Inhibition of autophagy as a novel treatment for neurofibromatosis type 1 tumors.

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutation of the NF1 gene that is associated with various symptoms, including the formation of benign tumors, called neurofibromas, within nerves. Drug treatments are currently limited. The mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib is used for a subset of plexiform neurofibromas (PNs) but is not always effective and can cause side effects. Therefore, there is a clear need to discover new drugs to target NF1-deficient tumor cells. Using a Drosophila cell model of NF1, we performed synthetic lethal screens to identify novel drug targets. We identified 54 gene candidates, which were validated with variable dose analysis as a secondary screen. Pathways associated with five candidates could be targeted using existing drugs. Among these, chloroquine (CQ) and bafilomycin A1, known to target the autophagy pathway, showed the greatest potential for selectively killing NF1-deficient Drosophila cells. When further investigating autophagy-related genes, we found that 14 out of 30 genes tested had a synthetic lethal interaction with NF1. These 14 genes are involved in multiple aspects of the autophagy pathway and can be targeted with additional drugs that mediate the autophagy pathway, although CQ was the most effective. The lethal effect of autophagy inhibitors was conserved in a panel of human NF1-deficient Schwann cell lines, highlighting their translational potential. The effect of CQ was also conserved in a Drosophila NF1 in vivo model and in a xenografted NF1-deficient tumor cell line grown in mice, with CQ treatment resulting in a more significant reduction in tumor growth than selumetinib treatment. Furthermore, combined treatment with CQ and selumetinib resulted in a further reduction in NF1-deficient cell viability. In conclusion, NF1-deficient cells are vulnerable to disruption of the autophagy pathway. This pathway represents a promising target for the treatment of NF1-associated tumors, and we identified CQ as a candidate drug for the treatment of NF1 tumors.

Dietary Amino Acids Promote Glucagon-like Hormone Release to Generate Novel Calcium Waves in Adipose Tissues.

Nutrient sensing and the subsequent metabolic responses are fundamental functions of animals, closely linked to diseases such as type 2 diabetes and various obesity-related morbidities. Among different metabolic regulatory signals, cytosolic Ca2+ plays pivotal roles in metabolic regulation, including glycolysis, gluconeogenesis, and lipolysis. Recently, intercellular calcium waves (ICWs), the propagation of Ca2+ signaling through tissues, have been found in different systems to coordinate multicellular responses. Nevertheless, our understanding of how ICWs are modulated and operate within living organisms remains limited. In this study, we explore the real-time dynamics, both in organ culture and free-behaving animals, of ICWs in Drosophila larval and adult adipose tissues. We identified Adipokinetic hormone (AKH), the fly functional homolog of mammalian glucagon, as the key factor driving Ca2+ activities in adipose tissue. Interestingly, we found that AKH, which is released in a pulsatile manner into the circulating hemolymph from the AKH-producing neurosecretory cells (APCs) in the brain, stimulates ICWs in the larval fat by a previously unrecognized gap-junction-independent mechanism to promote lipolysis. In the adult fat body, however, gap-junction-dependent random ICWs are triggered by a presumably uniformly diffused AKH. This highlights the stage-specific interplay of hormone secretion, extracellular diffusion, and intercellular communication in the regulation of Ca2+ dynamics. Additionally, we discovered that specific dietary amino acids activate the APCs, leading to increased intracellular Ca2+ and subsequent AKH secretion. Altogether, our findings identify that dietary amino acids regulate the release of AKH peptides from the APCs, which subsequently stimulates novel gap-junction-independent ICWs in adipose tissues, thereby enhancing lipid metabolism.

Wnt signaling modulates the response to DNA damage in the Drosophila wing imaginal disc by regulating the EGFR pathway.

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.

Gut tumors in flies alter the taste valence of an anti-tumorigenic bitter compound.

The sense of taste is essential for survival, as it allows animals to distinguish between foods that are nutritious from those that are toxic. However, innate responses to different tastants can be modulated or even reversed under pathological conditions. Here, we examined whether and how the internal status of an animal impacts taste valence by using Drosophila models of hyperproliferation in the gut. In all three models where we expressed proliferation-inducing transgenes in intestinal stem cells (ISCs), hyperproliferation of ISCs caused a tumor-like phenotype in the gut. While tumor-bearing flies had no deficiency in overall food intake, strikingly, they exhibited an increased gustatory preference for aristolochic acid (ARI), which is a bitter and normally aversive plant-derived chemical. ARI had anti-tumor effects in all three of our gut hyperproliferation models. For other aversive chemicals we tested that are bitter but do not have anti-tumor effects, gut tumors did not affect avoidance behaviors. We demonstrated that bitter-sensing gustatory receptor neurons (GRNs) in tumor-bearing flies respond normally to ARI. Therefore, the internal pathology of gut hyperproliferation affects neural circuits that determine taste valence postsynaptic to GRNs rather than altering taste identity by GRNs. Overall, our data suggest that increased consumption of ARI may represent an attempt at self-medication. Finally, although ARI's potential use as a chemotherapeutic agent is limited by its known toxicity in the liver and kidney, our findings suggest that tumor-bearing flies might be a useful animal model to screen for novel anti-tumor drugs.

