Mai M. Morimoto, Aljoscha Nern, Arthur Zhao, Edward M Rogers, Allan M. Wong, Mathew D Isaacson, Davi D. Bock, Gerald M. Rubin, Michael B. Reiser

Visual systems can exploit spatial correlations in the visual scene by using retinotopy, the organizing principle by which neighboring cells encode neighboring spatial locations. However, retinotopy is often lost, such as when visual pathways are integrated with other sensory modalities. How is spatial information processed in the absence of retinotopy? Here, we focused on visual looming responsive LC6 cells in Drosophila, a population whose dendrites collectively tile the visual field, but whose axons form a single glomerulus--a structure lacking retinotopic organization--in the central brain. We identified multiple glomerulus neurons and found that they respond to looming in different portions of the visual field, unexpectedly preserving spatial information. Through EM reconstruction of all LC6 synaptic inputs to the glomerulus, we found that LC6 and downstream cell types form circuits within the glomerulus that establish spatial readout of visual features and contralateral suppression--mechanisms that transform visual information for behavioral control. ### Competing Interest Statement The authors have declared no competing interest.

Karin D. Prummel, Helena L Crowell, Susan Nieuwenhuize, Eline C. Brombacher, Stephan Daetwyler, Charlotte Soneson, Jelena Kresoja-Rakic, Manuel Ronner, Agnese Kocere, Alexander Ernst, Zahra Labbaf, David E. Clouthier, Anthony B. Firulli, Hector Sanchez-Iranzo, Rebecca O'Rourke, Erez Raz, Nadia Mercader, Alexa Burger, Emanuela Felley-Bosco, Jan Huisken, Mark D Robinson, Christian Mosimann

The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration, and their dysregulation can result in congenital anomalies of the viscera, ventral wall defects, and mesothelioma tumors. Nonetheless, the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here, we combine genetic lineage tracing, in toto live imaging, and single-cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm (LPM). Our single-cell analysis uncovers a post-gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos, we chart the origin of mesothelial progenitors to the lateral-most, hand2-expressing LPM and confirm evolutionary conservation in mouse. Our time-lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation, documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally, hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium, we find de novo expression of LPM-associated transcription factors, and in particular of Hand2, indicating the re-initiation of a developmental transcriptional program in mesothelioma. Taken together, our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2, contributing to our understanding of mesothelial pathologies and mesothelioma.

Chloe Desiree Goldsmith, Damien Cohen, Anaelle Dubois, Maria-Guadaloupe Martinez, Kilian Petitjean, Anne Corlu, Hector Hernandez-Vargas, Isabelle Chemin

Methylation of viral DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues to why certain infections are cleared, or persist with or without progression to cancer. The detection 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. We used cas9 guided RNPs to specifically target and enrich in HBV DNA from infected PHH, prior to sequencing with nanopores. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species. Moreover, using the developed technique, we have provided the first de novo assembly of naive HBV DNA, as well as the first landscape of 5mCpG from naive HBV sequences.

Hongru Wang, Lenore Pipes, Rasmus Nielsen

Human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is most closely related, by average genetic distance, to two coronaviruses isolated from bats, RaTG13 and RmYN02. However, there is a segment of high amino acid similarity between human SARS-CoV-2 and a pangolin isolated strain, GD410721, in the receptor binding domain (RBD) of the spike protein, a pattern that can be caused by either recombination or by convergent amino acid evolution driven by natural selection. We perform a detailed analysis of the synonymous divergence, which is less likely to be affected by selection than amino acid divergence, between human SARS-CoV-2 and related strains. We show that the synonymous divergence between the bat derived viruses and SARS-CoV-2 is larger than between GD410721 and SARS-CoV-2 in the RBD, providing strong additional support for the recombination hypothesis. However, the synonymous divergence between pangolin strain and SARS-CoV-2 is also relatively high, which is not consistent with a recent recombination between them, instead it suggests a recombination into RaTG13. We also find a 14-fold increase in the dN/dS ratio from the lineage leading to SARS-CoV-2 to the strains of the current pandemic, suggesting that the vast majority of non-synonymous mutations currently segregating within the human strains have a negative impact on viral fitness. Finally, we estimate that the time to the most recent common ancestor of SARS-CoV-2 and RaTG13 or RmYN02 based on synonymous divergence, is 51.71 years (95% C.I., 28.11-75.31) and 37.02 years (95% C.I., 18.19-55.85), respectively. ### Competing Interest Statement The authors have declared no competing interest.

