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Perivascular macrophages (pvMs) are associated with cerebral vasculature and mediate brain drainage and immune regulation. Here, using reporter mouse models, whole brain and section immunofluorescence, flow cytometry, and single cell RNA sequencing, besides the Lyve1 + F4/80 + CD206 + CX3CR1 + pvMs, we identify a CX3CR1 – pvM population that shares phagocytic functions and location. Furthermore, the brain parenchyma vasculature mostly hosts Lyve1 + MHCII – pvMs with low to intermediate CD45 expression. Using the double Cx3cr1 GFP x Cx3cr1-Cre ; Rosa tdT reporter mice for finer mapping of the lineages, we establish that CD45 low CX3CR1 – pvMs are derived from CX3CR1 + precursors and require PU.1 during their ontogeny. In parallel, results from the Cxcr4-CreErt2 ; Rosa26 tdT lineage tracing model support a bone marrow-independent replenishment of all Lyve1 + pvMs in the adult mouse brain. Lastly, flow cytometry and 3D immunofluorescence analysis uncover increased percentage of pvMs following photothrombotic induced stroke. Our results thus show that the parenchymal pvM population is more heterogenous than previously described, and includes a CD45 low and CX3CR1 – pvM population. Perivascular macrophages (pvMs) are important for brain drainage and immune regulation. Here the authors analyse various reporter mouse strains for finer mapping of pvM subsets and lineage differentiation, and propose CX3CR1negative and CD45low as additional markers of intermediate pvMs for studying this heterogenous population.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, increased impulsivity and emotion dysregulation. Linkage analysis followed by fine-mapping identified variation in the gene coding for Latrophilin 3 (LPHN3), a putative adhesion-G protein-coupled receptor, as a risk factor for ADHD. In order to validate the link between LPHN3 and ADHD, and to understand the function of LPHN3 in the etiology of the disease, we examined its ortholog lphn3.1 during zebrafish development. Loss of lphn3.1 function causes a reduction and misplacement of dopamine-positive neurons in the ventral diencephalon and a hyperactive/impulsive motor phenotype. The behavioral phenotype can be rescued by the ADHD treatment drugs methylphenidate and atomoxetine. Together, our results implicate decreased Lphn3 activity in eliciting ADHD-like behavior, and demonstrate its correlated contribution to the development of the brain dopaminergic circuitry.

Traditionally, the formation of mineralized tissue has been studied in mammalian settings, both in vivo and in vitro. There are undeniable advantages of using murine systems, but there are also short-comings such as in vivo observation of chondrocytes and osteoblasts, and the inability to carry out forward genetic screens that would allow for an unbiased way to identify gene functions at the whole organism level. We have therefore turned to the zebrafish (Danio rerio), which complements mammalian systems and provides an alternative approach to the study of osteogenesis and mineralization. In this lecture I will provide examples of how genetic studies have enabled us to identify novel gene functions, and how studying embryonic skeletal development in zebrafish can provide mechanistic insight into the role of novel factors essential for osteoblast function and mineralization. Disclosure of Interest None Declared

Atypical E2F transcription factors (E2F7 and E2F8) function as key regulators of cell cycle progression and their inactivation leads to spontaneous cancer formation in mice. However, the mechanism of the tumor suppressor functions of E2F7/8 remain obscure. In this study we discovered that atypical E2Fs control tumor angiogenesis, one of the hallmarks of cancer. We genetically inactivated atypical E2Fs in epithelial and mesenchymal neoplasm and analyzed blood vessel formation in three different animal models of cancer. Tumor formation was either induced by application of 7,12-Dimethylbenz(a)anthracene/12-O-Tetradecanoylphorbol-13-acetate or by Myc/Ras overexpression. To our surprise, atypical E2Fs suppressed tumor angiogenesis in all three cancer models, which is in a sharp contrast to previous findings showing that atypical E2Fs promote angiogenesis during fetal development in mice and zebrafish. Real-time imaging in zebrafish displayed that fluorescent-labeled blood vessels showed enhanced intratumoral branching in xenografted E2f7/8 -deficient neoplasms compared with E2f7/8 -proficient neoplasms. DLL4 expression, a key negative inhibitor of vascular branching, was decreased in E2f7/ 8-deficient neoplastic cells, indicating that E2F7/8 might inhibit intratumoral vessel branching via induction of DLL4.

Summary The zebrafish is an excellent model organism for forward‐genetics, with attributes such as small size, rapid development and straightforward imaging enabling mutagenesis screens for a wide variety of phenotypes. For the majority of these screens over the last few decades, the mutations were mapped using bulk segregant analysis (BSA) to establish approximate chromosomal locations, followed by fine mapping using microsatellite markers on hundreds (or thousands) of embryos. This process is very time consuming despite the large clutch sizes of the zebrafish. Next‐generation sequencing (NGS) technologies have drastically improved the speed of this process, but there is no consensus on the best method for performing the BSA and fine‐mapping analysis on NGS data. Here we describe a simple statistical approach to this problem using difference‐in‐homozygosity as a single variable with a normal distribution. This approach was used to accurately map and identify the causative mutation in a zebrafish line with a recessive mineralization disorder.

Background The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates. Results We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1 Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development. Conclusions We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.

The dorsoventral pattern of vertebrate embryos is established and regulated by opposing gradients of ventralizing bone morphogenetic proteins (BMPs) and BMP antagonists (such as Chordin, Noggin and Follistatin in the frog embryo) secreted by a dorsal organizer. In the zebrafish, Danio rerio, mutations in a number of genes that affect dorsoventral patterning of the early embryo have been identified, but only two, dino and mercedes, are involved in dorsal specification, with dino mutants displaying the stronger, ventralized phenotype. Here we show that the dino phenotype is caused by a mutation in the zebrafish chordin gene, revealing that Chordin is an essential component of the dorsal organizer. Furthermore, we provide evidence that Chordin and BMPs are antagonistic, not only at the protein level but also at the transcription level.

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic‐specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination‐based ultramicroscopy to entire wholemount‐immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF‐C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development. Ultramicroscopy of wholemount mouse embryos uncovers the first, previously unknown lymphatic structures in mammals: the dorsal longitudinal lymphatic vessel and the ventral primordial thoracic duct, which eventually connect with the cardinal vein as previously described.

Proteolytical processing of the growth factor VEGFC through the concerted activity of CCBE1 and ADAMTS3 is required for lymphatic development to occur. How these factors act together in time and space, and which cell types produce these factors is not understood. Here we assess the function of Adamts3 and the related protease Adamts14 during zebrafish lymphangiogenesis and show both proteins to be able to process Vegfc. Only the simultaneous loss of both protein functions results in lymphatic defects identical to vegfc loss-of-function situations. Cell transplantation experiments demonstrate neuronal structures and/or fibroblasts to constitute cellular sources not only for both proteases but also for Ccbe1 and Vegfc. We further show that this locally restricted Vegfc maturation is needed to trigger normal lymphatic sprouting and directional migration. Our data provide a single-cell resolution model for establishing secretion and processing hubs for Vegfc during developmental lymphangiogenesis. How and where VEGF-C is processed in lymphangiogenesis is unclear. Here, the authors show that development of the zebrafish lymphatic system is locally restricted by Vegfc maturation causing lymphatic sprouting in certain regions, which is regulated by the metalloproteases ADAMTS3 and ADAMTS14.