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Identification of high sugar diet-induced dysregulated metabolic pathways in muscle using tissue-specific metabolic models in Drosophila.

Individual tissues perform highly specialized metabolic functions to maintain whole-body homeostasis. Although Drosophila serves as a powerful model for studying human metabolic diseases, a lack of tissue-specific metabolic models makes it challenging to quantitatively assess the metabolic processes of individual tissues and disease models in this organism. To address this issue, we reconstructed 32 tissue-specific genome-scale metabolic models (GEMs) using pseudo-bulk single cell transcriptomics data, revealing distinct metabolic network structures across tissues. Leveraging enzyme kinetics and flux analyses, we predicted tissue-dependent metabolic pathway activities, recapitulating known tissue functions and identifying tissue-specific metabolic signatures, as supported by metabolite profiling. Moreover, to demonstrate the utility of tissue-specific GEMs in a disease context, we examined the effect of a high sugar diet (HSD) on muscle metabolism. Together with 13C-glucose isotopic tracer studies, we identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a rate-limiting enzyme in response to HSD. Mechanistically, the decreased GAPDH activity was linked to elevated NADH/NAD+ ratio, caused by disturbed NAD+ regeneration rates, and oxidation of GAPDH. Furthermore, we introduced a pathway flux index to predict and validate additionally perturbed pathways, including fructose and butanoate metabolism. Altogether, our results represent a significant advance in generating quantitative tissue-specific GEMs and flux analyses in Drosophila, highlighting their use for identifying dysregulated metabolic pathways and their regulation in a human disease model.

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.

An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway.

Animals sense and respond to nutrient availability in their environments, a task coordinated in part by the mTOR complex 1 (mTORC1) pathway. mTORC1 regulates growth in response to nutrients and, in mammals, senses specific amino acids through specialized sensors that bind the GATOR1/2 signaling hub. Given that animals can occupy diverse niches, we hypothesized that the pathway might evolve distinct sensors in different metazoan phyla. Whether such customization occurs, and how the mTORC1 pathway might capture new inputs, is unknown. Here, we identify the Drosophila melanogaster protein Unmet expectations (CG11596) as a species-restricted methionine sensor that directly binds the fly GATOR2 complex in a fashion antagonized by S-adenosylmethionine (SAM). We find that in Dipterans GATOR2 rapidly evolved the capacity to bind Unmet and to thereby repurpose a previously independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes to expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise conserved system.

Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness.

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.

Sensing of dietary amino acids and regulation of calcium dynamics in adipose tissues through Adipokinetic hormone in Drosophila.

Nutrient sensing and the subsequent metabolic responses are fundamental functions of animals, closely linked to diseases such as type 2 diabetes and various obesity-related diseases. Drosophila melanogaster has emerged as an excellent model for investigating metabolism and its associated disorders. In this study, we used live-cell imaging to demonstrate that the fly functional homolog of mammalian glucagon, Adipokinetic hormone (AKH), secreted from AKH hormone-producing cells (APCs) in the corpora cardiaca, stimulates intracellular Ca 2+ waves in the larval fat body/adipose tissue to promote lipid metabolism. Further, we show that specific dietary amino acids activate the APCs, leading to increased intracellular Ca 2+ and subsequent AKH secretion. Finally, a comparison of Ca 2+ dynamics in larval and adult fat bodies revealed different mechanisms of regulation, highlighting the interplay of pulses of AKH secretion, extracellular diffusion of the hormone, and intercellular communication through gap junctions. Our study underscores the suitability of Drosophila as a powerful model for exploring real-time nutrient sensing and inter-organ communication dynamics.

Genetic manipulation of an Ixodes scapularis cell line.

Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.

Mechanistic characterization of a Drosophila model of paraneoplastic nephrotic syndrome.