Alexander A Cohen, Priyanthi N.P. Gnanapragasam, Yu E. Lee, Susan Ou, Leesa M. Kakutani, Jennifer R Keeffe, Christopher O. Barnes, Hung-Jen Wu, Mark Howarth, Anthony P. West, Michel C. Nussenzweig, Pamela J Bjorkman

Protection against SARS-CoV-2 and SARS-related zoonotic coronaviruses with pandemic potential is urgently needed. To evaluate immunization strategies, we made nanoparticles displaying the receptor-binding domain (RBD) of only SARS-CoV-2 (homotypic nanoparticles) or co-displaying the SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles; 4-8 distinct RBDs). Mice immunized with RBD-nanoparticles, but not soluble antigen, elicited cross-reactive antibody binding and neutralization responses, confirming increased immunogenicity from multimerization. Mosaic-RBD-nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs compared to sera from immunizations with homotypic SARS-CoV-2-RBD-nanoparticles or antibodies from COVID-19 convalescent human plasmas. Moreover, sera from mosaic-RBD-immunized mice neutralized heterologous pseudotyped coronaviruses equivalently or better after priming than sera from homotypic SARS-CoV-2-RBD-nanoparticle immunizations, demonstrating no loss of immunogenicity against any particular RBD resulting from co-display. Thus, a single immunization with mosaic-RBD-nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.

Fabiana Heredia, Yanel Volonté, Joana Pereirinha, Magdalena Fernandez-Acosta, Andreia P. Casimiro, Cláudia G. Belém, Filipe Viegas, Kohtaro Tanaka, Maite Arana, Gisele A. Cardoso, André Macedo, Malwina Kotowicz, Facundo H. Prado Spalm, Marcos J. Dibo, Raquel D. Monfardini, Tatiana T. Torres, César S. Mendes, Andres Garelli, Alisson M. Gontijo

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of fly larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling. ### Competing Interest Statement The authors have declared no competing interest.

Y. Sun, H Yue, C Copos, K Zhu, Y. Zhang, X. Gao, B Reid, F Lin, Min Zhao, A Mogilner

Motile cells migrate directionally in the electric field in a process known as galvanotaxis. Galvanotaxis is important in wound healing, development, cell division, and nerve growth. Different cell types migrate in opposite directions in electric fields, to either cathode, or anode, and the same cell can switch the directionality depending on chemical conditions. We previously reported that individual fish keratocyte cells sense electric fields and migrate to the cathode, while inhibition of PI3K reverses single cells to the anode. Many physiological processes rely on collective, not individual, cell migration, so here we report on directional migration of cohesive cell groups in electric fields. Uninhibited cell groups of any size move to the cathode, with speed decreasing and directionality increasing with the group size. Surprisingly, large groups of PI3K-inhibited cells move to the cathode, in the direction opposite to that of individual cells, which move to the anode, while such small groups are not persistently directional. In the large groups, cells' velocities are distributed unevenly: the fastest cells are at the front of the uninhibited groups, but at the middle and rear of the PI3K-inhibited groups. Our results are most consistent with the hypothesis, supported by the computational model, that cells inside and at the edge of the groups interpret directional signals differently. Namely, cells in the group interior are directed to the cathode independently of their chemical state. Meanwhile, edge cells behave like the individual cells: they are directed to the cathode/anode in uninhibited/PI3K-inhibited groups, respectively. As a result, all cells drive uninhibited groups to the cathode, but a mechanical tug-of-war between the inner and edge cells directs large PI3K-inhibited groups with cell majority in the interior to the cathode, while rendering small groups non-directional.

