Biology subjects

Rovira, M.

Publications and source records attributed to Rovira, M..

16 recordsLinked to original sources

bioRxiv2026

3D-bioprinted skeletal muscle with tailored myofibre architecture

Engineered skeletal muscle tissue (eSMT) contractile function strongly depends on the alignment and orientation of its myofibres, as force generation and transmission are intrinsically linked to its microstructural architecture. However, fabricating truly functional eSMT with controlled myofibre architecture remains a major challenge. Here, we present a method that combines extrusion-based 3D bioprinting with custom multi-pillar anchoring systems to guide myofibre orientation. Using this approach, we successfully fabricated functional eSMTs with parallel, fusiform, bipennate, and circular architectures. We then integrated these basic designs into a functional system comprising multiple 3D-bioprinted eSMTs replicating the morphology and myofibre architecture of facial musculature. By recapitulating native muscle architecture, this approach advances the engineering of functional eSMT for multiple biomedical applications and establishes a foundation for next-generation biohybrid robotics.

bioengineering↗

bioRxiv2026

Ciliogenic pancreatopathy reveals a link between ciliopathies and exocrine pancreatic disease

BackgroundWhile pancreatic cysts have been described in syndromic ciliopathies, the pancreas is not commonly recognized as a target organ. However, several ciliary gene knockout mouse models develop a pancreatic phenotype combining acinar atrophy and adipocyte accumulation, hereby called adipopancreatosis, suggesting a link between ciliary dysfunction and pancreatic disease. ObjectiveWe investigated whether mutations in ciliopathy-associated genes are linked to pancreatic dysfunction in humans. DesignWe analyzed a cohort of 341 patients with pediatric-onset pancreatic anomalies and characterized the pancreatic phenotype of new mouse models with conditional Nphp3 inactivation or bearing Nphp3 mutations recapitulating human mutations. In patients, pancreatic fat content was quantified using Dixon-MRI. ResultsMutations in the cilium-related HNF1B and NPHP3 were identified in patients presenting with both renal and pancreatic dysfunction. Nphp3 mutant mice developed acinar atrophy, adipopancreatosis, and moderate inflammation. Adipocytes in the pancreas exhibited a white adipocyte-like profile and likely originated from mesothelial-derived fibroblasts. Reduced numbers and altered length of ductal cilia were monitored. Interestingly, secretory canaliculi, typically unnoticed structures found within and between acinar cells and connected to the acinar lumen, exhibited a microcystic morphology. Consistent with the mouse phenotype, Dixon-MRI revealed significantly increased pancreatic fat content in patients with HNF1B and NPHP3 mutations. ConclusionWe describe a previously unrecognized pancreatic manifestation of ciliopathies, which we name ciliogenic pancreatopathy. Patients with known ciliopathy-causing mutations should be evaluated for this pancreatic condition, particularly those with kidney disease, as concomitant exocrine pancreatic insufficiency may further compromise renal function or the outcome of kidney graft. What is already known on this topicO_LICiliopathies, resulting from defects in primary cilia, are genetic disorders primarily affecting the kidney and liver. C_LIO_LIPancreatic cysts have been sporadically reported in syndromic ciliopathies. C_LIO_LIThe pancreas is not currently recognized as a major target organ of ciliary dysfunction. C_LIO_LIA clear link between ciliary gene mutations and pancreatic anomalies is still unknown. C_LIO_LIAnimal studies have suggested a possible association between ciliary dysfunction and pancreatic anomalies. C_LI What this study addsO_LIIdentifies HNF1B and NPHP3 mutations as genetic causes of a pancreatic phenotype characterized by acinar atrophy and adipose replacement (adipopancreatosis). C_LIO_LIDemonstrates the presence of defective ductal cilia and moderate inflammation in the pancreas of Nphp3 mutant mice. C_LIO_LIReveals that secretory canaliculi in the exocrine pancreas of Nphp3 mutant mice acquire a microcystic morphology. C_LIO_LIShows that patients with HNF1B or NPHP3 mutations have significantly increased pancreatic fat content by Dixon-MRI. C_LIO_LIDefines a new disease entity, ciliogenic pancreatopathy, as a pancreatic manifestation of ciliopathies. C_LI How this study might affect research, practice or policyO_LIEstablishes the pancreas as a novel and clinically relevant target of ciliopathies. C_LIO_LIExpands the phenotypic spectrum of HNF1B- and NPHP3-related diseases to include exocrine pancreatic dysfunction. C_LIO_LISuggests that patients with ciliopathy-causing mutations should be evaluated for exocrine pancreatic insufficiency. C_LIO_LIHighlights the need to consider pancreatic function monitoring in kidney disease and transplant settings. C_LIO_LIOpens new research avenues into the role of primary cilia in pancreatic homeostasis and disease. C_LI

cell biology↗

bioRxiv2025

Identification and Implications for Tumor Heterogeneity of a DNA Methylation-Based Signature Classifying Pancreatic Ductal Adenocarcinoma Based on their Cellular Origin

