Hematopoietic stem cells and more committed progenitors (collectively referred to as HSPCs) emerge from vessels during development, via endothelial-to-hematopoietic transition (EHT). Recently, using the zebrafish embryo, we showed that two EHT cell types emerge from the dorsal aorta, raising the question of their subsequent fate. To address this issue, we established a complex pipeline based on singlecell photoconversion and transgenic lines to characterize the abilities of EHT cell progenies to conquer hematopoietic organs and to obtain their transcriptomic profiles. We show that the two EHT cell types lead to partly differentially fated cells, with significant differences in thymus colonization and T-lymphoid lineage commitment. In addition, we investigated implantation of HSPCs in niches, with the support of HSPC signatures (gata2b and cd34/podocalyxin), retrieved from our single-cell datasets. This revealed, at unprecedented resolution, the homing of HSPCs in niches of entire early larvae, including the pronephros, the sub-aortic and caudal regions, as well as the area contacting the supra-intestinal artery. Our work provides new insights into fundamental aspects of HSPC fate acquisition, from their emergence to their homing in specific niches.

Hematopoietic stem cells emerge in the embryo from an aortic-derived tissue called the hemogenic endothelium (HE). The HE appears to give birth to cells of different nature and fate but the molecular principles underlying this complexity are largely unknown. Here we show, in the zebrafish embryo, that two cell types emerge from the aortic floor with radically different morphodynamics. With the support of live imaging, we bring evidence suggesting that the mechanics underlying the two emergence types rely, or not, on apicobasal polarity establishment. While the first type is characterized by reinforcement of apicobasal polarity and maintenance of the apical/luminal membrane until release, the second type emerges via a dynamic process reminiscent of trans-endothelial migration. Interfering with Runx1 function suggests that the balance between the two emergence types depends on tuning apicobasal polarity at the level of the HE. In support of this and unexpectedly, we show that Pard3ba – one of the four Pard3 proteins expressed in the zebrafish – is sensitive to interference with Runx1 activity, in aortic endothelial cells. This supports the idea of a signaling cross talk controlling cell polarity and its associated features, between aortic and hemogenic cells. In addition, using new transgenic fish lines that express Junctional Adhesion Molecules and functional interference, we bring evidence for the essential role of ArhGEF11/PDZ-RhoGEF in controlling the HE-endothelial cell dynamic interface, including cell-cell intercalation, which is ultimately required for emergence completion. Overall, we highlight critical cellular and dynamic events of the endothelial-to-hematopoietic transition that support emergence complexity, with a potential impact on cell fate.

Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fates. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the mammalian fetal liver, arise from the ventral part of caudal somites. We have now found that this ventral domain is the sclerotome, and that two markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-α, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that trunk somites are similarly dependent on Alcam and Pdgfr-α to produce mesenchymal cells that foster HSPC emergence from the aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis.

Hematopoietic stem and progenitor cells emerge from the aorta and migrate to the caudal hematopoietic tissue (CHT) of zebrafish larvae, the hematopoietic equivalent of the mammalian fetal liver, for their proliferation and differentiation. We previously reported that somite-derived stromal cells were a key component of the CHT niche. Here, we found that the cell adhesion protein Protocadherin 18a (Pcdh18a) is expressed in the stromal cell progenitors (SCPs) emigrating from somites toward the future CHT. Deletion of most of the Pcdh18a intracellular domain caused a decrease in the number of SCPs, the directionality of their migration, and the cell-contact mediated repulsion that normally occurs between migrating SCPs. These defects were followed by abnormal morphogenesis of the venous plexus that forms the CHT framework, and the inability of the CHT to function as a niche for hematopoietic stem and progenitor cells. Finally, we found that the extracellular domain of Pcdh18a mediates trans heterophilic adhesion of stromal cells to endothelial cells in vivo and thereby the reticular versus perivascular fate of SCPs. Thus, Pcdh18a expression in SCPs is essential for the proper development of the hematopoietic niche.

