CoNIC Challenge: Pushing the frontiers of nuclear detection, segmentation, classification and counting.

Nuclear detection, segmentation and morphometric profiling are essential in helping us further understand the relationship between histology and patient outcome. To drive innovation in this area, we setup a community-wide challenge using the largest available dataset of its kind to assess nuclear segmentation and cellular composition. Our challenge, named CoNIC, stimulated the development of reproducible algorithms for cellular recognition with real-time result inspection on public leaderboards. We conducted an extensive post-challenge analysis based on the top-performing models using 1,658 whole-slide images of colon tissue. With around 700 million detected nuclei per model, associated features were used for dysplasia grading and survival analysis, where we demonstrated that the challenge's improvement over the previous state-of-the-art led to significant boosts in downstream performance. Our findings also suggest that eosinophils and neutrophils play an important role in the tumour microevironment. We release challenge models and WSI-level results to foster the development of further methods for biomarker discovery.

AimSeg: A machine-learning-aided tool for axon, inner tongue and myelin segmentation.

Electron microscopy (EM) images of axons and their ensheathing myelin from both the central and peripheral nervous system are used for assessing myelin formation, degeneration (demyelination) and regeneration (remyelination). The g-ratio is the gold standard measure of assessing myelin thickness and quality, and traditionally is determined from measurements made manually from EM images-a time-consuming endeavour with limited reproducibility. These measurements have also historically neglected the innermost uncompacted myelin sheath, known as the inner tongue. Nonetheless, the inner tongue has been shown to be important for myelin growth and some studies have reported that certain conditions can elicit its enlargement. Ignoring this fact may bias the standard g-ratio analysis, whereas quantifying the uncompacted myelin has the potential to provide novel insights in the myelin field. In this regard, we have developed AimSeg, a bioimage analysis tool for axon, inner tongue and myelin segmentation. Aided by machine learning classifiers trained on transmission EM (TEM) images of tissue undergoing remyelination, AimSeg can be used either as an automated workflow or as a user-assisted segmentation tool. Validation results on TEM data from both healthy and remyelinating samples show good performance in segmenting all three fibre components, with the assisted segmentation showing the potential for further improvement with minimal user intervention. This results in a considerable reduction in time for analysis compared with manual annotation. AimSeg could also be used to build larger, high quality ground truth datasets to train novel deep learning models. Implemented in Fiji, AimSeg can use machine learning classifiers trained in ilastik. This, combined with a user-friendly interface and the ability to quantify uncompacted myelin, makes AimSeg a unique tool to assess myelin growth.

3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and long-term stability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application is limited due to their reliance on variable biological components. Our studies focused on the development of 3D liver tissue under defined conditions. In vitro generated 3D tissues exhibited stable phenotype for over 1 year in culture, providing an attractive resource for long-term in vitro studies. Moreover, 3D derived tissue provided critical liver support in two animal models, including immunocompetent recipients. Therefore, we believe that our study provides stable human tissue to better model liver biology 'in the dish', and in the future may permit the support of compromised liver function in humans.

Injection of embryonic stem cell derived macrophages ameliorates fibrosis in a murine model of liver injury.

Chronic liver injury can be caused by viral hepatitis, alcohol, obesity, and metabolic disorders resulting in fibrosis, hepatic scarring, and cirrhosis. Novel therapies are urgently required and previous work has demonstrated that treatment with bone marrow derived macrophages can improve liver regeneration and reduce fibrosis in a murine model of hepatic injury and fibrosis. Here, we describe a protocol whereby pure populations of therapeutic macrophages can be produced in vitro from murine embryonic stem cells on a large scale. Embryonic stem cell derived macrophages display comparable morphology and cell surface markers to bone marrow derived macrophages but our novel imaging technique revealed that their phagocytic index was significantly lower. Differences were also observed in their response to classical induction protocols with embryonic stem cell derived macrophages having a reduced response to lipopolysaccharide and interferon gamma and an enhanced response to IL4 compared to bone marrow derived macrophages. When their therapeutic potential was assessed in a murine, carbon tetrachloride-induced injury and fibrosis model, embryonic stem cell derived macrophages significantly reduced the amount of hepatic fibrosis to 50% of controls, down-regulated the number of fibrogenic myofibroblasts and activated liver progenitor cells. To our knowledge, this is the first study that demonstrates a therapeutic effect of macrophages derived in vitro from pluripotent stem cells in a model of liver injury. We also found that embryonic stem cell derived macrophages repopulated the Kupffer cell compartment of clodronate-treated mice more efficiently than bone marrow derived macrophages, and expressed comparatively lower levels of Myb and Ccr2, indicating that their phenotype is more comparable to tissue-resident rather than monocyte-derived macrophages.