Pieter J. K. Aukes, Sherry L. Schiff, Jason J Venkiteswaran, Richard J. Elgood, John Spoelstra

Dissolved Organic Matter (DOM) represents a mixture of organic molecules that vary due to different source materials and degree of processing. Characterizing how DOM composition evolves along the aquatic continuum can be difficult. Using a size-exclusion chromatography technique (LC-OCD), we assessed the variability in DOM composition from both surface and groundwaters across a number of Canadian ecozones (mean annual temperature spanning -10 to +6 C). A wide range in DOM concentration was found from 0.2 to 120 mg C/L. Proportions of different size-based groupings across ecozones were variable, yet similarities between specific water-body types, regardless of location, suggest commonality in the processes dictating DOM composition. A PCA identified 70% of the variation in LC-OCD derived DOM compositions could be explained by the water-body type. We find that DOM composition within a specific water-body type is similar regardless of the differences in climate or surrounding vegetation where the sample originated from.

Christopher P. Lapointe, Rosslyn Grosely, Alex G. Johnson, Jinfan Wang, Israel S. Fernández, Joseph D. Puglisi

SARS-CoV-2 recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 associates with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel. ### Competing Interest Statement The authors have declared no competing interest.

Nora Czekalla, Janine Baumann, David S Stolz, Annalina V Mayer, Johanna F Voges, Lena Rademacher, Frieder Michel Paulus, Sören Krach, Laura Müller-Pinzler

Being confronted with social-evaluative stress elicits a physiological and a psychological stress response. This calls for regulatory processes to manage negative affect and maintain self-related optimistic beliefs. The aim of the current study was to investigate the affect-regulating potential of self-related belief updating after exposure to social-evaluative stress, in comparison to non-social physical stress or no stress. We assessed self-related belief updating using trial-by-trial performance feedback and described the updating behavior in a mechanistic way using computational modeling. We found that social-evaluative stress was accompanied by an increase in cortisol and negative affect which was related to a shift in self-related belief updating towards the positive direction. This self-beneficial belief updating, which was absent after physical stress or control, was associated with a better recovery from stress-induced negative affect. This indicates that enhanced integration of positive self-related feedback can act as a coping strategy to deal with social-evaluative stress.

Madeline M Keenen, David Brown, Lucy D Brennan, Roman Renger, Harrison Khoo, Christopher R. Carlson, Bo Huang, Stephan W. Grill, Geeta J. Narlikar, Sy Redding

In mammals HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog's DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale. ### Competing Interest Statement The authors have declared no competing interest.

Daniel O'Hagan, Amy Ralston

Fluorescent proteins and epitope tags can reveal protein localization in cells and animals. However, the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. We present two approaches for higher throughput production of endogenous protein reporters. Our first approach makes use of a split fluorescent protein mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest was highly efficient in embryos, eliminating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. However, we report a threshold of detection using this approach. By contrast, the V5 epitope enabled high efficiency and higher sensitivity protein reporting. We describe complementary advantages and prospective applications of these two approaches. ### Competing Interest Statement The authors have declared no competing interest.

Micha Heilbron, Kristijan Armeni, Jan-Mathijs Schoffelen, Peter Hagoort, Floris P de Lange

Understanding spoken language requires transforming ambiguous stimulus streams into a hierarchy of increasingly abstract representations, ranging from speech sounds to meaning. It has been suggested that the brain uses predictive computations to guide the interpretation of incoming information. However, the exact role of prediction in language understanding remains unclear, with widespread disagreement about both the ubiquity of prediction, and the level of representation at which predictions unfold. Here, we address both issues by analysing brain recordings of participants listening to audiobooks, and using a state-of-the-art deep neural network (GPT-2) to quantify predictions in a fine-grained, contextual fashion. First, we establish clear evidence for predictive processing, confirming that brain responses to words are modulated by probabilistic predictions. Next, we factorised the model-based predictions into distinct linguistic dimensions, revealing dissociable neural signatures of syntactic, phonemic and semantic predictions. Finally, we show that high-level (word) predictions inform low-level (phoneme) predictions, supporting theories of hierarchical predictive processing. Together, these results underscore the ubiquity of prediction in language processing, and demonstrate that linguistic prediction is not implemented by a single system but occurs throughout the language network, forming a hierarchy of linguistic predictions across all levels of analysis.

