Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator.

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.

Optogenetic Signaling Activation in Zebrafish Embryos.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Regulation of Nodal signaling propagation by receptor interactions and positive feedback.

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.

Building a body is complicated. Cells must organise themselves head-to-tail, belly-to-back, and inside-to-outside. They do this by laying down a chemical map, which is made up of gradients of molecular signals, high in some places and lower in others. The amount of signal each cell receives helps to decide which part of the body it will become. One of the essential signals in developing vertebrates is Nodal. It helps cells to tell inside from outside and left from right. Cells detect Nodal using an activin receptor and co-receptor complex, which catch hold of passing Nodal proteins and transmit developmental signals into cells. An important model to study Nodal signals is the zebrafish embryo, but the identity of the activin receptors and their exact role in this organism has been unclear. To find out more, Preiß, Kögler, Mörsdorf et al. studied the activin receptors Acvr1 and Acvr2 in zebrafish embryos. The experiments revealed that two putative Acvr1 and four Acvr2 receptors were present during early development. To better understand their roles, Preiß et al. eliminated them one at a time, and in combination. Losing single activin receptors had no effect. But losing both Acvr1 receptors together stopped Nodal signalling and changed the distribution of the Nodal gradient. Loss of all Acvr2 receptors also caused developmental problems, but they were partly independent of Nodal. This suggests that Acvr1s seem to be able to transmit signals and to shape the Nodal gradient, and that Acvr2s might have another, so far unknown, role. Nodal signals guide the development of all vertebrates. Understanding how they work in a model species like zebrafish could shed light on their role in other species, including humans. A clearer picture could help to uncover what happens at a molecular level when development goes wrong.

A topological refactoring design strategy yields highly stable granulopoietic proteins.

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.

Sustained impact of an academic-practice partnership.

Academic-practice partnerships foster innovation and transition to nursing practice in healthcare systems. The purpose of this paper is to describe the impact of a public-private academic-practice partnership for Doctor of Nursing Practice (DNP) education designed to transform a large healthcare system's nursing workforce and model of care. The conceptual framework is organized around Rogers's (2003) principles of diffusion of innovation in organizations. A logic model illuminates how inputs, activities, outputs and outcomes resulted in sustained impact for graduates, the college and the healthcare organization. Partnership outcomes include education of baccalaureate and master's-prepared employed nurses (n = 95) in a DNP program for advanced practice nursing (APN) roles in the healthcare system; dissemination of scholarship; and revision of the healthcare system's research approval process. Sustained impact includes advancement of DNP-prepared graduates to complex leadership and practice roles; development of new programs and advanced practice roles based on scholarly project findings; expansion of population-specific patient programs; and extension of continuum- and access-to-care models in the healthcare organization. Recommendations include continuing development of academic-practice partnerships for transition to practice and advancement of roles and levels of champions to achieve sustained impact of academic-practice partnerships in healthcare organizations.

Design of novel granulopoietic proteins by topological rescaffolding.

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

Optogenetic investigation of BMP target gene expression diversity.

Signaling molecules activate distinct patterns of gene expression to coordinate embryogenesis, but how spatiotemporal expression diversity is generated is an open question. In zebrafish, a BMP signaling gradient patterns the dorsal-ventral axis. We systematically identified target genes responding to BMP and found that they have diverse spatiotemporal expression patterns. Transcriptional responses to optogenetically delivered high- and low-amplitude BMP signaling pulses indicate that spatiotemporal expression is not fully defined by different BMP signaling activation thresholds. Additionally, we observed negligible correlations between spatiotemporal expression and transcription kinetics for the majority of analyzed genes in response to BMP signaling pulses. In contrast, spatial differences between BMP target genes largely collapsed when FGF and Nodal signaling were inhibited. Our results suggest that, similar to other patterning systems, combinatorial signaling is likely to be a major driver of spatial diversity in BMP-dependent gene expression in zebrafish.

Optogenetic approaches to investigate spatiotemporal signaling during development.

Embryogenesis is coordinated by signaling pathways that pattern the developing organism. Many aspects of this process are not fully understood, including how signaling molecules spread through embryonic tissues, how signaling amplitude and dynamics are decoded, and how multiple signaling pathways cooperate to pattern the body plan. Optogenetic approaches can be used to address these questions by providing precise experimental control over a variety of biological processes. Here, we review how these strategies have provided new insights into developmental signaling and discuss how they could contribute to future investigations.

A conserved regulatory program initiates lateral plate mesoderm emergence across chordates.