A combination of light and electron microscopy has revealed further details about the location and interactions of hematopoietic stem and progenitor cells.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here, we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. We found a variety of different configurations of HSPCs and surrounding niche cells, suggesting there could be functional heterogeneity in sites of HSPC lodgement. Our approach also allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglion cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types. Editor's evaluation The manuscript reports on an extensive body of work, achieving the still highly challenging identification of HSPCs within the ultrastructure of their niche. The study highlights the heterogeneous nature of HSC-niche interactions, which is consistent with heterogeneity identified through genomic and functional studies. The work presented is of high interest to the field. https://doi.org/10.7554/eLife.64835.sa0 Decision letter eLife's review process Introduction Hematopoietic stem and progenitor cells (HSPCs) give rise to all blood cell types throughout the life of an organism (Orkin and Zon, 2008). HSPCs reside in a complex microenvironment called the niche that is made up of many different kinds of support cells, including various types of mesenchymal stromal cells (MSCs) and endothelial cells (ECs) (Pinho and Frenette, 2019). However, our understanding of HSPC interactions with niche cells has been limited to low-resolution light microscopy. Recently developed transgenic reporter lines have allowed identification of well-defined endogenous HSPCs in both mouse and zebrafish model organisms (Acar et al., 2015; Chen et al., 2016; Christodoulou et al., 2020; Tamplin et al., 2015). The dynamic behavior of HSPCs in the niche of mouse and zebrafish can be observed using live imaging (Bixel et al., 2017; Christodoulou et al., 2020; Itkin et al., 2016; Koechlein et al., 2016; Lo Celso et al., 2009; Spencer et al., 2014; Tamplin et al., 2015). Yet, it remains challenging to observe HSPC-niche interactions at high resolution. Certain events during hematopoietic ontogeny, such as the colonization of the fetal bone marrow, have so far only been studied in fixed tissues because they are difficult to access (Coşkun et al., 2014). Our goal is to better define the ultrastructure of single endogenous HSPCs deep in niche tissue. We have taken advantage of the transparency and external development of the zebrafish larva to study the earliest migration events of HSPCs into the presumptive adult kidney marrow (KM) niche. We considered using two different correlative light and electron microscopy (CLEM) techniques (Karreman et al., 2016) to resolve the ultrastructure of HSPCs. For the first approach, to confirm cell identity we used a label-based approach similar to what was done using an APEX2-Venus-CAAX fusion protein (Hirabayashi et al., 2018) that could be visualized both with light microscopy and high contrast in EM imaging. A similar method was applied in zebrafish using an APEX-GBP (GFP Binding Protein) fusion to resolve GFP+ transgene expression on electron micrographs (Ariotti et al., 2015). Building on these previous studies, we used genetically encoded APEX2 engineered peroxidase (Lam et al., 2015), together with a fluorescent protein, to track single HSPCs as they migrated into and lodged in the larval KM during the earliest colonization stages. For the second approach, we used improved software alignments to merge datasets across different imaging platforms and could correlate single cells without the need for endogenous labels. We found clusters of HSPCs around the glomerulus as previously described, as well as single HSPCs lodged in a perivascular niche. The heterogeneity between sites of HSPC lodgement and their surrounding niche cells supports the model that functionally distinct sites exist within the hematopoietic microenvironment (Zhang et al., 2021). These large 3D CLEM datasets also allowed us to identify previously uncharacterized dopamine beta-hydroxylase (dbh) positive ganglion-like cells in the larval kidney niche that were in direct contact with HSPCs. Results Colonization of the larval kidney niche by circulating HSPCs To characterize the larval kidney HSPC niche in the zebrafish, we first wanted to determine the location of all HSPCs in the anterior KM. We used HSPC-specific Runx:mCherry+ transgenic zebrafish larvae (Tamplin et al., 2015) that were fixed at 5 days post fertilization (dpf) for anti-mCherry immunofluorescence, then optically cleared using benzyl alcohol/benzyl benzoate (BABB). This technique allowed visualization and quantification of all mCherry+ HSPCs in the larvae. We observed bilateral clusters of ~50 HSPCs, for a total of ~100 HSPCs, in the region of the anterior KM at 5 dpf (Figure 1—figure supplement 1A-D). During early HSPC colonization of the KM at 4 dpf we observed ~50 total HSPCs, which increased by ~100% from 4 to 5–6 dpf, and then again by ~50% from 5–6 to 7–8 dpf (Figure 1—figure supplement 1E). We performed phospho-histone H3 (PH3) antibody labeling to determine if HSPCs were highly proliferative. On average, we only found one to two mitotic HSPCs in the KM at 5 dpf (Figure 1—figure supplement 1F-J). Previous studies of hematopoietic cells in the larval KM at 7 dpf also showed low levels of proliferation (van Rooijen et al., 2009). These data suggest that the increase in HSPC numbers during the early stages of KM colonization results from arrival via circulation, that is HSPCs that originated in the caudal hematopoietic tissue (CHT) and dorsal aorta (Murayama et al., 2006), and not extensive proliferation of resident HSPCs. To follow the dynamics of HSPC colonization in the KM niche, we performed time-lapse live imaging at 4 and 5 dpf. Although the early zebrafish larva is transparent, imaging live KM using point scanning confocal microscopy is challenging due to the relatively slow acquisition time and depth of the tissue. To rapidly capture HSPC colonization events throughout the entire depth of the larval KM, we performed light sheet fluorescence microscopy (Huisken and Stainier, 2009). Using this technique, we could rapidly acquire a Z stack through the entire KM in less than 30 s (>200 slices with 1 μm spacing). Consistent with our observations of fixed embryos, a depth-coded projection of light sheet time-lapse images showed bilateral Runx:mCherry+ HSPC clusters (‘green’ cells on left and ‘blue’ cells on right; Figure 1—figure supplement 2; Figure 1—video 1). Together with the cdh17:GFP reporter line for pronephric tubules (Zhou et al., 2010), we localized Runx:mCherry+ HSPCs within the anterior kidney region, mediolateral to the proximal pronephric tubules (Figure 1—figure supplement 3). To determine the location of these HSPC clusters relative to the vasculature, we imaged HSPC-specific Runx:mCherry together with the flk:ZsGreen vascular-specific transgenic reporter (Cross et al., 2003). HSPC clusters were located between lateral dorsal aortae and cardinal veins (Figure 1—figure supplement 4). Light sheet live imaging of the KM niche at 5 dpf allowed us to directly visualize the