BACKGROUND:Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD+ in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD+ levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants.RESULTS:Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD+ levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos.CONCLUSIONS:Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD+ levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.

BMP signaling has a conserved function in patterning the dorsal-ventral body axis in Bilateria and the directive axis in anthozoan cnidarians. So far, cnidarian studies have focused on the role of different BMP signaling network components in regulating pSMAD1/5 gradient formation. Much less is known about the target genes downstream of BMP signaling. To address this, we generated a genome-wide list of direct pSMAD1/5 target genes in the anthozoan Nematostella vectensis, several of which were conserved in Drosophila and Xenopus. Our ChIP-seq analysis revealed that many of the regulatory molecules with documented bilaterally symmetric expression in Nematostella are directly controlled by BMP signaling. We identified several so far uncharacterized BMP-dependent transcription factors and signaling molecules, whose bilaterally symmetric expression may be indicative of their involvement in secondary axis patterning. One of these molecules is zswim4-6, which encodes a novel nuclear protein that can modulate the pSMAD1/5 gradient and potentially promote BMP-dependent gene repression.

Enhancing cytokine-based therapies by systematically tuning how an agonist associates its receptor is emerging as a powerful new concept in drug discovery. Here, we report the design and characterization of agonists that tune the granulocyte-colony stimulating factor receptor (G-CSFR) activity, which is central for the proliferation and granulocytic differentiation of hematopoietic stem cells. Using design agonists, we study the impact of varying the receptor-binding affinity and dimerization geometry on receptor association, downstream signaling, and cellular response. Hence, we achieved agonists with altered signaling specificities that are hyper-thermostable, can outcompete the native ligand (G-CSF), and bias granulopoietic differentiation over triggering proliferation. Furthermore, the design agonists differentially modulate the kinetics and amplitudes of signal transduction pathways, and gene expression patterns. Unlike G-CSF, they achieve selective activation of hematopoietic functions with minimal undesired immunomodulatory effects. These findings demonstrate the potential of dissecting the complex G-CSFR signaling, and open ways for new therapeutic applications. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Signaling pathways feature multiple interacting ligand and receptor variants, which are thought to act in a combinatorial manner to elicit different cellular responses. Transcriptome analyses now suggest that many signaling pathways use their components in combinations that are surprisingly often shared between otherwise dissimilar cell states.

AbstractEnhancing cytokine-based therapies by systematically tuning how an agonist associates its receptor is emerging as a powerful new concept in drug discovery. Here, we report the design and characterization of agonists that tune the granulocyte-colony stimulating factor receptor (G-CSFR) activity, which is central for the proliferation and granulocytic differentiation of hematopoietic stem cells. Using design agonists, we study the impact of varying the receptor-binding affinity and dimerization geometry on receptor association, downstream signaling, and cellular response. Hence, we achieved agonists with altered signaling specificities that are hyper-thermostable, can outcompete the native ligand (G-CSF), and bias granulopoietic differentiation over triggering proliferation. Furthermore, the design agonists differentially modulate the kinetics and amplitudes of signal transduction pathways, and gene expression patterns. Unlike G-CSF, they achieve selective activation of gene sets with hematopoietic functions with minimal unwanted effects on immunomodulatory signaling. These findings demonstrate the potential of dissecting the complex G-CSFR signaling, and open up ways for new therapeutic applications for designed cytokines.Graphical abstract

In protein design, the energy associated with a huge number of sequence-conformer perturbations has to be routinely estimated. Hence, enhancing the throughput and accuracy of these energy calculations can profoundly improve design success rates and enable tackling more complex design problems. In this work, we explore the possibility of tensorizing the energy calculations and apply them in a protein design framework. We use this framework to design enhanced proteins with anti-cancer and radio-tracing functions. Particularly, we designed multispecific binders against ligands of the epidermal growth factor receptor (EGFR), where the tested design could inhibit EGFR activity in vitro and in vivo. We also used this method to design high-affinity Cu2+ binders that were stable in serum and could be readily loaded with copper-64 radionuclide. The resulting molecules show superior functional properties for their respective applications and demonstrate the generalizable potential of the described protein design approach.

During animal development, embryos undergo complex morphological changes over time. Differences in developmental tempo between species are emerging as principal drivers of evolutionary novelty, but accurate description of these processes is very challenging. To address this challenge, we present here an automated and unbiased deep learning approach to analyze the similarity between embryos of different timepoints. Calculation of similarities across stages resulted in complex phenotypic fingerprints, which carry characteristic information about developmental time and tempo. Using this approach, we were able to accurately stage embryos, quantitatively determine temperature-dependent developmental tempo, detect naturally occurring and induced changes in the developmental progression of individual embryos, and derive staging atlases for several species de novo in an unsupervised manner. Our approach allows us to quantify developmental time and tempo objectively and provides a standardized way to analyze early embryogenesis.

We developed EmbryoNet, a deep learning tool that can automatically identify and classify developmental defects caused by perturbations of signaling pathways in vertebrate embryos. The tool could help to elucidate the mechanisms of action of pharmaceuticals, potentially transforming the drug discovery process.

Evolutionarily conserved signaling pathways are essential for early embryogenesis, and reducing or abolishing their activity leads to characteristic developmental defects. Classification of phenotypic defects can identify the underlying signaling mechanisms, but this requires expert knowledge and the classification schemes have not been standardized. Here we use a machine learning approach for automated phenotyping to train a deep convolutional neural network, EmbryoNet, to accurately identify zebrafish signaling mutants in an unbiased manner. Combined with a model of time-dependent developmental trajectories, this approach identifies and classifies with high precision phenotypic defects caused by loss of function of the seven major signaling pathways relevant for vertebrate development. Our classification algorithms have wide applications in developmental biology and robustly identify signaling defects in evolutionarily distant species. Furthermore, using automated phenotyping in high-throughput drug screens, we show that EmbryoNet can resolve the mechanism of action of pharmaceutical substances. As part of this work, we freely provide more than 2 million images that were used to train and test EmbryoNet.

By combining classical embryological experiments, advanced imaging, and active-particle simulations, a new study reveals how the coupling of signaling and active mechanical forces triggers the collective ordered internalization of mesendoderm cells during gastrulation in zebrafish embryos.

During vertebrate embryogenesis, the germ layers are patterned by secreted Nodal signals. In the classical model, Nodals elicit signaling by binding to a complex comprising Type I/II Activin receptors (Acvr) and the co-receptor Tdgf1. However, it is currently unclear whether receptor binding can also affect the distribution of Nodals themselves through the embryo, and it is unknown which of the putative Acvr paralogs mediate Nodal signaling in zebrafish. Here, we characterize three Type I (Acvr1) and four Type II (Acvr2) homologs and show that – except for Acvr1c – all receptor-encoding transcripts are maternally deposited and present during zebrafish embryogenesis. We generated mutants and used them together with combinatorial morpholino knockdown and CRISPR F0 knockout (KO) approaches to assess compound loss-of-function phenotypes. We discovered that the Acvr2 homologs function partly redundantly and partially independently of Nodal to pattern the early zebrafish embryo, whereas the Type I receptors Acvr1b-a and Acvr1b-b redundantly act as major mediators of Nodal signaling. By combining quantitative analyses with expression manipulations, we found that feedback-regulated Type I receptors and co-receptors can directly influence the diffusion and distribution of Nodals, providing a mechanism for the spatial restriction of Nodal signaling during germ layer patterning.

Protein therapeutics frequently face major challenges, including complicated production, instability, poor solubility, and aggregation. De novo protein design can readily address these challenges. Here, we demonstrate the utility of a topological refactoring strategy to design novel granulopoietic proteins starting from the granulocyte-colony stimulating factor (G-CSF) structure. We change a protein fold by rearranging the sequence and optimising it towards the new fold. Testing four designs, we obtain two that possess nanomolar activity, the most active of which is highly thermostable and protease-resistant, and matches its designed structure to atomic accuracy. While the designs possess starkly different sequence and structure from the native G-CSF, they show specific activity in differentiating primary human haematopoietic stem cells into mature neutrophils. The designs also show significant and specific activity in vivo. Our topological refactoring approach is largely independent of sequence or structural context, and is therefore applicable to a wide range of protein targets.

The hindered diffusion model postulates that the movement of a signaling molecule through an embryo is affected by tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstrated in vivo. Here, we visualize extracellular movement and binding of individual molecules of the activator-inhibitor signaling pair Nodal and Lefty in live developing zebrafish embryos using reflected light-sheet microscopy. We observe that diffusion coefficients of molecules are high in extracellular cavities, whereas mobility is reduced and bound fractions are high within cell-cell interfaces. Counterintuitively, molecules nevertheless accumulate in cavities, which we attribute to the geometry of the extracellular space by agent-based simulations. We further find that Nodal has a larger bound fraction than Lefty and shows a binding time of tens of seconds. Together, our measurements and simulations provide direct support for the hindered diffusion model and yield insights into the nanometer-to-micrometer-scale mechanisms that lead to macroscopic signal dispersal.