Umakanta Swain, Urmila Sehrawat, Avital Sarusi-Portuguez, Gilgi Friedlander, Ron Rotkopf, Charlotte Ebert, Tamar Paz-Elizur, Rivka Dikstein, Thomas Carell, Nicholas Geacintov, Zvi Livneh

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here we focus on its multilayer regulation of translesion DNA synthesis (TLS), in which DNA lesions are bypassed by error-prone DNA polymerases. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase {eta} and RAD18 E3 ligase, which guides the polymerase to replication stalling sites, and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD, and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase {eta}, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

Haichao Guo, Habtamu Ayalew, Anand Seethepalli, Kundan Dhakal, Marcus D Griffiths, Xue-feng Ma, Larry M. York

The root economics space is a useful framework for plant ecology, but rarely considered for crop ecophysiology. In order to understand root trait integration in winter wheat, we combined functional phenomics with trait economic theory utilizing genetic variation, high-throughput phenotyping, and multivariate analyses. We phenotyped a diversity panel of 276 genotypes for root respiration and architectural traits using a novel high-throughput method for CO2 flux and the open-source software RhizoVision Explorer for analyzing scanned images. We uncovered substantial variation for specific root respiration (SRR) and specific root length (SRL), which were primary indicators of root metabolic and construction costs. Multiple linear regression estimated that lateral root tips had the greatest SRR, and the residuals of this model were used as a new trait. SRR was negatively correlated with plant mass. Network analysis using a Gaussian graphical model identified root weight, SRL, diameter, and SRR as hub traits. Univariate and multivariate genetic analyses identified genetic regions associated with aspects of the root economics space, with underlying gene candidates. Combining functional phenomics and root economics is a promising approach to understand crop ecophysiology. We identified root traits and genomic regions that could be harnessed to breed more efficient crops for sustainable agroecosystems. ### Competing Interest Statement The authors have declared no competing interest.

Wei-Tang Chang, Stephanie Kristina Langella, Yichuan Tang, Han c Zhang, Pew-Thian Yap, Kelly Sullivan Giovanello, Weili Lin

The hippocampus is critical for learning and memory and may be separated into anatomically-defined hippocampal subfields (aHPSFs). Many studies have shown that aHPSFs, and their respective functional networks, are differentially vulnerable to a variety of disorders. Hippocampal functional networks, particularly during resting state, are generally analyzed using aHPSFs as seed regions, with the underlying assumption that the function within a subfield is homogeneous, yet heterogeneous between subfields. However, several prior studies that have utilized aHPSFs and assessed brain-wide cortical connectivity have observed similar resting-state functional connectivity profiles between aHPSFs. Alternatively, data-driven approaches offer a means to investigate hippocampal functional organization without a priori assumptions. However, insufficient spatial resolution may lead to partial volume effects at the boundaries of hippocampal subfields, resulting in a number of caveats concerning the reliability of the results. Hence, we developed a functional Magnetic Resonance Imaging (fMRI) sequence on a 7T MR scanner achieving 0.94 mm isotropic resolution with a TR of 2s and brain-wide coverage to 1) investigate whether hippocampal functional segmentation with ultrahigh-resolution data demonstrate similar anatomical, lamellar structures in the hippocampus, and 2) define and compare the brain-wide FC associated with fine-grained aHPSFs and functionally-defined hippocampal subfields (fHPSFs). Using a spatially restricted hippocampal Independent Component Analysis (ICA) approach, this study showed that fHPSFs were arranged along the longitudinal axis of the hippocampus that were not comparable to the lamellar structures of aHPSFs. Contrary to the anatomically defined hippocampal subfields which are bilaterally symmetrical, 13 out of 20 fHPSFs were unilateral. For brain-wide FC, the fHPSFs rather than aHPSFs revealed that a number of fHPSFs connected specifically with some of the functional networks. The visual and sensorimotor networks preferentially connected with different portions of CA1, CA3 and CA4/DG. The DMN was also found to connect more extensively with posterior subfields rather than anterior subfields. Finally, the frontoparietal network (FPN) was anticorrelated with the head portion of CA1. The investigation of functional networks associated with the fHPSFs may enhance the sensitivity of biomarkers for a range of neurological disorders, as network-based approaches take into account disease-related alterations in brain-wide interconnections rather than measuring the regional changes of hippocampus.