Paraneoplastic syndromes occur in cancer patients and originate from dysfunction of organs at a distance from the tumor or its metastasis. A wide range of organs can be affected in paraneoplastic syndromes; however, the pathological mechanisms by which tumors influence host organs are poorly understood. Recent studies in the fly uncovered that tumor secreted factors target host organs, leading to pathological effects. In this study, using a Drosophila gut tumor model, we characterize a mechanism of tumor-induced kidney dysfunction. Specifically, we find that Pvf1, a PDGF/VEGF signaling ligand, secreted by gut tumors activates the PvR/JNK/Jra signaling pathway in the principal cells of the kidney, leading to mis-expression of renal genes and paraneoplastic renal syndrome-like phenotypes. Our study describes an important mechanism by which gut tumors perturb the function of the kidney, which might be of clinical relevance for the treatment of paraneoplastic syndromes.

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-characterizsed 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.

Lipophorin receptors genetically modulate neurodegeneration caused by reduction of Psn expression in the aging Drosophila brain.

Mutations in the Presenilin (PSEN) genes are the most common cause of early-onset familial Alzheimer's disease (FAD). Studies in cell culture, in vitro biochemical systems, and knockin mice showed that PSEN mutations are loss-of-function mutations, impairing γ-secretase activity. Mouse genetic analysis highlighted the importance of Presenilin (PS) in learning and memory, synaptic plasticity and neurotransmitter release, and neuronal survival, and Drosophila studies further demonstrated an evolutionarily conserved role of PS in neuronal survival during aging. However, molecular pathways that interact with PS in neuronal survival remain unclear. To identify genetic modifiers that modulate PS-dependent neuronal survival, we developed a new DrosophilaPsn model that exhibits age-dependent neurodegeneration and increases of apoptosis. Following a bioinformatic analysis, we tested top ranked candidate genes by selective knockdown (KD) of each gene in neurons using two independent RNAi lines in Psn KD models. Interestingly, 4 of the 9 genes enhancing neurodegeneration in Psn KD flies are involved in lipid transport and metabolism. Specifically, neuron-specific KD of lipophorin receptors, lpr1 and lpr2, dramatically worsens neurodegeneration in Psn KD flies, and overexpression of lpr1 or lpr2 does not alleviate Psn KD-induced neurodegeneration. Furthermore, lpr1 or lpr2 KD alone also leads to neurodegeneration, increased apoptosis, climbing defects, and shortened lifespan. Lastly, heterozygotic deletions of lpr1 and lpr2 or homozygotic deletions of lpr1 or lpr2 similarly lead to age-dependent neurodegeneration and further exacerbate neurodegeneration in Psn KD flies. These findings show that LpRs modulate Psn-dependent neuronal survival and are critically important for neuronal integrity in the aging brain.

Expression Atlas update: insights from sequencing data at both bulk and single cell level.

Expression Atlas (www.ebi.ac.uk/gxa) and its newest counterpart the Single Cell Expression Atlas (www.ebi.ac.uk/gxa/sc) are EMBL-EBI's knowledgebases for gene and protein expression and localisation in bulk and at single cell level. These resources aim to allow users to investigate their expression in normal tissue (baseline) or in response to perturbations such as disease or changes to genotype (differential) across multiple species. Users are invited to search for genes or metadata terms across species or biological conditions in a standardised consistent interface. Alongside these data, new features in Single Cell Expression Atlas allow users to query metadata through our new cell type wheel search. At the experiment level data can be explored through two types of dimensionality reduction plots, t-distributed Stochastic Neighbor Embedding (tSNE) and Uniform Manifold Approximation and Projection (UMAP), overlaid with either clustering or metadata information to assist users' understanding. Data are also visualised as marker gene heatmaps identifying genes that help confer cluster identity. For some data, additional visualisations are available as interactive cell level anatomograms and cell type gene expression heatmaps.

Finding information about uncharacterized Drosophila melanogaster genes.

Genes that have been identified in the genome but remain uncharacterized with regards to function offer an opportunity to uncover novel biological information. Novelty is exciting but can also be a barrier. If nothing is known, how does one start planning and executing experiments? Here, we provide a recommended information-mining workflow and a corresponding guide to accessing information about uncharacterized Drosophila melanogaster genes, such as those assigned only a systematic coding gene identifier. The available information can provide insights into where and when the gene is expressed, what the function of the gene might be, whether there are similar genes in other species, whether there are known relationships to other genes, and whether any other features have already been determined. In addition, available information about relevant reagents can inspire and facilitate experimental studies. Altogether, mining available information can help prioritize genes for further study, as well as provide starting points for experimental assays and other analyses.

Protein phosphatase 1 regulates core PCP signaling.

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

Molecular and functional characterization of the Drosophila melanogaster conserved smORFome.

Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and ∼182 are conserved in plants. We observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts-indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. We conduct an embryonic ribosome profiling experiment and find support for translation of 137 of these smORFs during embryogenesis. We further embark on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity.