A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species.

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

Functional implications of NHR-210 enrichment in C. elegans cephalic sheath glia: insights into metabolic and mitochondrial disruptions in Parkinson's disease models.

Glial cells constitute nearly half of the mammalian nervous system's cellular composition. The glia in C. elegans perform majority of tasks comparable to those conducted by their mammalian equivalents. The cephalic sheath (CEPsh) glia, which are known to be the counterparts of mammalian astrocytes, are enriched with two nuclear hormone receptors (NHRs)-NHR-210 and NHR-231. This unique enrichment makes the CEPsh glia and these NHRs intriguing subjects of study concerning neuronal health. We endeavored to assess the role of these NHRs in neurodegenerative diseases and related functional processes, using transgenic C. elegans expressing human alpha-synuclein. We employed RNAi-mediated silencing, followed by behavioural, functional, and metabolic profiling in relation to suppression of NHR-210 and 231. Our findings revealed that depleting nhr-210 changes dopamine-associated behaviour and mitochondrial function in human alpha synuclein-expressing strains NL5901 and UA44, through a putative target, pgp-9, a transmembrane transporter. Considering the alteration in mitochondrial function and the involvement of a transmembrane transporter, we performed metabolomics study via HR-MAS NMR spectroscopy. Remarkably, substantial modifications in ATP, betaine, lactate, and glycine levels were seen upon the absence of nhr-210. We also detected considerable changes in metabolic pathways such as phenylalanine, tyrosine, and tryptophan biosynthesis metabolism; glycine, serine, and threonine metabolism; as well as glyoxalate and dicarboxylate metabolism. In conclusion, the deficiency of the nuclear hormone receptor nhr-210 in alpha-synuclein expressing strain of C. elegans, results in altered mitochondrial function, coupled with alterations in vital metabolite levels. These findings underline the functional and physiological importance of nhr-210 enrichment in CEPsh glia.

Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila.

Background: Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods: We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results: We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions: Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans.

The Polarity/Protusion model of UNC-6/Netrin function in axon repulsion does not rely on a gradient of UNC-6/Netrin. Instead, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge. UNC-5 then inhibits growth cone protrusion ventrally based upon this polarity, resulting in dorsally-biased protrusion and dorsal migration away from UNC-6/Netrin. While previous studies have shown that UNC-5 inhibits growth cone protrusion by destabilizing actin, preventing microtubule + end entry, and preventing vesicle fusion, the signaling pathways involved are unclear. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.

Misregulation of bromotyrosine compromises fertility in male Drosophila.

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.

Improvement in Tol2 transposon for efficient large-cargo capacity transgene applications in cultured cells and zebrafish ( Danio rerio).

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer ( QBI SP163, ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish ( Danio rerio). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.

Chickens with a Truncated Light Chain Transgene Express Single-Domain H Chain-Only Antibodies.

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.

Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, Ags. B cells also reside and develop in the thymus, although their functions are less clear. During "thymic involution," a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ∼1% of human neonatal thymocytes but up to ∼10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. In this article, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- versus postinvolution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells postinvolution. We also performed RNA sequencing on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is, to our knowledge, the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution and establishing the highly genetically manipulatable zebrafish model as a template for involution studies.

Ergosterol promotes neurite outgrowth, inhibits amyloid-beta synthesis, and extends longevity: In vitro neuroblastoma and in vivo Caenorhabditis elegans evidence.

AIMS: Alzheimer's disease (AD), the most common neurodegenerative disorder associated with aging, is characterized by amyloid-ß (Aß) plaques in the hippocampus. Ergosterol, a mushroom sterol, exhibits neuroprotective activities; however, the underlying mechanisms of ergosterol in promoting neurite outgrowth and preventing Aß-associated aging have never been investigated. We aim to determine the beneficial activities of ergosterol in neuronal cells and Caenorhabditis elegans (C. elegans). MATERIALS AND METHODS: The neuritogenesis and molecular mechanisms of ergosterol were investigated in wild-type and Aß precursor protein (APP)-overexpressing Neuro2a cells. The anti-amyloidosis properties of ergosterol were determined by evaluating in vitro Aß production and the potential inhibition of Aß-producing enzymes. Additionally, AD-associated transgenic C. elegans was utilized to investigate the in vivo attenuating effects of ergosterol. KEY FINDINGS: Ergosterol promoted neurite outgrowth in Neuro2a cells through the upregulation of the transmembrane protein Teneurin-4 (Ten-4) mRNA and protein expressions, phosphorylation of the extracellular signal-regulated kinases (ERKs), activity of cAMP response element (CRE), and growth-associated protein-43 (GAP-43). Furthermore, ergosterol enhanced neurite outgrowth in transgenic Neuro2A cells overexpressing either the wild-type APP (Neuro2a-APPwt) or the Swedish mutant APP (Neuro2a-APPswe) through the Ten-4/ERK/CREB/GAP-43 signaling pathway. Interestingly, ergosterol inhibited Aß synthesis in Neuro2a-APPwt cells. In silico analysis indicated that ergosterol can interact with the catalytic sites of ß- and γ-secretases. In Aß-overexpressing C. elegans, ergosterol decreased Aß accumulation, increased chemotaxis behavior, and prolonged lifespan. SIGNIFICANCE: Ergosterol is a potential candidate compound that might benefit AD patients by promoting neurite outgrowth, inhibiting Aß synthesis, and enhancing longevity.