BackgroundPancreatic ductal adenocarcinoma (PDAC) arises from distinct cellular origins, yet the extent to which DNA methylation patterns from normal pancreatic cells are preserved in tumor cells remains unclear. Identifying cell-of-origin signatures may enhance PDAC classification and therapeutic stratification. ObjectiveTo determine whether DNA methylation signatures in normal acinar and ductal pancreatic cells are retained in PDAC cell lines and to develop a robust classifier for distinguishing tumor origins. DesignWe performed DNA methylation profiling using the Illumina Infinium Mouse MethylationEPIC array on normal acinar and ductal cells and their PDAC derivatives in genetically engineered mouse models (GEMMs). Differential methylation analysis, and hierarchical clustering were used to identify and validate a conserved cell-of-origin DNA methylation signature. A logistic regression model was developed for classification. ResultsWe identified 178 CpG sites that remain preserved during tumorigenesis and effectively distinguished acinar- and ductal-derived PDAC cell lines. This signature was validated across independent sample sets, primary tumors, and orthotopic allografts. It successfully classified cell lines of unknown origin, including PDAC samples from KPC mice, and revealed the impact of oncogenic mutations on tumor fate. A logistic regression model supported these findings, confirming the robustness of the classification approach. Furthermore, the cell of origin influenced key PDAC characteristics, including treatment response, highlighting its potential role in molecular subtyping and patient stratification. ConclusionA preserved DNA methylation signature during pancreatic carcinogenesis distinguishes PDAC origins and influences tumor behavior. These results highlight the potential of DNA methylation profiling for tumor classification and personalized treatment strategies. They also raise important questions about the relevance of KPC mice as a preclinical model and the mechanisms driving PDAC heterogeneity. What is already known on this topicO_LIHuman PDAC exhibits significant heterogeneity, with molecular subtyping (classical vs basal-like) providing some insights into tumor behavior and clinical outcomes. C_LIO_LIMouse acinar and ductal cells can give rise to PDAC, influencing tumor characteristics and survival outcomes. C_LIO_LIDNA methylation is a powerful tool for tracing cellular identity and distinguishing cancer subtypes based on epigenetic profiles. C_LI What this study addsO_LIA cell-of-origin methylation signature is preserved during mouse carcinogenesis and across diverse experimental settings, providing a reliable tool for tumor classification. C_LIO_LIA DNA methylation-based classification system reliably distinguishes between acinar- and ductal-origin PDAC, filling a critical gap in methods to trace tumor lineage. C_LIO_LIAcinar- and ductal-derived PDACs exhibit distinct methylation patterns that correlate with differences in tumor behavior, such as chemoresistance, highlighting the biological relevance of cellular origin in PDAC. C_LIO_LIThe study provides new insights into how cell-of-origin influences PDAC heterogeneity and could lead to more precise therapeutic strategies tailored to the tumors lineage. C_LI How this study might affect research, practice or policyO_LIThis study provides a new, reliable method for classifying PDAC based on its cellular origin, which could significantly improve tumor classification in both preclinical and clinical settings, aiding in more accurate diagnoses and prognostic predictions. C_LIO_LIThe identification of distinct methylation patterns linked to tumor behavior offers valuable insights for developing personalized treatment strategies, as therapies could be tailored based on the tumors cellular origin and associated molecular characteristics. C_LIO_LIThe preservation of cell-of-origin methylation signature suggests the potential for developing a universal biomarker for PDAC classification, which could guide future clinical trials, therapeutic targeting, and patient stratification in PDAC care. C_LI

cancer biology↗

bioRxiv2025

Spatial map of native duct cell populations in human pancreas and their representation in pancreatic cancers