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

Nodal and BMP dispersal during early zebrafish development.

The secreted TGF-ß superfamily signals Nodal and BMP coordinate the patterning of vertebrate embryos. Nodal specifies endoderm and mesoderm during germ layer formation, and BMP specifies ventral fates and patterns the dorsal/ventral axis. Five major models have been proposed to explain how the correct distributions of Nodal and BMP are achieved within tissues to orchestrate embryogenesis: source/sink, transcriptional determination, relay, self-regulation, and shuttling. Here, we discuss recent experiments probing these signal dispersal models, focusing on early zebrafish development.

Scale-invariant patterning by size-dependent inhibition of Nodal signalling.

Individuals can vary substantially in size, but the proportions of their body plans are often maintained. We generated smaller zebrafish by removing 30% of their cells at the blastula stages and found that these embryos developed into normally patterned individuals. Strikingly, the proportions of all germ layers adjusted to the new embryo size within 2 hours after cell removal. As Nodal-Lefty signalling controls germ-layer patterning, we performed a computational screen for scale-invariant models of this activator-inhibitor system. This analysis predicted that the concentration of the highly diffusive inhibitor Lefty increases in smaller embryos, leading to a decreased Nodal activity range and contracted germ-layer dimensions. In vivo studies confirmed that Lefty concentration increased in smaller embryos, and embryos with reduced Lefty levels or with diffusion-hindered Lefty failed to scale their tissue proportions. These results reveal that size-dependent inhibition of Nodal signalling allows scale-invariant patterning.

TGF-ß uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition.

The best characterized signaling pathway downstream of transforming growth factor ß (TGF-ß) is through SMAD2 and SMAD3. However, TGF-ß also induces phosphorylation of SMAD1 and SMAD5, but the mechanism of this phosphorylation and its functional relevance is not known. Here, we show that TGF-ß-induced SMAD1/5 phosphorylation requires members of two classes of type I receptor, TGFBR1 and ACVR1, and establish a new paradigm for receptor activation where TGFBR1 phosphorylates and activates ACVR1, which phosphorylates SMAD1/5. We demonstrate the biological significance of this pathway by showing that approximately a quarter of the TGF-ß-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the ID genes. Finally, we show that TGF-ß-induced epithelial-to-mesenchymal transition requires signaling via both the SMAD3 and SMAD1/5 pathways, with SMAD1/5 signaling being essential to induce ID1. Therefore, combinatorial signaling via both SMAD pathways is essential for the full TGF-ß-induced transcriptional program and physiological responses.

Nodal patterning without Lefty inhibitory feedback is functional but fragile.

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhibitor Lefty is required for development. We find that zebrafish lefty mutants exhibit excess Nodal signaling and increased specification of mesendoderm, resulting in embryonic lethality. Strikingly, development can be fully restored without feedback: Lethal patterning defects in lefty mutants can be rescued by ectopic expression of lefty far from its normal expression domain or by spatially and temporally uniform exposure to a Nodal inhibitor drug. While drug-treated mutants are less tolerant of mild perturbations to Nodal signaling levels than wild type embryos, they can develop into healthy adults. These results indicate that patterning without inhibitory feedback is functional but fragile.

Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling.

Toddler/Apela/Elabela is a conserved secreted peptide that regulates mesendoderm development during zebrafish gastrulation. Two non-exclusive models have been proposed to explain Toddler function. The 'specification model' postulates that Toddler signaling enhances Nodal signaling to properly specify endoderm, whereas the 'migration model' posits that Toddler signaling regulates mesendodermal cell migration downstream of Nodal signaling. Here, we test key predictions of both models. We find that in toddler mutants Nodal signaling is initially normal and increasing endoderm specification does not rescue mesendodermal cell migration. Mesodermal cell migration defects in toddler mutants result from a decrease in animal pole-directed migration and are independent of endoderm. Conversely, endodermal cell migration defects are dependent on a Cxcr4a-regulated tether of the endoderm to mesoderm. These results suggest that Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling and indirectly affects endodermal cell migration via Cxcr4a-signaling.

Dynamics of BMP signaling and distribution during zebrafish dorsal-ventral patterning.

