Max Planck Institute for Molecular Biomedicine

Tendons are sparsely vascularized connective tissues that link muscles to bones, withstanding some of the highest mechanical stresses in the body. Mechanical overloading and tissue hypervascularity are implicated in tendinopathy, a common musculoskeletal disorder, yet their mechanistic roles remain unclear. Here, we identify hypoxia-inducible factor 1α (HIF1α) as not only a marker but also a driver of tendinopathy. Histological and multiomics evaluation of human tendinopathic samples revealed extensive extracellular matrix remodeling, including pathological collagen cross-linking coinciding with active hypoxic signaling. Hypothesizing a causal contribution of hypoxia signaling, we generated mice with tenocyte-targeted deletions of the von Hippel-Lindau (Vhl) gene, which controls hypoxia signaling by regulating HIFα degradation. Vhl inactivation was sufficient to induce pathological hallmarks of tendinopathy, such as collagen matrix disorganization, cross-linking, altered mechanics, and neurovascular ingrowth. This phenotype was HIF1α dependent given that codeleting HIF1α rescued tendon morphology and mechanics. Moreover, deleting vascular endothelial growth factor A (Vegfa) alongside VHL effectively suppressed neovascularization but failed to rescue extracellular matrix abnormalities or restore mechanical function, emphasizing a direct role of HIF1α in driving tendon disease independently of angiogenesis. Mechanistically, we found that HIF1α activation was strain dependent in primary cultured human tendon cells and induced by mechanical overload in murine tendon explants. Furthermore, genetically removing Hif1α from tenocytes prevented aberrant tendon remodeling in response to chronic overload. These findings position HIF1α signaling as a central driver of tendinopathy that acts through a maladaptive tissue response to chronic overload, providing mechanistic insights that could be leveraged for therapeutic approaches.

Abstract Study question What does a secreted oocyte-specific protein, ZP2, do inside mouse embryos during cleavage? Summary answer Contrary to prevalent notion, ZP2 protein synthesis is not silenced after ovulation, but goes on in preimplantation embryos to support the morula-blastocyst transition What is known already A tenet of reproductive biology is that the expression of oocyte-specific genes must remain silent in embryos. Among such genes, those of the ZP encode glycoproteins that are secreted until end of oogenesis and tasked with extracellular roles during folliculogenesis and fertilization. No further synthesis of ZP proteins is thought to occur after ovulation. However, already since the mouse gene knockout was reported in 2001 it was noticed that ZP2 null oocytes failed to develop to term even when fertilized correctly (PMID 11245577). Likewise, rabbit oocytes in which ZP4 was mutated at pronuclear stage featured defective embryogenesis (PMID 31635692). Study design, size, duration This is a treatment vs control study conducted in wild-type mice. For profiling of ZP2 protein expression,zygotes were produced in vivo and cultured 4 days in the presence of natural vs isotopic amino acids, and compared to embryos grown in vivo. For analysis of ZP2 function, zygotic knock-down of ZP2 was compared with that of a protein not present in wild-type embryos (green fluorescent protein, GFP), followed by transfer of both groups to uterus. Participants/materials, setting, methods B6C3F1 female mice were stimulated with gonadotropins and mated to CD1 males to produce zygotes. These were labeled continuously in vitro or in vivo using isotopic Arginine + Lysine, and sampled at consecutive stages for monoclonal immunofluorescence and mass spectrometry. To achieve knockdown of ZP2 during embryogenesis, zygotes were microinjected with Trim-Away reagents (PMID 29153837) and/or morpholino oligonucleotide anti-ZP2, followed by scoring of cleavage rates, blastocyst quality (germ layers) and transcriptome and proteome compositions. Main results and the role of chance The most abundant protein of the mouse zona pellucida, ZP2, not only was still present inside embryos up to blastocyst, but also became significantly more abundant in the 4-8 cell stage, as determined by direct immunofluorescence with monoclonal antibody (+25%, p < 0.05, Wilcoxon test). Mass spectrometry after isotopic labeling of protein synthesis during embryo culture confirmed that ZP2 incorporated the label, meaning: de novo protein synthesis of an oocyte-specific gene product in embryos. Zygotic knockdown via morpholino oligonucleotides and Trim-Away resulted in 50% reduction of ZP2 abundance at the 8-cell stage (p < 0.05, Wilcoxon test), accompanied by growth retardation at the 8-cell and morula stage. Embryos with depletion of ZP2 formed blastocysts with significantly smaller trophectoderm and primitive endoderm compartments. These effects were not observed in controls subjected to mock knockdown (GFP). Consistent with these morphometric data, transcriptome and proteome analyses returned ‘embryonic lethality’ and ‘embryonic growth retardation’ among the over-represented gene ontology terms of ZP2 (but not GFP) knockdown embryos. Thus, intra-embryonic ZP2 appears necessary to keep up the pace of cleavage progression and secure blastocyst fitness. This discovery can explain the historical conundrum of why ZP2 null oocytes formed blastocysts and yet these could not develop to birth (PMID 11245577). Limitations, reasons for caution Although there is sufficient evidence for expanding on the tasks of ZP proteins in mouse development, human oocytes contain ZP4 in addition to ZP1-3. While the novel tasks of ZP2 in mice hinge on de novo protein synthesis, it is unclear if this occurs also in humans. Wider implications of the findings Our study provides novel insight into the pathogenic mechanism of ZP gene mutations, adding a novel intracellular route to the longstanding extracellular roles. If ZP proteins function inside human oocytes and embryos, then using ICSI to overcome problems of thick or thin zona pellucida remains ineffective. Trial registration number No