Hitesh Mistry

Despite the efforts of many within the drug development and clinical community surrogate biomarkers for patient survival have remained elusive in Oncology. This failure in part is attributable to there being a paucity of clinical trials showing a treatment effect on patient survival. Given this issue an alternative system to explore the surrogacy potential of biomarkers are large preclinical xenograft studies i.e. panel of patient derived xenografts or mouse clinical trials. In this study we explored the surrogacy potential of tumour burden biomarkers, current size of tumour and how its changed, preclinically in a large patient derived xenograft database which contains a diverse number of drugs/treatments (n=61) and xenografts (n=245). We found that of the possible 1830 two-arm mouse trials, 1103 showed a treatment effect on the preclinical end-point, disease progression, (p<0.05). Of these only in 30% did tumour burden markers fully capture the treatment effect on disease progression times i.e. satisfied a key condition for surrogacy. These results highlight that preclinically it is very challenging to find a surrogate marker based purely on measures of tumour burden.

Samuel E Ross, Allegra Angeloni, Alex de Mendoza, Ozren Bogdanovic

In vertebrates, DNA methylation predominantly occurs at CG dinucleotides even though widespread non-CG methylation (mCH) has been reported in mammalian embryonic and neural cells. Unlike in mammals, where mCH is found enriched at CAC/G trinucleotides and is tissue-restricted, we find that zebrafish embryos as well as adult somatic and germline tissues display robust methylation enrichment at TGCT positions associated with mosaic satellite repeats. These repeats reside in H3K9me3-marked heterochromatin and display mCH reprogramming coincident with zygotic genome activation. Altogether, this work provides insight into a novel form of vertebrate mCH and highlights the substrate diversity of vertebrate DNA methyltransferases. ### Competing Interest Statement The authors have declared no competing interest.

Yue Wu, Kathryn S. Burch, Andrea Ganna, Paivi Pajukanta, Bogdan Pasaniuc, Sriram Sankararaman

Genetic correlation is an important parameter in efforts to understand the relationships among complex traits. Current methods that analyze individual genotype data for estimating genetic correlation are challenging to scale to large datasets. Methods that analyze summary data, while being computationally efficient, tend to yield estimates of genetic correlation with reduced precision. We propose SCORE, a randomized method of moments estimator of genetic correlation that is both scalable and accurate. SCORE obtains more precise estimates of genetic correlations relative to summary-statistic methods that can be applied at scale achieving a 50% reduction in standard error relative to LD-score regression (LDSC) and a 26% reduction relative to high-definition likelihood (HDL) (averaged over all simulations).The efficiency of SCORE enables computation of genetic correlations on the UK biobank dataset consisting of ~300K individuals and ~500K SNPs in a few hours (orders of magnitude faster than methods that analyze individual data such as GCTA). Across 780 pairs of traits in 291,273 unrelated white British individuals in the UK Biobank, SCORE identifies significant genetic correlations between 200 additional pairs of traits over LDSC (beyond the 245 pairs identified by both).

Nicola Baker, Carolina M.C Catta-Preta, Rachel Neish, Jovana Sadlova, Ben Powell, Eliza V.C. Alves-Ferreira, Vincent Geohegan, Juliana B.T. Carnielli, Katherine Newling, Charlotte Hughes, Barbora Vojtkova, Jayanthi Anand, Andrei Mihut, Pegine Walrad, Laurence G. Wilson, Jon W. Pitchford, Petr Volf, Jeremy C Mottram

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here we applied kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 were dispensable, 44 protein kinase genes were refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering revealed functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.