locations and dynamics of early HSPC niche colonization. To observe the interaction between single HSPCs as they arrive in the larval KM niche, we imaged the Runx:GFP transgenic line together with flk:mCherry to label vessels (Figure 1—video 2). As previously described, Runx:GFP is a more restricted marker of HSPCs and is expressed in the cytoplasm, allowing cellular morphology to be resolved, while Runx:mCherry more broadly labels the progenitor pool and is only localized to the nucleus, making it more appropriate for quantifying single cells (Tamplin et al., 2015; Figure 1—figure supplement 5A). Upon arrival within the anterior kidney region, rare circulating Runx:GFP+ HSPCs were seen interacting with and lodging in the perivascular niche (Figure 1A). We resolved single lodged HSPCs surrounded by ECs in a pocket-like structure, as we observed previously in the zebrafish CHT and mouse fetal liver (Tamplin et al., 2015; Figure 1B). Figure 1 with 7 supplements see all Download asset Open asset Single hematopoietic stem and progenitor cells (HSPCs) lodge in a perivascular region of the larval kidney niche. (A) Snapshot of single optical sections (XY, XZ, YZ planes) from light sheet live image of a Runx:GFP;flk:mCherry double transgenic zebrafish larva. A single Runx:GFP+ HSPC (white arrowhead) is lodged in a perivascular region lateral to the dorsal aorta (DA). (B) Detail of optical sections (1 µm steps) through the single lodged Runx:GFP+ HSPC in (A). mCherry+ endothelial cells contact the HSPC and form a surrounding pocket. The +5 µm section is also shown in XZ and YZ planes. Abbreviations: DA, dorsal aorta; SeA, intersegmental artery; SeV, intersegmental vein; PCV, posterior cardinal vein; CCV, common cardinal vein; ISVs, intersegmental vessels; D, dorsal; V, ventral; A, anterior; P, posterior. We also examined expression of the cd41:GFP HSPC reporter line in the region of the anterior KM and found substantial overlap with Runx:mCherry+ HSPCs (Figure 1—figure supplement 5A). The widespread labeling of the 5 dpf HSPC pool by the cd41:GFP reporter, and its cytoplasmic expression that reveals cellular morphology, makes it a valuable tool for further analysis of HSPCs in the larval KM. Furthermore, cd41:GFP+ HSPCs were visible in EC pockets, confirming that they had lodged in the KM niche (Figure 3—figure supplement 1A). Together, the Runx:mCherry, Runx:GFP, and cd41:GFP reporter lines show similar lodgement in the larval KM niche, and represent an array of valuable tools to interrogate the interaction of HSPCs with various niche cell types. HSPCs in the larval KM niche form direct contacts with an MSC and multiple ECs After characterizing the specific location of HSPCs in the larval KM niche, and their lodgement in EC pocket structures that are also formed in the CHT (Tamplin et al., 2015), we sought to identify the specific types of contacts that form between HSPCs and niche cells. We considered that tight junctions may form between HSPCs and ECs and would be marked by tight junction protein 1 (tjp1), the scaffolding protein also known as zonula occludens-1 (ZO-1) that has two orthologues in zebrafish (tjp1a and tjp1b) (Anderson et al., 1988; Stevenson et al., 1986). We injected Oregon Green dextran into the circulation of Runx:mCherry transgenic larvae to label the vessel lumen, followed by fixation and immunofluorescence with anti-ZO-1 and anti-mCherry antibodies. We observed expression of ZO-1 broadly on ECs, as well as localization between Runx:mCherry HSPCs and surrounding niche cells (Figure 2A and B). These data suggest that tight junctions form at the contact points between HSPCs and the niche. Figure 2 Download asset Open asset Hematopoietic stem and progenitor cells (HSPCs) lodged in the larval kidney niche make direct contacts with endothelial cells (ECs) and mesenchymal stromal cells (MSCs). (A) Single optical section from confocal image of larval kidney (fixed) shows Runx:mCherry+ HSPCs (magenta) lodged in the perivascular niche. Oregon Green dye labels the vessel lumen. Blue dotted lines surround the dorsal aorta (DA) and red dotted lines surround the glomerulus (G). Tight junction protein is marked by zonula occludens-1 (ZO-1) (white). (B) High-resolution optical sections (0.5 µm steps) through the boxed regions in (A) show ZO-1+ contact points between mCherry+ HSPCs and the niche (yellow arrowheads). (C) Orthogonal slices (XY and YZ planes) from live light sheet 3D volume of larval kidney niche. Single cd41:GFP+ HSPCs (green) is in contact or in close proximity (yellow arrowhead) to cxcl12:DsRed2+ MSCs (magenta). The white dotted line represents the DA. (D) Quantification of distances measured between GFP+ HSPC and DsRed2+ MSCs shows ~60% of HSPCs are in contact with MSCs, and the remaining are within 9 µm. Numbers above the columns indicate the cell numbers counted in each group (from n=8 embryos). Abbreviations: D, dorsal; V, ventral; A, anterior; P, posterior. We then went on to characterize additional niche cell types that are present in the larval KM. We performed imaging using the cd41:GFP HSPC transgenic reporter line together with cxcl12:DsRed2 (Glass et al., 2011) to label MSCs (Figure 2C). We measured the distance between HSPCs and MSCs and observed 57% (n=16/28) of HSPCs were in direct contact with an MSC, 29% were <5 μm (n=8/28), and 14% (n=4/28) were <10 μm away (Figure 2D). These data from the larval KM niche are very similar to previous observations from the CHT showing that the majority of HSPCs are in contact with, or close proximity to, an MSC (Tamplin et al., 2015). Together, our results demonstrate that HSPC lodgement in the larval KM niche occurs close to or in contact with a single MSC, and that HSPCs are in contact with multiple ECs, similar to what we observed previously in the CHT. A CLEM approach to characterize the ultrastructure of HSPCs in the larval KM niche Next we wanted to explore the ultrastructure of HSPCs and their surrounding cells in the larval KM niche. One approach to analyze the ultrastructure of an entire region of tissue is serial section electron microscopy (EM), a technique that has been used to resolve the projectome of the complete zebrafish larval brain (Hildebrand et al., 2017). We previously used CLEM based on anatomical landmarks to match the position of fluorescently labeled HSPCs in the CHT niche between confocal and SBEM datasets (Tamplin et al., 2015). However, we found this approach was difficult to apply in the KM because the tissue is much larger and denser than the CHT (Cell Image Library [CIL] accession numbers CIL:54845 and CIL:54850). To confirm precise correlation of single cells between light and EM imaging modalities, we developed two distinct approaches depending on the goals of the experiment. In Workflow #1, we wanted to track lodgement of HSPCs in the larval KM niche using time-lapse live imaging, followed by high-contrast DAB (3,3'-diaminobenzidine) staining with genetically encoded APEX2 to label single endogenous HSPCs in EM sections. In Workflow #2, we wanted to correlate the position of fluorescently labeled cells in existing transgenic reporter lines across confocal and EM datasets. Using these workflows, we were able to