Comparison of baseline cataract rates in AB and TL wildtype zebrafish strains.

Zebrafish are an outstanding model for assessing the involvement of genes in paediatric cataracts. Gene discovery for cataracts is enhanced by manipulation of the genome of zebrafish embryos and comparing the phenotypes of mutant progeny with the wildtype embryos. However, wildtype laboratory fish can also develop cataracts, potentially confounding the results. In this study, we compared the baseline cataract rate between two commonly used wildtype laboratory strains, AB and TL, and also an outbred transgenic line with mCherry reporter. We assessed a total of 805 lens images of fish at 4 days post-fertilisation for cataracts and scored each cataract observed as mild, moderate or severe. We found that the AB strain had a cataract rate of 16.2%, TL had 8.9%, and mCherry had 0.7% and these rates were significantly different. We found that TL strain had a lower rate of mild cataracts than AB fish, however, the rate of moderate and severe phenotypes in the AB and the TL strain was similar. Overall, we showed that the baseline cataract rate varies significantly between the strains housed in a single facility and conclude that baseline rates of cataracts should be assessed when planning experiments to assess the genetic causes of cataracts.

Expanding the Drosophila toolkit for dual control of gene expression.

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

In order for researchers to understand how organisms develop and function, they often switch specific genes on or off in certain tissues or at selected times. This can be achieved using genetic tools called binary expression systems. In the fruit fly ­ a popular organism for studying biological processes ­ the most common is the GAL4/UAS system. In this system, a protein called GAL4 is expressed in a specific organ or tissue where it activates a UAS element ­ a genetic sequence that is inserted in front of the gene that is to be switched on. This can also include genes inserted into the fruit fly encoding fluorescent proteins or stretches of DNA coding for factors that can silence specific genes. For example, fruit flies expressing GAL4 protein specifically in nerve cells and a UAS element in front of a gene for a fluorescent protein will display fluorescent nerve cells, which can then be examined using fluorescence microscopy. Studying how organs communicate with one other can require controlled expression of multiple genes at the same time. In fruit flies, other binary expression systems that are analogous to the GAL4/UAS system (known as LexA/LexAop and QF/QUAS) can be used in tandem. For example, to study gut-brain communication, the GAL4/UAS system might be used to switch on the gene for an insulin-like protein in the gut, with one of the other systems controlling the expression of its corresponding receptor in the brain. However, these experiments are currently difficult because, while there are thousands of GAL4/UAS genetic lines, there are only a few LexA/LexAop and QF/QUAS genetic lines. To address this lack of resources, Zirin et al. produced a range of genetically engineered fruit flies containing the LexA/LexAop and QF/QUAS binary expression systems. The flies expressed LexA or QF in each of the major fly organs, including the brain, heart, muscles, and gut. A fluorescent reporter gene linked to the LexAop or QUAS elements, respectively, was then used to test the specificity to single organs and compare the different systems. In some organs the LexA/LexAop system was more reliable than the QF/QUAS system. However, both systems could be successfully combined with genetic elements to switch on a fluorescent reporter gene or switch off a gene of interest in the intended organ. The resources developed by Zirin et al. expand the toolkit for studying fruit fly biology. In future, it will be important to understand the differences between GAL4, LexA and QF systems, and to increase the number of fruit fly lines containing the newer binary expression systems.

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality.

BACKGROUND: CTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer-promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries. CTCFs of various organisms contain an unstructured N-terminal dimerization domain (DD) and clusters comprising eleven zinc-finger domains of the C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCFs share sequence homology in only five C2H2 domains that specifically bind to a conserved 15 bp motif. RESULTS: Previously, we demonstrated that CTCFs from different organisms carry unstructured N-terminal dimerization domains (DDs) that lack sequence homology. Here we used the CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene and generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected by the deletion of the N-terminal DD. Additionally, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression levels than those of the dCTCF variants, it efficiently bound to chromatin similarly to the wild type (wt) protein. CONCLUSIONS: Our findings suggest that one of the evolutionarily conserved functions of the unstructured N-terminal dimerization domain is to recruit dCTCF to its genomic sites in vivo.

Complement networks in gene-edited pig xenotransplantation: enhancing transplant success and addressing organ shortage.