BackgroundA resemblance of pancreatic tumors to their native tissue architecture remains largely unexplored, while it may reveal novel insights into the healthy and diseased tissue. ObjectiveThis study aims at generating a spatially resolved map of human pancreatic duct cell populations in the native tissue and in tumors, i.e. pancreatic ductal adenocarcinoma (PDAC) and adenosquamous cancer of the pancreas (ASCP). DesignNew datasets were acquired with several spatial transcriptomics platforms and were integrated with public single-cell RNAseq datasets and validated by multiplex immunofluorescence. Cell lines and primary human cell cultures were genetically manipulated. ResultsGroups of Keratin-5+ cells in larger ducts have a gene signature reminiscent of stem cells and (supra)basal cells from other tissues. At single cell resolution, this group comprises {Delta}Np63+ basal cells (BAS) and {Delta}Np63- supra-basally residing luminal-B cells (LUM-B). The latter express previously unreported MUC4 and MUC16. Additionally, we identified three other luminal cell populations in the ducts (LUM-A, -C, -D). In cancer, BAS and LUM-B signatures associate with basal-like (BL) PDAC, and correlate with lower survival. However, PDAC exhibits a random spatial pattern and fragmented native expression programs while ASCP preserve the identity of LUM-B and BAS in a spatially unmixed pattern. {Delta}Np63 drives cell plasticity to BAS, conserved from the native tissue to cancer. ConclusionSpatially distinct duct cell populations are revealed, and the extent of preservation of the native cell identities in pancreatic cancer underpins distinct tumor identities. This warrants a separate consideration in research and therapy. What is already known on this topicWhile tumors in the pancreas can exhibit basal-like and squamous features resembling tumors of other tissues, their resemblance to the native duct cells, and possible subpopulations thereof, had not been studied in detail. What this study addsOne basal and four luminal spatially distinct cell populations exist in human pancreatic ducts. These are best conserved in ASCP while PDAC shows loss of the native cell identity program. How this study might affect research, practice or policyOur study demonstrates fundamental differences between the tumor cells of the BL PDAC versus the rare ASCP, highlighting the necessity for a clear distinction between them in research and in tumor-specific treatments. Meanwhile, the research community should acknowledge the spatial and functional heterogeneity of human duct cells, including in their experimental models.

cancer biology↗

bioRxiv2024

ΔNP63 defines an exocrine-committed multipotent progenitor subset in the murine pancreas

Cellular plasticity underpins heterogeneity in embryogenic progenitor cells and cancer cells. The transcription factor deltaNp63 ({Delta}Np63) has been implicated in regulating cellular plasticity in several epithelial tissues. Despite a recently established role in steering plasticity of pancreatic cancer, {Delta}Np63 remains unstudied in pancreatic development. Using murine single-cell sequencing data and RNA and protein in situ stainings, we assessed the spatio-temporal expression of Trp63 and {Delta}NP63 in the embryonic pancreas. {Delta}NP63 demonstrates a transient and spatially restricted expression in the multipotent pancreatic progenitor (MPP) compartment delineating pro-exocrine progenitor cells. Lineage tracing of TP63+ cells marks a subset of MPPs and descendant exocrine acinar and centro-acinar/terminal duct cells. Lack of {Delta}NP63 in knock-out mice leads to hypotrophic exocrine acini with reduced levels of differentiation markers. In summary, {Delta}Np63 confers heterogeneity within the MPP compartment, supporting exocrine cell development. These new insights in developmental plasticity have potential implications for pancreatic regeneration and cancer.

developmental biology↗

bioRxiv2024

Medieval genomes from eastern Iberia illuminate the role of Morisco mass deportations in dismantling a long-standing genetic bridge with North Africa

BackgroundThe Islamic influence on the Iberian Peninsula left an enduring legacy culturally and linguistically, however the demographic impact is less well understood. This study aims to explore the dynamics of gene flow and population structure in eastern Iberia from the early to late Medieval period through ancient DNA. ResultsOur comprehensive genomic analysis uncovered gene flow from various Mediterranean regions into Iberia before the Islamic period, supporting a pre-existing pan-Mediterranean homogenization phenomenon during the Roman Empire. North African ancestry is present but sporadic in late antiquity genomes but becomes consolidated during the Islamic period. We uncovered one of the earliest dated Islamic burials in Spain, which showed high levels of inbreeding. For the first time we also prove the persistence of North African ancestry in a Christian cemetery until the 17th century, in addition to evidence of slave traffick from the Maghreb. ConclusionsThis study reveals the complex interaction between political events and cultural shifts that influenced the population of eastern Iberia. It highlights the existence of a slave trade and underscores the lasting impact of historical events, such as the Expulsion of the Moriscos in 1609 CE, on the regions genetic and cultural landscape through mass population displacement and replacement.

evolutionary biology↗

bioRxiv2024

A single-cell atlas of the murine pancreatic ductal tree identifies novel cell populations with potential implications in pancreas regeneration and exocrine pathogenesis.