observe HSPC lodgement in the KM, then resolve the ultrastructure of those same cells together with the surrounding niche. The first step in Workflow #1 was to generate a transgenic construct that expressed mCherry for light microscopy, and APEX2 as a genetic tag that allowed electron-dense contrast on target subcellular structures (Figure 3A). Our rationale was that mCherry+ HSPCs are also APEX2+ and will be identifiable by both fluorescence imaging and EM, respectively. We fused APEX2 with H2B and mito tags for localization to the nucleus and the mitochondrial matrix, respectively. To drive expression of this construct in HSPCs, we cloned these elements under control of the draculin (drl) promoter, generating the drl:mito-APEX2_p2A_APEX2-H2B_p2A_mCherry transgene, hereafter referred to as drl:APEX2-mCherry. The drl promoter is a marker of vascular and hematopoietic lineages (Herbomel et al., 1999; Mosimann et al., 2015), and we chose it because of its high HSPC expression level compared to other available promoters, such as Runx1+23 (Tamplin et al., 2015; Nottingham et al., 2007) or cd41 (Ma et al., 2011). Furthermore, it was previously confirmed that drl:GFP+ HSPCs almost completely overlap with Runx:mCherry+ HSPCs from embryo to adult (Henninger et al., 2017; Mosimann et al., 2015). Figure 3 with 4 supplements see all Download asset Open asset Correlative light and electron microscopy (CLEM) Workflow #1 to genetically encode a label in endogenous hematopoietic stem and progenitor cells (HSPCs) for live tracking by light microscopy and high-contrast resolution in serial block-face scanning electron microscopy (SBEM) sections. (A) Fusion construct encoding p2A-linked proteins mito-APEX2, APEX2-H2B, and mCherry that localize to the mitochondria, nucleus, and cytoplasm, respectively. The draculin promoter was used to transiently drive strong mosaic expression in HSPCs. Random insertion in the genome was by Tol2-mediated transgenesis. (B) Tol2 draculin:mito-APEX2_p2A_APEX2-H2B_p2A_mCherry (drl:APEX2-mCherry) fusion construct was injected together with tol2 mRNA in one cell wild type zebrafish embryos. (C) At 5 days post fertilization (dpf), embryos with circulating mCherry+ HSPCs were visually screened and retro-orbitally injected with alpha bungarotoxin to paralyze the embryo, and Oregon Green dye to label the vasculature. (D) Dye-injected mCherry+ double positive embryos were visually screened and used for light sheet microscopy (example shows a 439 × 439 × 115 µm3 volume of the anterior kidney marrow (KM); ISVs, intersegmental vessels; yellow dotted line, DA, dorsal aorta; gut AF, gut autofluorescence). (E) Brightfield example of a single embryo after fixation and DAB (3,3'-diaminobenzidine) staining to label APEX2+ HSPCs that are located within the dotted box (dotted line marks DA, dorsal aorta; Y, yolk; D, dorsal; V, ventral; A, anterior; P, posterior). (F) After embedding, the sample was oriented and trimmed based on images acquired using micro-computed tomography (microCT) (example shows orthogonal sections in three planes, N; notochord, G; glomerulus, S; swim bladder). (G) Single plane from ~3000 sections of SBEM data (example shows a 233 × 331 × 213 µm3 volume; s1-s5, somites 1–5; G, glomerulus; PD, pneumatic duct). To track and correlate single cells through multiple imaging modalities, we required sparse labeling of HSPCs. Therefore, we generated transient F0 transgenics with a mosaically labeled HSPC pool. Although a caveat of F0 mosaic transgenics in zebrafish is the inherent variability of labeling between embryos, this allowed us to select embryos that had similar numbers of lodged HSPCs in the KM niche compared to other HSPC reporter lines. We observed only one to two rare HSPCs surrounded by EC pockets in the KM niche of each cd41:GFP or Runx:GFP transgenic larvae, and selected F0 drl:APEX2-mCherry larvae with similar HSPC numbers (Figures 1 and 3, Figure 3—figure supplement 1). Both cd41:GFP+ and selected F0 drl:APEX2+ larvae had similar numbers of positive HSPCs in the mediolateral clusters of the anterior KM niche (Figure 3—figure supplement 1B). We gained further confirmation of HSPC-specific expression in F0 drl:APEX2-mCherry+ larvae by injection of the drl:APEX2-mCherry construct into cd41:GFP transgenic embryos, and observed ~15% of HSPCs were both mCherry+;APEX2+ and GFP+ (data not shown). These data demonstrate that transient expression of the drl:APEX2-mCherry construct in F0 larvae can generate sparse labeling of HSPCs in the KM niche with similar frequency to other stable HSPC-specific reporter lines. To generate larvae for CLEM Workflow #1, we injected drl:APEX2-mCherry construct together with tol2 transposase into single-cell stage wild type zebrafish embryos (Figure 3B). At 5 dpf, larvae with circulating mCherry+ HSPCs were injected retro-orbitally with alpha-bungarotoxin and dextran-conjugated Oregon Green dye, to paralyze the larvae and label the vasculature, respectively (Figure 3C). Once immobilized and mounted for light sheet live imaging, optical sections were acquired through the entire depth of the KM. A short time-lapse was performed to confirm an HSPC was lodged in the KM and not circulating (~30 min; Figure 3—video 1). Single lodged HSPCs could be identified relative to the surrounding tissues (Figure 3D), and larvae were immediately fixed after imaging. Larvae were stained with DAB to label APEX2+ cells for identification by brightfield microscopy (Figure 3E). Larvae were treated with osmium tetroxide and embedded for micro-computed tomography (microCT; Figure 3F and Figure 3—figure supplement 2; Figure 3—video 2). This intermediate microCT step allowed the larvae to be oriented and trimmed to select a discrete region of interest (ROI) for SBEM (Figure 3G and Figure 3—figure supplement 2). Last, automated SBEM generated over 3000 high-resolution sections of the ROI (e.g., XY = 10 nm/pixel, Z=70 nm/pixel; Figure 3—figure supplement 2; Figure 4—video 1). Using focal charge compensation (Deerinck et al., 2018) which effectively eliminates specimen charging, we obtained a high-resolution SBEM dataset without the need for excessive post-processing alignment. Together, these experimental steps in Workflow #1 allowed us to label an endogenous HSPC that was tracked live, then stained for high-contrast detection in a large SBEM dataset. To correlate the position of a single labeled HSPC across multiple imaging modalities, we performed 3D software alignment of both light sheet and SBEM datasets. First, we identified anatomical features as landmarks in the SBEM data, such as the somites, glomerulus, and pronephric tubules (Figure 4—figure supplement 1). We also observed clustered hematopoietic cells around the glomerulus in the same region as seen by light sheet microscopy (compare Figure 1—figure supplement 3 and Figure 4—figure supplement 2). We merged 3D rendered light sheet and SBEM datasets using image analysis software (Imaris) and aligned matching anatomical features in all three planes (Figure 4A–C and Figure 4—figure supplement 3). By performing these 3D alignments, we could locate a single APEX2+ cell in the SBEM dataset that was <5 μm from the corresponding mCherry+ HSPC