Abstract VEcadherin is a major component of endothelial adherens junctions and pivotal to the regulation of vascular barrier integrity. Whereas two phosphorylation sites of VEcadherin (Y685 and Y731) are known to be relevant for the regulation of endothelial junctions in vivo, several others were suggested to be relevant based on in vitro studies. Here, we analyze for two of these, serine 665 (S665) and tyrosine 658 (Y658), whether they are relevant for the induction of vascular permeability in vivo. To this end, we generated and characterized two point-mutated VEcadherin knockin mouse lines where either S665 was replaced by valine (S665V) or Y658 by phenylalanine (Y658F). We found that the induction of vascular permeability by histamine or VEGF in the skin was clearly reduced in S665V mice, whereas Y658F mice showed a normal increase of permeability. In line with this, we found that histamine-induced endocytosis was impaired for the VEcadherin-S665V mutant, but not for the Y658F mutant. Comparing the regulation of VEcadherin phosphorylation at S665, Y658 and Y685, we found that only phosphorylation of S665 and Y685 were strongly induced by inflammatory mediators, while phosphorylation of Y658 increased weakly. Interestingly, phosphorylation of S665 and Y685 occurred with different kinetics, but independent of each other. Collectively, our results demonstrate that Y658 is irrelevant for vascular leak formation under inflammatory conditions and establish S665 of VEcadherin as an important phosphorylation site regulating the induction of endothelial permeability in vivo.

Abstract Vascular endothelial growth factor (VEGF), a major regulator of blood vessel formation, is naturally bound to heparan sulfate proteoglycans in the extracellular matrix (ECM). Yet, how the physical presentation of VEGF by the matrix impacts its signaling potential remains fully unknown. To address this question, we have developed a tunable heparin-containing hydrogel model that recapitulates natural VEGF binding modes with full and independent control over physical properties. Using this model, we show that the degree of heparin flexibility in the hydrogel network determines the mobility of bound VEGF and, in turn, its ability to interact with VEGF receptor 2. We demonstrate that VEGF mobility is driven by a relay mechanism, in which VEGF molecules directly switch from one heparin chain to another. When strong electrostatic interactions between heparin and other hydrogel constituents immobilize the sugar backbone, this relay mechanism is impeded, in turn reducing VEGF’s ability to reach its target receptor at the cell membrane, thereby reducing its bioactivity. This work identifies heparin flexibility within the ECM as a previously unknown regulator of the microenvironment, which will not only contribute to a better mechanistic understanding of how the ECM regulates growth factor bioactivity, but it will also provide an important design criterion for the development of tissue-engineered biomaterials that require vascularization.

During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 activate the pluripotency-related genes through the putative OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory toward the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.

Abstract Two types of osteoblasts are required to assemble the zebrafish embryonic skeleton: classical osteoblasts homologous to the mammalian cell, and notochord sheath cells, which serve as non-classical osteoblasts. The gene entpd5a is critically required for ossification via both types of osteoblasts. Despite the indispensability of zebrafish models in vertebrate research, the genetic regulation of bone formation, as well as mechanisms of transcriptional control of entpd5a, remain largely unknown. Here, using a newly generated transgenic line, we isolate classical and non-classical osteoblasts from zebrafish embryos and performed both ATAC-seq and RNA-seq. We analysed results independently and integratively to understand those chromatin dynamics and accompanying transcriptomic changes that occur in different skeletal cell types. We show that although Dlx family factors are playing important roles in classical osteoblast regulation, Hox family factors are involved in governing spinal ossification via non-classical osteoblasts. We further present a resource-driven analysis of the entpd5a promoter, experimentally validating the ATAC-seq dataset and proposing mechanisms of regulating the complex entpd5a expression pattern during zebrafish osteogenesis. Our results thus provide a necessary comprehensive resource for the field of bone development and indicate spatio-temporally regulated promoter/enhancer interactions taking place in the entpd5a locus.

Stem cells are a hallmark of animal multicellularity. Sox and POU transcription factors are associated with stemness and were believed to be animal innovations, reported absent in their unicellular relatives. Here we describe unicellular Sox and POU factors. Choanoflagellate and filasterean Sox proteins have DNA-binding specificity similar to mammalian Sox2. Choanoflagellate—but not filasterean—Sox can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSCs) through interacting with the mouse POU member Oct4. In contrast, choanoflagellate POU has a distinct DNA-binding profile and cannot generate iPSCs. Ancestrally reconstructed Sox proteins indicate that iPSC formation capacity is pervasive among resurrected sequences, thus loss of Sox2-like properties fostered Sox family subfunctionalization. Our findings imply that the evolution of animal stem cells might have involved the exaptation of a pre-existing set of transcription factors, where pre-animal Sox was biochemically similar to extant Sox, whilst POU factors required evolutionary innovations.