Background and aimsPancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. MethodsWe used scRNA-seq to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. ResultsWe have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro, including Wnt-responsive-population, ciliated-population and FLRT3+ cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples, highlighting a putative role of WNT-responsive, IFN-responsive and EMT-populations in pancreatic exocrine pathogenesis as their expression increases in chronic pancreatitis and PanIN lesions. ConclusionsIn light of our discovery of previously unidentified ductal populations, we unmask the potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis.

cell biology↗

bioRxiv2024

Implications of noncoding regulatory functions in the development of insulinomas

Insulinomas are rare neuroendocrine tumours arising from the pancreatic {beta}-cells. While retaining the ability to produce insulin, insulinomas feature aberrant proliferation and altered hormone secretion resulting in failure to maintain glucose homeostasis. With the aim of uncovering the role of noncoding regulatory regions and their aberrations to the development of these tumors, we coupled epigenetic and gene expression profiling with whole-genome sequencing. As a result, we mapped H3K27ac sites in the tumoral tissue and unraveled overlapping somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development and epigenetic modifying genes, including key components of the polycomb complex. Chromatin remodeling is apparent as insulinoma-selective regions are mostly clustered in regulatory domains, shared across patients and containing a specific set of regulatory sequences dominated by the binding motif of the transcription factor SOX17. Moreover, a large fraction of these regions are H3K27me3-repressed in unaffected {beta}-cells, suggesting that tumoral transition is coupled with derepression of {beta}-cell polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements and transcription factors that alter {beta}-cell function and fate in their progression to pancreatic neuroendocrine tumors and a framework to identify coding and noncoding driver mutations.

genomics↗

bioRxiv2023

A single-cell transcriptomic atlas reveals resident dendritic-like cells in the zebrafish brain parenchyma

Recent studies have highlighted the heterogeneity of the immune cell compartment within the steady-state murine and human CNS. However it is not known whether this diversity is conserved among non mammalian vertebrates, especially in the zebrafish, a model system with increasing translational value. Here, we reveal the complexity of the immune landscape of the adult zebrafish brain. Using single-cell transcriptomics, we characterized these different immune cell subpopulations, including cell types that have not been -or have only been partially-characterized in zebrafish so far. By histology, we found that, despite microglia being the main immune cell type in the parenchyma, the zebrafish brain is also populated by a distinct myeloid population that shares a gene signature with mammalian dendritic cells (DC). Notably, zebrafish DC-like cells rely on batf3, a gene essential for the development of conventional DC1 in the mouse. Using specific fluorescent reporter lines that allowed us to reliably discriminate DC-like cells from microglia, we quantified brain myeloid cell defects in commonly used irf8-/-, csf1ra-/- and csf1rb-/- mutant fish, revealing previously unappreciated distinct microglia and DC-like phenotypes. Overall, our results suggest a conserved heterogeneity of brain immune cells across vertebrate evolution and also highlights zebrafish-specific brain immunity characteristics.

neuroscience↗

bioRxiv2022

Iron accumulation drives fibrosis, senescence, and the senescence-associated secretory phenotype

Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). However, the mechanisms involved in the conversion of damaged cells into senescent cells remain incompletely understood. Here, we report that multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries, through the release of iron, are efficient in triggering senescence and fibrosis. Interestingly, the accumulation of iron is an intrinsic property of senescent cells that does not require an abnormal surge in extracellular iron. Upon damage, cells initiate an iron accumulation response with abundant ferritin-bound iron within lysosomes and high levels of labile iron, the latter being a main driver of senescence-associated ROS and SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging (MRI) is a powerful non-invasive method to assess fibrotic burden in the kidneys of mice and patients with renal fibrosis. Our findings establish a central role for iron accumulation in senescence and fibrogenesis.

physiology↗

bioRxiv2022

Zebrafish Galectin 3 binding protein (Lgals3bp) is the target antigen of the microglial 4C4 monoclonal antibody