imaged in the light sheet volume (Figure 4D). Furthermore, the APEX2+ cell had dark nuclear staining with much higher contrast than any of the surrounding cells, confirming we had identified the same APEX2+;mCherry+ HSPC across multiple imaging modalities (Figure 4D). By correlating 3D light sheet and SBEM data, we could confirm lodgement of a single HSPC in the larval KM niche, allowing us to further define the ultrastructure of this rare cell relative to its surrounding cells in the niche. Figure 4 with 4 supplements see all Download asset Open asset 3D alignment of light sheet and serial block-face scanning electron microscopy (SBEM) datasets localizes a single rare hematopoietic stem and progenitor cell (HSPC) across multiple imaging modalities. (A) Single Z plane from light sheet imaging of drl:APEX2-mCherry+ transgenic larva showing the lodged mCherry+ HSPC (white arrowhead). (B) Global alignment of 3D rendered models generated from light sheet and SBEM datasets using Imaris software. (C) Orthogonal views of the white boxed region within B shows a 3D view of the alignment between light sheet and SBEM datasets. White arrowhead points to the single lodged HSPC in the aligned light sheet and SBEM datasets. (C’) APEX2+ HSPC in SBEM data. (C’’) mCherry+ HSPC in light sheet data. Green: Injected Oregon Green dextran dye marking vessels. Magenta: Runx:mCherry+ HSPCs and autofluorescence in gut. (D) Detail of the alignment shows mCherry+ HSPC and APEX2+ HSPC are <5 µm apart (dotted white line). Abbreviations: ISVs, intersegmental vessels; D, dorsal; V, ventral; A, anterior; P, posterior. 3D modeling of SBEM data reveals all cells in contact with an endogenous HSPC To reconstruct the spatial relationships of the single APEX2+ HSPC relative to its surrounding niche cells, we performed extensive tracing of cell membranes within the SBEM data using 3D modeling software (IMOD) (Kremer et al., 1996). ECs are generally elongated cells with a large nucleus and little cytoplasm, while MSCs can be distinguished by their granular cytoplasm and a nucleus that occupies three-fourths of the cell volume (Tamplin et al., 2015). This morphological analysis revealed that the lodged APEX2+;mCherry+ HSPC (Figure 5A) was enclosed in a pocket of five ECs, and attached to a single MSC, that all directly contact the surface of the HSPC (Figure 5B; Figure 5—video 1). This same configuration of cells was seen previously in the CHT (Tamplin et al., 2015), demonstrating that this cellular structure is also conserved in the larval KM niche. Within other sections of the SBEM dataset, we observed the clusters of hematopoietic cells posterior to the glomerulus that were also seen with light sheet imaging (compare Figure 1—figure supplement 3 and Figure 5—figure supplement 1). Figure 5 with 4 supplements see all Download asset Open asset Hematopoietic stem and progenitor cells (HSPCs) lodge in a multicellular niche in the perivascular kidney marrow (KM). The ultrastructure of a single APEX2+ HSPC (white arrow) and its surrounding niche cells are modeled using 3D SBEM (00:15 from Figure 4—video 1). (A) The APEX2+ HSPC is lodged in the perivascular KM niche. (Ai) Surrounding tissues are labeled; the HSPC is anterior to the pneumatic duct, dorsal to intestine and pancreas, and ventral to the somites and pronephric tubule. (Aii) Higher magnification shows the APEX2+ HSPC is only two-cell diameters from the vessel lumen (white area). (Aiii) Full resolution detail of the APEX2+ HSPC showing high-contrast labeling of the nucleus (APEX2-H2B), mitochondria (mito-APEX2; white arrowhead), and extracellular space dorsal to the cell. (B) (i–iii) SBEM sections at different levels through the APEX2+ HSPC (white arrows) as shown in the schematic (iv). The HSPC is simultaneously in contact with multiple niche cells: five endothelial cells (EC1–5), 1 mesenchymal stromal cell (MSC), and a ganglion-like (GL) cell. Two unlabeled APEX2 negative putative HSPCs were lodged in the same niche (HSPC2 and HSPC3). HSPC2 is attached to HSPC3, and the APEX2+ HSPC (Biii; asterisk). (C) 3D rendered models of the APEX2+ HSPC (solid green) in contact with niche cells. 3D contours are in the same colors as outlines in (B). The APEX2+ HSPC is directly contacted by: (i) five ECs; (ii) one MSC; (iii) one HSPC, and a chain of GL-like cells. (iv) The GL-like cell is part of a long continuous chain of similar cells that extends through the niche. Scale bars: 5 µm unless otherwise labeled. Abbreviations: D, dorsal; V, ventral; A, anterior; P, posterior. A significant advantage of SBEM datasets is that they provide a complete 3D picture of the cellular composition of a tissue that is not dependent on prior knowledge of transgenic or immunolabeled markers. Known transgenic markers allowed us to characterize HSPC-EC (Figure 1) and HSPC-MSC (Figure 2C and D) interactions, but not discover novel HSPC-niche cellular interactions. Careful analysis of our SBEM dataset and the cells in direct contact with the APEX2+;mCherry+ HSPC identified ganglion-like cells in the larval KM (Figure 5B and C; Figure 5—video 1). The ability to move through all adjacent sections of the 3D SBEM dataset allowed us to follow the length of these ganglion-like cells and discover that it was part of a chain of at least eight morphologically similar cells that extended throughout the larval KM niche. Further tracing of the neighboring cells in contact with the APEX2+;mCherry+ HSPC revealed two unlabeled cells with the distinctive morphology of putative HSPCs (i.e., scant cytoplasm, large round nucleus, ruffled membrane; HSPC2 and HSPC3; Figure 5B and C). These other two putative HSPCs were APEX2 negative, suggesting they were not progeny derived by division from the APEX2+;mCherry+ HSPC, and were more likely independent HSPC clones that had lodged in the same niche. All three HSPCs were in direct contact with the chain of ganglion-like cells that we found extends through the larval KM niche (Figure 5C; Figure 5—video 1). This 3D SBEM dataset allowed identification of all surrounding cells in contact with the APEX2+;mCherry+ HSPC, and strikingly showed that a single endogenous HSPC can be in direct physical contact with as many as eight other cells. Finally, given this multicellular HSPC niche structure we observed, we reasoned that an unlabeled HSPC in an independent dataset should be identifiable based on location and morphology alone. We generated a second SBEM dataset that also had a single APEX2+;mCherry+ HSPC, however it was found in a vessel lumen in the larval KM niche and attached to the vessel wall (Figure 5—figure supplement 2). We searched the perivascular regions of the larval KM niche within this second SBEM dataset and found two unlabeled putative HSPCs. Not only did both putative HSPCs share a distinct morphology, but following 3D modeling of all surrounding niche cells in contact with the HSPCs, we found each one was also in its own pocket of five ECs, and attached to a single MSC, exactly as we had observed previously in the CHT (Tamplin et al., 2015), and with the APEX2+;mCherry+ labeled HSPC (compare Figure 5C and Figure 5—figure supplement 3). Furthermore, one of the unlabeled putative HSPCs was