The shortage of organs for transplantation emphasizes the urgent need for alternative solutions. Xenotransplantation has emerged as a promising option due to the greater availability of donor organs. However, significant hurdles such as hyperacute rejection and organ ischemia-reperfusion injury pose major challenges, largely orchestrated by the complement system, and activated immune responses. The complement system, a pivotal component of innate immunity, acts as a natural barrier for xenotransplantation. To address the challenges of immune rejection, gene-edited pigs have become a focal point, aiming to shield donor organs from human immune responses and enhance the overall success of xenotransplantation. This comprehensive review aims to illuminate strategies for regulating complement networks to optimize the efficacy of gene-edited pig xenotransplantation. We begin by exploring the impact of the complement system on the effectiveness of xenotransplantation. Subsequently, we delve into the evaluation of key complement regulators specific to gene-edited pigs. To further understand the status of xenotransplantation, we discuss preclinical studies that utilize gene-edited pigs as a viable source of organs. These investigations provide valuable insights into the feasibility and potential success of xenotransplantation, offering a bridge between scientific advancements and clinical application.

A pig kidney supporting human physiology.

Because of the global shortage of donor kidneys, xenotransplantation emerges as a potential solution for individuals with kidney failure who face challenges in securing a suitable donor kidney. A study featured in this month's issue of Kidney International assesses the kidney physiology of a porcine kidney transplanted into a brain-dead human with kidney failure, demonstrating life-sustaining physiological function for 7 days. Together with preclinical nonhuman primate studies, decedent models provide complementary data for development of clinical kidney xenotransplantation.

Differential impact of diesel exhaust particles on glutamatergic and dopaminergic neurons in Caenorhabditis elegans: A neurodegenerative perspective.

The growing body of evidence links exposure to particulate matter pollutants with an increased risk of neurodegenerative diseases. In the present study, we investigated whether diesel exhaust particles can induce neurobehavioral alterations associated with neurodegenerative effects on glutamatergic and dopaminergic neurons in Caenorhabditis elegans (C. elegans). Exposure to DEP at concentrations of 0.167 µg/cm2 and 1.67 µg/cm2 resulted in significant developmental delays and altered locomotion behaviour. These effects were accompanied by discernible alterations in the expressions of antioxidant genes sod-3 and gst-4 observed in transgenic strains. Behaviour analysis demonstrated a significant reduction in average speed (p < 0.001), altered paths, and decreased swimming activities (p < 0.01), particularly at mid and high doses. Subsequent assessment of neurodegeneration markers in glutamatergic (DA1240) and dopaminergic (BZ555) transgenic worms revealed notable glutamatergic neuron degeneration at 0.167 µg/cm2 (∼30 % moderate, ∼20 % advanced) and 1.67 µg/cm2 (∼28 % moderate, ∼24 % advanced, p < 0.0001), while dopaminergic neurons exhibited structural deformities (∼16 %) without significant degeneration in terms of blebs and breaks. Furthermore, in silico docking simulations suggest the presence of an antagonistic competitive inhibition induced by DEP in the evaluated neuro-targets, stronger for the glutamatergic transporter than for the dopaminergic receptor from the comparative binding affinity point of view. The results underscore DEP's distinctive neurodegenerative effects and suggest a link between locomotion defects and glutamatergic neurodegeneration in C. elegans, providing insights into environmental health risks assessment.

Spinocerebellar Ataxia Type 3 Pathophysiology-Implications for Translational Research and Clinical Studies.

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado-Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed.

Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

Genetic interference with HvNotch provides new insights into the role of the Notch-signalling pathway for developmental pattern formation in Hydra.

The Notch-signalling pathway plays an important role in pattern formation in Hydra. Using pharmacological Notch inhibitors (DAPT and SAHM1), it has been demonstrated that HvNotch is required for head regeneration and tentacle patterning in Hydra. HvNotch is also involved in establishing the parent-bud boundary and instructing buds to develop feet and detach from the parent. To further investigate the functions of HvNotch, we successfully constructed NICD (HvNotch intracellular domain)-overexpressing and HvNotch-knockdown transgenic Hydra strains. NICD-overexpressing transgenic Hydra showed a pronounced inhibition on the expression of predicted HvNotch-target genes, suggesting a dominant negative effect of ectopic NICD. This resulted in a "Y-shaped" phenotype, which arises from the parent-bud boundary defect seen in polyps treated with DAPT. Additionally, "multiple heads", "two-headed" and "ectopic tentacles" phenotypes were observed. The HvNotch-knockdown transgenic Hydra with reduced expression of HvNotch exhibited similar, but not identical phenotypes, with the addition of a "two feet" phenotype. Furthermore, we observed regeneration defects in both, overexpression and knockdown strains. We integrated these findings into a mathematical model based on long-range gradients of signalling molecules underlying sharply defined positions of HvNotch-signalling cells at the Hydra tentacle and bud boundaries.