Erratum: Author Correction: Injection of embryonic stem cell-derived macrophages ameliorates fibrosis in a murine model of liver injury.

[This corrects the article DOI: 10.1038/s41536-017-0017-0.].

Analysis of Coronary Vessels in Cleared Embryonic Hearts.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

Rho-kinase-dependent actin turnover and actomyosin disassembly are necessary for mouse spinal neural tube closure.

The cytoskeleton is widely considered essential for neurulation, yet the mouse spinal neural tube can close despite genetic and non-genetic disruption of the cytoskeleton. To investigate this apparent contradiction, we applied cytoskeletal inhibitors to mouse embryos in culture. Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure. In contrast, inhibiting Rho kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) or blocking F-actin disassembly prevented closure, with apical F-actin accumulation and adherens junction disturbance in the neuroepithelium. Cofilin-1-null embryos yielded a similar phenotype, supporting the hypothesis that there is a key role for actin turnover. Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect. We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly. In contrast, actomyosin assembly and myosin ATPase activity are not limiting for closure.

The CXCL12/CXCR4 Axis Plays a Critical Role in Coronary Artery Development.

The chemokine CXCL12 and its receptor CXCR4 have many functions during embryonic and post-natal life. We used murine models to investigate the role of CXCL12/CXCR4 signaling in cardiac development and found that embryonic Cxcl12-null hearts lacked intra-ventricular coronary arteries (CAs) and exhibited absent or misplaced CA stems. We traced the origin of this phenotype to defects in the early stages of CA stem formation. CA stems derive from the peritruncal plexus, an encircling capillary network that invades the wall of the developing aorta. We showed that CXCL12 is present at high levels in the outflow tract, while peritruncal endothelial cells (ECs) express CXCR4. In the absence of CXCL12, ECs were abnormally localized and impaired in their ability to anastomose with the aortic lumen. We propose that CXCL12 is required for connection of peritruncal plexus ECs to the aortic endothelium and thus plays a vital role in CA formation.

The neural milieu of the developing choroid plexus: neural stem cells, neurons and innervation.

The choroid plexus produces cerebrospinal fluid and plays an important role in brain homeostasis both pre and postnatally. In vitro studies have suggested that cells from adult choroid plexus have stem/progenitor cell-like properties. Our initial aim was to investigate whether such a cell population is present in vivo during development of the choroid plexus, focusing mainly on the chick choroid plexus. Cells expressing neural markers were indeed present in the choroid plexus of chick and also those of rodent and human embryos, both within their epithelium and mesenchyme. ß3-tubulin-positive cells with neuronal morphology could be detected as early as at E8 in chick choroid plexus and their morphological complexity increased with development. Whole mount immunochemistry demonstrated the presence of neurons throughout choroid plexus development and they appeared to be mainly catecholaminergic, as indicated by tyrosine-hydroxylase reactivity. The presence of cells co-labeling for BrdU and the neuroblast marker, doublecortin, in organotypic choroid plexus cultures supported the hypothesis that neurogenesis can occur from neural precursors within the developing choroid plexus. Furthermore, we found that extrinsic innervation is present in the developing choroid plexus, unlike previously suggested. Altogether, our data are consistent with the presence of neural progenitors within the choroid plexus, suggest that at least some of the choroid plexus neurons are born locally, and show for the first time that choroid plexus innervation occurs prenatally. Hence, we propose the existence of a complex neural regulatory network within the developing choroid plexus that may play a crucial role in modulating its function during development as well as throughout life.

The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes.

BACKGROUND: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. METHODS AND RESULTS: Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. CONCLUSIONS: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.

Mutations in SNX14 cause a distinctive autosomal-recessive cerebellar ataxia and intellectual disability syndrome.

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.