Zusammenfassung Hintergrund Von Mensch zu Mensch übertragene Infektionskrankheiten können eine Berufskrankheit(BK)-Nr. 3101 bedingen, wenn sie bei Versicherten auftreten, die infolge der Ausübung ihrer Arbeitstätigkeit in bestimmten Bereichen einer gegenüber der allgemeinen Bevölkerung wesentlich erhöhten Infektionsgefahr ausgesetzt sind. Ziel der Arbeit Es erfolgt die Darstellung der besonderen medizinischen und versicherungsrechtlichen Aspekte einer beruflichen MRSA-Kolonisation am Beispiel eines Gutachtenfalles und Auswertung der BK-Verdachtsanzeigen (BK-Nr. 3101) der gewerblichen Berufsgenossenschaften und Unfallversicherungsträger. Patienten und Methoden Die Geschäftsergebnisse und BK-Dokumentation der gewerblichen Berufsgenossenschaften und Unfallversicherungsträger 2007 bis 2012 wurden zur BK-Nr. 3101 ausgewertet sowie das Patientenkollektiv berufsdermatologischer Begutachtungsfälle 2007 bis 2012 der Hautklinik Universitätsklinikum Erlangen zum Vorliegen von Mensch zu Mensch übertragener Infektionskrankheiten der Haut retrospektiv untersucht. Ergebnisse Von Mensch zu Mensch übertragene Infektionskrankheiten der Haut sind in der berufsdermatologischen Begutachtung selten. Von den 2007 bis 2012 erfassten BK-Verdachtsanzeigen waren 2,6 % BK-Nr. 3101-Verdachtsfälle; 4,2 % aller anerkannten BKen entfielen auf BK-Nr. 3101-Fälle, darunter 9 Fälle von MRSA (Methicillin-resistenter Staphylococcus aureus). Eine symptomlose MRSA-Besiedlung wird im Gegensatz zur manifesten Infektionskrankheit nicht als BK-Nr. 3101 anerkannt. Bakterielle Superantigene können eine atopische Dermatitis (AD) triggern. Im Einzelfall kann eine AD infolge einer beruflich erworbenen MRSA-Ansteckung auftreten und eine BK-Nr. 3101 begründen. Diskussion Ein frühzeitiger Nachweis einer MRSA-Kolonisation und Eradikation sind notwendig für eine Rehabilitation. Der Umgang mit Hauterkrankungen als Folge einer Infektionskrankheit im Berufskrankheitenverfahren wird dargelegt.