BackgroundTwo decades ago, the fish-specific monoclonal antibody 4C4 was found to be highly reactive to zebrafish microglia, the macrophages of the central nervous system. This has resulted in 4C4 being widely used, in combination with available fluorescent transgenic reporters to identify and isolate microglia. However, the target protein of 4C4 remains unidentified, which represents a major caveat. In addition, whether the 4C4 expression pattern is strictly restricted to microglial cells in zebrafish has never been investigated. ResultsHaving demonstrated that 4C4 is able to capture its native antigen from adult brain lysates, we used immunoprecipitation/mass-spectrometry, coupled to recombinant expression analyses, to identify its target. The cognate antigen was found to be a paralog of Galectin 3 binding protein (Lgals3bpb), known as MAC2-binding protein in mammals. Notably, 4C4 did not recognize other paralogs, demonstrating specificity. Moreover, our data show that Lgals3bpb expression, while ubiquitous in microglia, also identifies leukocytes in the periphery, including populations of gut and liver macrophages. ConclusionsThe 4C4 monoclonal antibody recognizes Lgals3bpb, a predicted highly glycosylated protein whose function in the microglial lineage is currently unknown. Identification of Lgals3bpb as a new pan-microglia marker will be fundamental in forthcoming studies using the zebrafish model.

immunology↗

bioRxiv2021

Hnf1b-CreER, a model used for fate mapping pancreatic lineages, achieves efficient Cre-mediated recombination in duct and islet δ cells

The Hnf1b-CreERT2 BAC transgenic (Tg(Hnf1b-cre/ERT2)1Jfer) has been used extensively to trace the progeny of pancreatic ducts in developmental, regeneration, or cancer models. Hnf1b-CreERT2 transgenics have been used to show that cells from that form a duct-like plexus in the embryonic pancreas are bipotent duct-endocrine progenitors, whereas adult mouse duct cells are not a common source of {beta} cells in various regenerative settings. The interpretation of such genetic lineage tracing studies is critically dependent on a correct understanding of the cell type specificity of recombinase activity with each reporter system. We have re-examined the performance of Hnf1b-CreERT2 with a Rosa26-RFP reporter transgene. This showed inducible recombination of up to 96% adult duct cells, a much higher efficiency than previously used reporter transgenes. Despite this high duct-cell excision, recombination in and {beta} cells remained very low, similar to previously used reporters. However, nearly half of somatostatin-expressing {delta} cells showed reporter activation, which was due to Cre expression in {delta} cells rather than to duct to {delta} cell conversions. The high recombination efficiency in duct cells indicates that the Hnf1b-CreERT2 model can be useful for both ductal fate mapping and genetic inactivation studies. The recombination in {delta} cells does not modify the interpretation of studies that failed to show duct conversions to other cell types, but needs to be considered if this model is used in studies that aim to modify the plasticity of pancreatic duct cells.

developmental biology↗

bioRxiv2021

The transcriptional repressor REST is a major negative regulator of endocrine differentiation during pancreas organogenesis

Understanding genomic regulatory mechanisms of pancreas differentiation is relevant to the pathophysiology of diabetes mellitus, and to the development of replacement therapies. Numerous transcription factors promote {beta} cell differentiation, although less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrine gene programs in the embryonic pancreas. However, pancreatic Rest knock-out mice failed to show increased numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we now observe a marked increase in the formation of pancreatic endocrine cells. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts, and induced {beta}-cell specific genes in human adult duct-derived organoids. Finally, we define REST genomic programs that suppress pancreatic endocrine differentiation. These results establish a crucial role of REST as a negative regulator of pancreatic endocrine differentiation.

developmental biology↗

bioRxiv2020

Discovery and 3D imaging of a novel {triangleup}Np63-expressing basal cell type in human pancreatic ducts with implications in disease