During vertebrate embryogenesis, dorsal-ventral patterning is controlled by the BMP/Chordin activator/inhibitor system. BMP induces ventral fates, whereas Chordin inhibits BMP signaling on the dorsal side. Several theories can explain how the distributions of BMP and Chordin are regulated to achieve patterning, but the assumptions regarding activator/inhibitor diffusion and stability differ between models. Notably, 'shuttling' models in which the BMP distribution is modulated by a Chordin-mediated increase in BMP diffusivity have gained recent prominence. Here, we directly test five major models by measuring the biophysical properties of fluorescently tagged BMP2b and Chordin in zebrafish embryos. We found that BMP2b and Chordin diffuse and rapidly form extracellular protein gradients, Chordin does not modulate the diffusivity or distribution of BMP2b, and Chordin is not required to establish peak levels of BMP signaling. Our findings challenge current self-regulating reaction-diffusion and shuttling models and provide support for a graded source-sink mechanism underlying zebrafish dorsal-ventral patterning.

Paracrine Activin-A Signaling Promotes Melanoma Growth and Metastasis through Immune Evasion.

The secreted growth factor Activin-A of the transforming growth factor ß family and its receptors can promote or inhibit several cancer hallmarks including tumor cell proliferation and differentiation, vascularization, lymphangiogenesis and inflammation. However, a role in immune evasion and its relationship with tumor-induced muscle wasting and tumor vascularization, and the relative contributions of autocrine versus paracrine Activin signaling remain to be evaluated. To address this, we compared the effects of truncated soluble Activin receptor IIB as a ligand trap, or constitutively active mutant type IB receptor versus secreted Activin-A or the related ligand Nodal in mouse and human melanoma cell lines and tumor grafts. We found that although cell-autonomous receptor activation arrested tumor cell proliferation, Activin-A secretion stimulated melanoma cell dedifferentiation and tumor vascularization by functional blood vessels, and it increased primary and metastatic tumor burden and muscle wasting. Importantly, in mice with impaired adaptive immunity, the tumor-promoting effect of Activin-A was lost despite sustained vascularization and cachexia, suggesting that Activin-A promotes melanoma progression by inhibiting antitumor immunity. Paracrine Activin-A signaling emerges as a potential target for personalized therapies, both to reduce cachexia and to enhance the efficacy of immunotherapies.

Measuring protein stability in living zebrafish embryos using fluorescence decay after photoconversion (FDAP).

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Morphogen transport.

The graded distribution of morphogens underlies many of the tissue patterns that form during development. How morphogens disperse from a localized source and how gradients in the target tissue form has been under debate for decades. Recent imaging studies and biophysical measurements have provided evidence for various morphogen transport models ranging from passive mechanisms, such as free or hindered extracellular diffusion, to cell-based dispersal by transcytosis or cytonemes. Here, we analyze these transport models using the morphogens Nodal, fibroblast growth factor and Decapentaplegic as case studies. We propose that most of the available data support the idea that morphogen gradients form by diffusion that is hindered by tortuosity and binding to extracellular molecules.

Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system.

Biological systems involving short-range activators and long-range inhibitors can generate complex patterns. Reaction-diffusion models postulate that differences in signaling range are caused by differential diffusivity of inhibitor and activator. Other models suggest that differential clearance underlies different signaling ranges. To test these models, we measured the biophysical properties of the Nodal/Lefty activator/inhibitor system during zebrafish embryogenesis. Analysis of Nodal and Lefty gradients revealed that Nodals have a shorter range than Lefty proteins. Pulse-labeling analysis indicated that Nodals and Leftys have similar clearance kinetics, whereas fluorescence recovery assays revealed that Leftys have a higher effective diffusion coefficient than Nodals. These results indicate that differential diffusivity is the major determinant of the differences in Nodal/Lefty range and provide biophysical support for reaction-diffusion models of activator/inhibitor-mediated patterning.

Morphogen gradients: from generation to interpretation.

Morphogens are long-range signaling molecules that pattern developing tissues in a concentration-dependent manner. The graded activity of morphogens within tissues exposes cells to different signal levels and leads to region-specific transcriptional responses and cell fates. In its simplest incarnation, a morphogen signal forms a gradient by diffusion from a local source and clearance in surrounding tissues. Responding cells often transduce morphogen levels in a linear fashion, which results in the graded activation of transcriptional effectors. The concentration-dependent expression of morphogen target genes is achieved by their different binding affinities for transcriptional effectors as well as inputs from other transcriptional regulators. Morphogen distribution and interpretation are the result of complex interactions between the morphogen and responding tissues. The response to a morphogen is dependent not simply on morphogen concentration but also on the duration of morphogen exposure and the state of the target cells. In this review, we describe the morphogen concept and discuss the mechanisms that underlie the generation, modulation, and interpretation of morphogen gradients.