Hematopoiesis leads to the formation of blood and immune cells. Hematopoietic stem cells emerge during development, from vascular components, via a process called the endothelial-to-hematopoietic transition (EHT). Here, we reveal essential biomechanical features of the EHT, using the zebrafish embryo imaged at unprecedented spatio-temporal resolution and an algorithm to unwrap the aorta into 2D-cartography. We show that the transition involves anisotropic contraction along the antero-posterior axis, with heterogenous organization of contractile circumferential actomyosin. The biomechanics of the contraction is oscillatory, with unusually long periods in comparison to other apical constriction mechanisms described so far in morphogenesis, and is supported by the anisotropic reinforcement of junctional contacts. Finally, we show that abrogation of blood flow impairs the actin cytoskeleton, the morphodynamics of EHT cells, and the orientation of the emergence. Overall, our results underline the peculiarities of the EHT biomechanics and the influence of the mechanical forces exerted by blood flow.

There is increasing recognition that agricultural landscapes meet multiple societal needs and demands beyond provision of economic and environmental goods and services. Accordingly, there have been significant calls for the inclusion of societal, amenity and cultural values in agri-environmental landscape indicators to assist policy makers in monitoring the wider impacts of land-based policies. However, capturing the amenity and cultural values that rural agrarian areas provide, by use of such indicators, presents significant challenges. The EU social awareness of landscape indicator represents a new class of generalized social indicator using a top-down methodology to capture the social dimensions of landscape without reference to the specific structural and cultural characteristics of individual landscapes. This paper reviews this indicator in the context of existing agri-environmental indicators and their differing design concepts. Using a stakeholder consultation approach in five case study regions, the potential and limitations of the indicator are evaluated, with a particular focus on its perceived meaning, utility and performance in the context of different user groups and at different geographical scales. This analysis supplements previous EU-wide assessments, through regional scale assessment of the limitations and potentialities of the indicator and the need for further data collection. The evaluation finds that the perceived meaning of the indicator does not vary with scale, but in common with all mapped indicators, the usefulness of the indicator, to different user groups, does change with scale of presentation. This indicator is viewed as most useful when presented at the scale of governance at which end users operate. The relevance of the different sub-components of the indicator are also found to vary across regions. (C) 2015 Elsevier Ltd. All rights reserved.

