Publications

Human cytomegalovirus (HCMV) infection causes severe illness in newborns and immunocompromised patients. Since treatment options are limited there is an unmet need for new therapeutic approaches. Defensins are cationic peptides, produced by various human tissues, which serve as antimicrobial effectors of the immune system. Furthermore, some defensins are proteolytically cleaved, resulting in the generation of smaller fragments with increased activity. Together, this led us to hypothesize that defensin-derived peptides are natural human inhibitors of virus infection with low toxicity. We screened several human defensin HNP4- and HD5-derived peptides and found HD5(1-9) to be antiviral without toxicity at high concentrations. HD5(1-9) inhibited HCMV cellular attachment and thereby entry and was active against primary as well as a multiresistant HCMV isolate. Moreover, cysteine and arginine residues were identified to mediate the antiviral activity of HD5(1-9). Altogether, defensin-derived peptides, in particular HD5(1-9), qualify as promising candidates for further development as a novel class of HCMV entry inhibitors.

Embryonic development is a largely self-organizing process, in which the adult body plan arises from a ball of cells with initially nearly equal potency. The reaction-diffusion theory first proposed by Alan Turing states that the initial symmetry in embryos can be broken by the interplay between two diffusible molecules, whose interactions lead to the formation of patterns. The reaction-diffusion theory provides a valuable framework for self-organized pattern formation, but it has been difficult to relate simple two-component models to real biological systems with multiple interacting molecular species. Recent studies have addressed this shortcoming and extended the reaction-diffusion theory to realistic multi-component networks. These efforts have challenged the generality of previous central tenets derived from the analysis of simplified systems and guide the way to a new understanding of self-organizing processes. Here, we discuss the challenges in modeling multi-component reaction-diffusion systems and how these have recently been addressed. We present a synthesis of new pattern formation mechanisms derived from these analyses, and we highlight the significance of reaction-diffusion principles for developmental and synthetic pattern formation.

Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.

Diffusion is essential for biochemical processes because it dominates molecular movement on small scales. Enzymatic reactions, for example, require fast exchange of substrate and product molecules in the local environment of the enzyme to ensure efficient turnover. On larger spatial scales, diffusion of secreted signaling proteins is thought to limit the spatial extent of tissue differentiation during embryonic development. While it is possible to measure diffusion in vivo, specifically interfering with diffusion processes and testing diffusion models directly remains challenging. The development of genetically encoded nanobodies that bind specific proteins has provided the opportunity to alter protein localization and reduce protein mobility. Here, we extend the nanobody toolbox with a membrane-tethered low-affinity diffusion regulator that can be used to tune the effective diffusivity of extracellular molecules over an order of magnitude in living embryos. This opens new avenues for future applications to functionally interfere with diffusion-dependent processes.

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.

The secreted TGF-β 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.

In order to contribute to the appropriate tissues during development, cells need to know their position within the embryo. This positional information is conveyed by gradients of signaling molecules, termed morphogens, that are produced in specific regions of the embryo and induce concentration-dependent responses in target tissues. Positional information is remarkably robust, and embryos often develop with the correct proportions even if large parts of the embryo are removed. In this Review, we discuss classical embryological experiments and modern quantitative analyses that have led to mechanistic insights into how morphogen gradients adapt, scale and properly pattern differently sized domains. We analyze these experimental findings in the context of mathematical models and synthesize general principles that apply to multiple systems across species and developmental stages.

Fluorescence Recovery After Photobleaching (FRAP) and inverse FRAP (iFRAP) assays can be used to assess the mobility of fluorescent molecules. These assays measure diffusion by monitoring the return of fluorescence in bleached regions (FRAP), or the dissipation of fluorescence from photoconverted regions (iFRAP). However, current FRAP/iFRAP analysis methods suffer from simplified assumptions about sample geometry, bleaching/photoconversion inhomogeneities, and the underlying reaction-diffusion kinetics. To address these shortcomings, we developed the software PyFRAP, which fits numerical simulations of three-dimensional models to FRAP/iFRAP data and accounts for bleaching/photoconversion inhomogeneities. Using PyFRAP we determined the diffusivities of fluorescent molecules spanning two orders of magnitude in molecular weight. We measured the tortuous effects that cell-like obstacles exert on effective diffusivity and show that reaction kinetics can be accounted for by model selection. These applications demonstrate the utility of PyFRAP, which can be widely adapted as a new extensible standard for FRAP analysis.

The best characterized signaling pathway downstream of transforming growth factor β (TGF-β) is through SMAD2 and SMAD3. However, TGF-β 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-β-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-β-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the ID genes. Finally, we show that TGF-β-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-β-induced transcriptional program and physiological responses.

Turing’s theory of pattern formation is a universal model for self-organization, applicable to many systems in physics, chemistry, and biology. Essential properties of a Turing system, such as the conditions for the existence of patterns and the mechanisms of pattern selection, are well understood in small networks. However, a general set of rules explaining how network topology determines fundamental system properties and constraints has not been found. Here we provide a first general theory of Turing network topology, which proves why three key features of a Turing system are directly determined by the topology: the type of restrictions that apply to the diffusion rates, the robustness of the system, and the phase relations of the molecular species.

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.

The H2.0-like homeobox transcription factor (HLX) regulates hematopoietic differentiation and is overexpressed in Acute Myeloid Leukemia (AML), but the mechanisms underlying these functions remain unclear. We demonstrate here that HLX overexpression leads to a myeloid differentiation block both in zebrafish and human hematopoietic stem and progenitor cells (HSPCs). We show that HLX overexpression leads to downregulation of genes encoding electron transport chain (ETC) components and upregulation of PPARδ gene expression in zebrafish and human HSPCs. HLX overexpression also results in AMPK activation. Pharmacological modulation of PPARδ signaling relieves the HLX-induced myeloid differentiation block and rescues HSPC loss upon HLX knockdown but it has no effect on AML cell lines. In contrast, AMPK inhibition results in reduced viability of AML cell lines, but minimally affects myeloid progenitors. This newly described role of HLX in regulating the metabolic state of hematopoietic cells may have important therapeutic implications.

The secreted growth factor Activin-A of the transforming growth factor β 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.