ObjectiveAn aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) exists, driven by {Delta}Np63. In other epithelia, {Delta}Np63+ basal cells have stem cell capacity and can be at the origin of tumors. In the pancreas, basal cells have not been identified. DesignWe assessed basal cell markers in human and mouse pancreas, chronic pancreatitis and PDAC, and developed a 3D imaging protocol (FLIP-IT) to study sizeable samples at single cell resolution. We generated organoid cultures of ducts from Sox9-eGFP reporter mice. ResultsIn normal human pancreas, rare {Delta}Np63+ cells exist in ducts that expand in chronic pancreatitis. {Delta}Np63+ cells express KRT19 and canonical basal markers (KRT5, KRT14 and S100A2) but lack markers of duct cells such as CA19.9 and SOX9. In addition, {Delta}Np63+ cells pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views of the ductal tree in formalin fixed paraffin embedded samples show that basal cells are localized on the basal membrane of medium to large ducts and expand as multilayer dome-like structures in chronic pancreatitis. In mice, {Delta}Np63 expression is induced when culturing organoids from Sox9-low ductal cells but could not be found in normal pancreas nor in models of pancreatitis or pancreatic cancer. ConclusionWe discovered a novel ductal cell population in normal human pancreas similar to basal cells in other tissues. Using FLIP-IT, we provide unprecedented 3D visualization of these cells in archival clinical specimens. {Delta}Np63+ cells may play an important role in pancreatic tissue regeneration and cancer. SUMMARY BOXWhat is already known about this subject? O_LI{Delta}Np63 has a central role in determining the basal-like subtype of pancreatic ductal adenocarcinoma (PDAC). C_LIO_LIDifferent to other tissues with basal cancers, the normal pancreas reportedly does not contain ({Delta}Np63-expressing) basal cells. C_LIO_LICurrent protocols face severe limitations for marker-based identification and 3D imaging of individual (rare) cells in archival pancreatic samples. C_LI What are the new findings? O_LIWe report a rare and atypical pancreatic duct cell that expresses {Delta}Np63, other basal cell markers and g.i. stem cell markers. C_LIO_LIThe number of these basal cells increases in diseases such as chronic pancreatitis and pancreatic cancer. C_LIO_LIWe provide an easy to implement protocol for 3D clearing and high-resolution imaging of sizeable samples of (fresh or FFPE) human pancreas or an entire mouse pancreas. C_LIO_LIExcept after culturing medium to large ducts as organoids, we fail to detect basal cells in mouse experimental pancreatic models. C_LI How might it impact on clinical practice in the foreseeable future? O_LIExtrapolating from knowledge in other organs, basal cells in the pancreas may have a stem cell/progenitor role, including in diseases such as (basal) pancreatic cancer. C_LIO_LIUse of the 3D imaging protocol in archival clinical specimens will allow unprecedented insights in pancreatic histopathology. C_LIO_LIFor above mentioned diseases, we caution for findings in experimental mouse models that may not (fully) recapitulate the etiopathogenesis. C_LI

pathology↗

bioRxiv2020

Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes

Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. Here, we analyzed mpeg1:GFP-expressing macrophages in csf1r-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, Csf1r deficiency disrupts embryonic to adult macrophage development. Csf1r-deficient zebrafish are viable and permit analyzing the consequences of macrophage loss throughout life.

developmental biology↗

bioRxiv2019

The macrophage-expressed gene (mpeg) 1 identifies a subpopulation of B cells in the adult zebrafish

The mononuclear phagocytic system (MPS) consists of many cells, in particular macrophages, scattered throughout the body. However, there is increasing evidence for the heterogeneity of tissue-resident macrophages, leading to a pressing need for new tools to discriminate MPS subsets from other hematopoietic lineages. Mpeg1.1 is an evolutionary conserved gene encoding perforin-2, a pore-forming protein associated with host defense against pathogens. Zebrafish mpeg1.1:GFP and mpeg1.1:mCherry reporters were originally established to specifically label macrophages. Since, more than 100 peer-reviewed publications have made use of mpeg1.1-driven transgenics for in vivo studies, providing new insights into key aspects of macrophage ontogeny, activation and function. However, while the macrophage-specific expression pattern of the mpeg1.1 promoter has been firmly established in the zebrafish embryo, it is currently not known whether this specificity is maintained through adulthood. Here we report direct evidence that beside macrophages, a subpopulation of B-lymphocytes is marked by mpeg1.1 reporters in most adult zebrafish organs. These mpeg1.1+ lymphoid cells endogenously express mpeg1.1 and can be separated from mpeg1.1+ macrophages by virtue of their light-scatter characteristics using FACS. Remarkably, our analyses also revealed that B-lymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1-positive population in irf8null myeloid-defective mutants, which were previously reported to recover tissue-resident macrophages in adulthood. One notable exception are skin macrophages, whose development and maintenance appear to be independent from irf8, similar to mammals. Collectively, our findings demonstrate that irf8 functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage-specific markers to study the MPS in adult zebrafish. SUMMARY SENTENCEMpeg1 is not a restricted macrophage marker, but also labels B cells in the adult zebrafish. Therefore, previously identified irf8-independent macrophages likely consist of B lymphocytes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/836098v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@128a738org.highwire.dtl.DTLVardef@1fa6097org.highwire.dtl.DTLVardef@db61b2org.highwire.dtl.DTLVardef@eb2e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