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.

Bin/Amphiphysin/Rvs ( BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third alpha-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third alpha-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors.

BACKGROUND:This study evaluates the psychometric properties of the Child and Parent versions of the German CFQ-R (Cystic Fibrosis Questionnaire Revised), a disease-specific measure of Health-Related Quality of Life (HRQoL) in children with cystic fibrosis (CF). Self-Rating is combined with proxy-rating by parents in the use of the questionnaire.METHODS:136 children with CF (6 - 13 years) and their parents were recruited to evaluate internal consistency (Cronbach's alpha) and validity, 20 children and parents to examine reproducibility (ICC).RESULTS:Cronbach's alpha is high in all but two dimensions of the Child version (alpha = 0.23-0.77) and for all dimensions of the Parent version (alpha = 0.69-0.89). For both questionnaires, reproducibility is moderate to high (ICC = 0.50-0.94). Factor analysis shows loadings of >0.4 in the majority of items. Higher HRQoL is reported by children with mild disease compared to those with moderate/severe disease and by boys compared to girls. Convergence between self-rating and proxy-rating depends on the dimension.CONCLUSION:The German CFQ-R, Child and Parent versions, are reliable and valid measures of HRQoL. They should be administered in combination as both, child and parent, provide important information. The measure offers a new patient-reported outcome for clinical purposes as well as for national and international studies in schoolchildren.

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools.

A toner supplying device is constructed such that, at each side of a pair of supporting plates 39 rotatably about a rotating center C, a toner supply roller 20, an upper and lower auger rollers 35 and 34, and a developing roller 19 are integrally supported, and each supporting plate 39 is made rotatable about a supporting shaft 1e whereby to allow the developing roller 19 to come into contact with a photosensitive drum 12. Accordingly, the toner supply roller 20 and the developing roller 19 can integrally be provided as maintaining a proper positional relation therebetween. With such a simple and inexpensive structure, the developing roller 19 is allowed to come into contact with the photosensitive drum 12, thereby enabling development using toner on an electrostatic latent image.

For decades potassium dichromate has been the most important allergen in the construction industry. In Scandinavian countries the prevalence of potassium dichromate sensitization declined following the introduction of low-chromate cement. In contrast, analysis of our register in Northern Bavaria and the data of workers compensation board show no significant decline in potassium dichromate sensitization in the German construction industry during the 1990s. In 1993, German legislation provided an Approved Code of Practice and the cement manufacturers committed themselves to reduce the level of water-soluble chromates to less than 2 ppm in bag cement. Since 2000 this agreement has also included ready-mixed concrete. It remains to be seen, if sensitization against potassium dichromate will decline in the upcoming years. Based on our data, it would be desirable if only low-chromate cement was used in the construction industry in Germany as in Scandinavia.

Present evidence convincingly indicates that workers with occupational skin disease are more frequently affected by atopic skin diathesis than the general working population. Population-based studies estimating the impact of atopic skin diathesis on occupational skin disease in various occupations have not been reported to date. We analyzed data of all initial reports of occupational skin diseases recorded in the register of occupational skin diseases in northern Bavaria, Germany, from 1990 to 1999. The main outcome measure was the attributable risk of atopic skin diathesis on occupational skin disease within 24 occupational groups that are most hazardous to the skin. Of the 5285 registered cases, 3730 had a confirmed occupational causation. Among these, 1366 workers (37%) presented an atopic skin diathesis. Assuming a prevalence of atopic skin diathesis of 20% in the total population, we found that 21.6% (95% confidence interval 19.4; 23.7) of occupational skin disease cases within 24 occupational groups may be ascribed to this endogenous risk factor. The attributable risk of atopic skin diathesis helped to explain a large proportion of occupational skin diseases. Empirical evidence supports the importance of surveying atopic skin diathesis as part of an occupational skin disease prevention strategy.

We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.