3 weeks postnatally, terminal tubule cells at the acinar-intercalated duct junction were poorly developed or absent in Tlx1-/- mice. Granular convoluted ducts in adult mutant mice were decreased, and epidermal growth factor and nerve growth factor expression were reduced. Along with normal acinar cell proteins, adult acinar cells of Tlx1-/- mice continued to express neonatal proteins and expressed parotid proteins not normally present in submandibular glands. Sublingual gland mucous acinar and serous demilune cell differentiation were altered. Tlx1 is necessary for proper differentiation of submandibular and sublingual gland acinar cells, and granular convoluted ducts. The mechanism(s) underlying Tlx1 regulation of salivary gland development and differentiation remains unknown.