The plasma membrane (PM) must be overcome by viruses during entry and release. Furthermore, the PM represents the cellular communication compartment and the immune system interface. Hence, viruses have evolved sophisticated strategies to remodel the PM, for instance to avoid immune sensing and clearance of infected cells. We performed a comprehensive analysis of cell surface dysregulation by two human-pathogenic viruses, human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1), in primary macrophages, which are classical antigen-presenting cells and orchestrators of the immune system. Scanning ion conductance microscopy revealed a loss of roughness and an overall smooth phenotype of HCMV-infected macrophages, in contrast to HIV-1 infection. This phenotype was also evident on the molecular level. When we screened for cell surface receptors modulated by HCMV, 42 of 332 receptors tested were up-or downregulated, whereas HIV-1 affected only 7 receptors. In particular CD164, CD84, and CD180 were targeted by HCMV. Mechanistically, HCMV induced transcriptional silencing of these receptors in an interferon (IFN)-independent manner, and expression was reduced not only by lab adapted HCMV but also by clinical HCMV isolates. Altogether, our plasma membrane profiling of human macrophages provides clues to understand how viruses evade the immune system and identified novel cell surface receptors targeted by HCMV. IMPORTANCE The PM is a key component that viruses have to cope with. It is a barrier for infection and egress and is critically involved in antiviral immune signaling. We hence asked the question how two immunomodulatory viruses, HIV-1 and HCMV, dysregulate this compartment in infected macrophages, relevant in vivo targets of both viruses. We employed a contact-free microscopic technique to image the PM of infected cells and performed a phenotypic flow cytometry-based screen to identify receptor modulations on a molecular level. Our results show that HIV-1 and HCMV differentially manipulate the PM of macrophages. While HIV-1-mediated changes are relatively subtle, HCMV induces major alterations of the PM. We identify novel immune receptors manipulated by HCMV and define mechanisms of how HCMV interferes with receptor expression. Altogether, our study reveals differential strategies of how two human-pathogenic viruses manipulate infected cells and identifies potential novel pathways of HCMV immune evasion.

Abstract The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Protein therapeutics are clinically developed and used as minorly engineered forms of their natural templates. This direct adoption of natural proteins in therapeutic contexts very frequently faces major challenges, including instability, poor solubility, and aggregation, which may result in undesired clinical outcomes. In contrast to classical protein engineering techniques, de novo protein design enables the introduction of radical sequence and structure manipulations, which can be used to address these challenges. In this work, we test the utility of two different design strategies to design novel granulopoietic proteins, using structural information from human granulocyte-colony stimulating factor (hG-CSF) as a template. The two strategies are: (1) An epitope rescaffolding where we migrate a tertiary structural epitope to simpler, idealised, proteins scaffolds (Fig. 1A-C), and (2) a topological refactoring strategy, where we change the protein fold by rearranging connections across the secondary structures and optimised the designed sequence of the new fold (Fig. 1A,D,E). Testing only eight designs, we obtained novel granulopoietic proteins that bind to the G-CSF receptor, have nanomolar activity in cell-based assays, and were highly thermostable and protease-resistant. NMR structure determination showed three designs to match their designed coordinates within less than 2.5 Å. While the designs possessed starkly different sequence and structure from the native G-CSF, they showed very specific activity in differentiating primary human haematopoietic stem cells into fully mature granulocytes. Morever, one design shows significant and specific activity in vivo in zebrafish and mice. These results are prospectively directing us to investigate the role of dimerisation geometry of G-GCSF receptor on activation magnitude and downstream signalling pathways. More broadly, the results also motivate our ongoing work on to design other heamatopoietic agents. In conclusion, our findings highlight the utility of computational protein design as a highly effective and guided means for discovering nover receptor modulators, and to obtain new mechanistic information about the target molecule. Figure 1. Two different strategies to generate superfolding G-CSF designs. (A) X-ray structure of G-CSF (orange) bound to its cognate receptor (red) through its binding epitope (blue). According to the epitope rescaffolding strategy, (B) the critical binding epitope residues were disembodied and used as a geometric search query against the entire Protein Data Bank (PDB) to retrieve structurally compatible scaffolds. The top six compatible scaffolds structures are shown in cartoon representation. (C) The top two templates chosen for sequence design, were a de novo designed coiled-coil and a four-helix bundle with unknown function. The binding epitopes were grafted, and the scaffolds were optimised to rigidly host the guest epitope. (D-E) According to the topological refactoring strategy (D) the topology of the native G-CSF was rewired from around the fixed binding epitope, and then was further mutated to idealise the core residues (blue volume (E)) and residues distal from the binding epitope (orange crust (E)). Both strategies aimed at simplifying the topology, reducing the size, and rigidifying the bound epitope conformation through alternate means. Figure 1 Disclosures No relevant conflicts of interest to declare.