Vascularisation is not necessary for gut colonisation by enteric neural crest cells.

The vasculature and nervous system share striking similarities in their networked, tree-like architecture and in the way they are super-imposed in mature organs. It has previously been suggested that the intestinal microvasculature network directs the migration of enteric neural crest cells (ENCC) along the gut to promote the formation of the enteric nervous system (ENS). To investigate the inter-relationship of migrating ENCC, ENS formation and gut vascular development we combined fate-mapping of ENCC with immunolabelling and intravascular dye injection to visualise nascent blood vessel networks. We found that the enteric and vascular networks initially had very distinct patterns of development. In the foregut, ENCC migrated through areas devoid of established vascular networks. In vessel-rich areas, such as the midgut and hindgut, the distribution of migrating ENCC did not support the idea that these cells followed a pre-established vascular network. Moreover, when gut vascular development was impaired, either genetically in Vegfa(120/120) or Tie2-Cre;Nrp1(fl/-) mice or using an in vitro Wnt1-Cre;Rosa26(Yfp/+) mouse model of ENS development, ENCC still colonised the entire length of the gut, including the terminal hindgut. These results demonstrate that blood vessel networks are not necessary to guide migrating ENCC during ENS development. Conversely, in miRet(51) mice, which lack ENS in the hindgut, the vascular network in this region appeared to be normal suggesting that in early development both networks form independently of each other.

Disease-associated missense mutations in the EVH1 domain disrupt intrinsic WASp function causing dysregulated actin dynamics and impaired dendritic cell migration.

Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.

Human γδ T lymphocytes are licensed for professional antigen presentation by interaction with opsonized target cells.

Activated human blood γδ T cells have also been previously demonstrated to behave as professional APCs, although the processes that control APC function have not been characterized. n this study, we show that the acquisition of potent APC function by human blood γδ T cells is achieved after physical interaction with an Ab-coated target cell, a process that we refer to as licensing. In cancer models, licensing of γδ T cells by tumor-reactive mAbs promotes the uptake of tumor Ags and professional presentation to tumor-reactive αß T cells. We propose that licensing by Ab is a mechanism whereby the adaptive properties of γδ T cells are induced by their innate functions in a spatially and temporally controlled manner.

Increased Wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans.

Wingless (Wnt)/ß-catenin signaling plays an essential role during normal development, is a critical regulator of stem cells, and has been associated with cancer in many tissues. Here we demonstrate that genetic expression of a degradation-resistant mutant form of ß-catenin in early Rathke's pouch (RP) progenitors leads to pituitary hyperplasia and severe disruption of the pituitary-specific transcription factor 1-lineage differentiation resulting in extreme growth retardation and hypopituitarism. Mutant mice mostly die perinatally, but those that survive weaning develop lethal pituitary tumors, which closely resemble human adamantinomatous craniopharyngioma, an epithelial tumor associated with mutations in the human ß-catenin gene. The tumorigenic effect of mutant ß-catenin is observed only when expressed in undifferentiated RP progenitors, but tumors do not form when committed or differentiated cells are targeted to express this protein. Analysis of affected pituitaries indicates that expression of mutant ß-catenin leads to a significant increase in the total numbers of pituitary progenitor/stem cells as well as in their proliferation potential. Our findings provide insights into the role of the Wnt pathway in normal pituitary development and demonstrate a causative role for mutated ß-catenin in an undifferentiated RP progenitor in the genesis of murine and human craniopharyngioma.

Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability.

Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury.

Bio-electrospraying primary cardiac cells: in vitro tissue creation and functional study.

Manifestations of myocardial infarctions have been recognized as one of the major killers in the Western world. Therefore, advancing and developing novel cardiac tissue repair and replacement therapeutics have great implications to our health sciences and well-being. There are several approaches for forming cardiac tissues, non-jet-based and jet-based methodologies. A unique advantage of jet-based approaches is the possibility to handle living cells with a matrix for cell distribution and deposition in suspension, either as single or heterogeneous cell populations. Our previous studies on bio-electrospraying of cardiac cells have shown great promise. Here, we show for the first time the ability to bio-electrospray the three major cell types of the myocardium, both independently and simultaneously, for forming a fully functional cardiac tissue. Several samples are characterized in vitro and found to be indistinguishable in comparison to controls. Thus, we are describing a swiftly emerging novel biotechnique for direct cardiac tissue generation. Moreover, the present investigations pave the way for the development and optimization of a bio-patterning approach for the fabrication of biologically viable cardiac tissue grafts for the potential treatment of severe heart failure after myocardial infarction.

Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling.

Large brain size is one of the defining characteristics of modern humans. Seckel syndrome (MIM 210600), a disorder of markedly reduced brain and body size, is associated with defective ATR-dependent DNA damage signaling. Only a single hypomorphic mutation of ATR has been identified in this genetically heterogeneous condition. We now report that mutations in the gene encoding pericentrin (PCNT)--resulting in the loss of pericentrin from the centrosome, where it has key functions anchoring both structural and regulatory proteins--also cause Seckel syndrome. Furthermore, we find that cells of individuals with Seckel syndrome due to mutations in PCNT (PCNT-Seckel) have defects in ATR-dependent checkpoint signaling, providing the first evidence linking a structural centrosomal protein with DNA damage signaling. These findings also suggest that other known microcephaly genes implicated in either DNA repair responses or centrosomal function may act in common developmental pathways determining human brain and body size.

Microcephalin coordinates mitosis in the syncytial Drosophila embryo.

Microcephalin (MCPH1) is mutated in primary microcephaly, an autosomal recessive human disorder of reduced brain size. It encodes a protein with three BRCT domains that has established roles in DNA damage signalling and the cell cycle, regulating chromosome condensation. Significant adaptive evolutionary changes in primate MCPH1 sequence suggest that changes in this gene could have contributed to the evolution of the human brain. To understand the developmental role of microcephalin we have studied its function in Drosophila. We report here that Drosophila MCPH1 is cyclically localised during the cell cycle, co-localising with DNA during interphase, but not with mitotic chromosomes. mcph1 mutant flies have a maternal effect lethal phenotype, due to mitotic arrest occurring in early syncytial cell cycles. Mitotic entry is slowed from the very first mitosis in such embryos, with prolonged prophase and metaphase stages; and frequent premature separation as well as detachment of centrosomes. As a consequence, centrosome and nuclear cycles become uncoordinated, resulting in arrested embryonic development. Phenotypic similarities with abnormal spindle (asp) and centrosomin (cnn) mutants (whose human orthologues are also mutated in primary microcephaly), suggest that further studies in the Drosophila embryo may establish a common developmental and cellular pathway underlying the human primary microcephaly phenotype.

Otx2 regulates subtype specification and neurogenesis in the midbrain.

The transcription factor Otx2 is required to determine mesencephalic versus metencephalic (cerebellum/pons) territory during embryogenesis. This function of Otx2 primarily involves positioning and maintaining the mid-hindbrain organizer at the border between midbrain and anterior hindbrain. Otx2 expression is maintained long after this organizer is established. We therefore generated conditional mutants of Otx2 using the Cre/loxP system to study later roles during rostral brain development. For inactivation of Otx2 in neuronal progenitor cells, we crossed Otx2(flox/flox) animals with Nestin-Cre transgenic animals. In Nestin-Cre/+; Otx2(flox/flox) embryos, Otx2 activity was lost from the ventral midbrain starting at embryonic day 10.5 (E10.5). In these mutant embryos, the mid-hindbrain organizer was properly positioned at E12.5, although Otx2 is absent from the midbrain. Hence, the Nestin-Cre/+; Otx2(flox/flox) animals represent a novel mouse model for studying the role of Otx2 in the midbrain, independently of abnormal development of the mid-hindbrain organizer. Our data demonstrate that Otx2 controls the development of several neuronal populations in the midbrain by regulating progenitor identity and neurogenesis. Dorsal midbrain progenitors ectopically expressed Math1 and generate an ectopic cerebellar-like structure. Similarly, Nkx2.2 ectopic expression ventrally into tegmentum progenitors is responsible for the formation of serotonergic neurons and hypoplasia of the red nucleus in the midbrain. In addition, we discovered a novel role for Otx2 in regulating neurogenesis of dopaminergic neurons. Altogether, these results demonstrate that Otx2 is required from E10.5 onward to regulate neuronal subtype identity and neurogenesis in the midbrain.