Biology subjects

Goessling, W.

Publications and source records attributed to Goessling, W..

12 recordsLinked to original sources

bioRxiv2026

Hepatic CD8+TOX+ T-cells are a hallmark of autoimmune hepatitis

Autoimmune hepatitis (AIH) is a chronic progressive liver disease that despite suggestive serum autoantibodies or plasma cell enrichment, remains functionally a diagnosis of exclusion. Whether the broader cellular composition of the liver might enable improved specificity of diagnosis has not been systematically tested. We prospectively recruited patients undergoing a clinically-indicated liver biopsy for suspected AIH and performed single-nucleus RNA sequencing (snRNA-seq) on biopsy tissue to map the cellular landscape of AIH and its diagnostic mimics. Unsupervised clustering on cell-type abundances alone largely separated AIH from non-AIH samples. Among individual populations, a subset of CD8 T-cells marked by high TOX and PD1 expression was the most discriminating feature: its enrichment perfectly distinguished AIH by both snRNA-seq and in situ density (AUC = 1.00), outperforming plasma cell abundance (AUC = 0.83). CD8TOX T-cell enrichment may therefore be the histologic lesion that marks the diagnosis of AIH.

pathology↗

bioRxiv2024

Single-cell multi-omic analysis of fibrolamellar carcinoma reveals rewired cell-to-cell communication patterns and unique vulnerabilities

Fibrolamellar carcinoma (FLC) is a rare malignancy disproportionately affecting adolescents and young adults with no standard of care. FLC is characterized by thick stroma, which has long suggested an important role of the tumor microenvironment. Over the past decade, several studies have revealed aberrant markers and pathways in FLC. However, a significant drawback of these efforts is that they were conducted on bulk tumor samples. Consequently, identities and roles of distinct cell types within the tumor milieu, and the patterns of intercellular communication, have yet to be explored. In this study we unveil cell-type specific gene signatures, transcription factor networks, and super-enhancers in FLC using a multi-omics strategy that leverages both single-nucleus ATAC-seq and single-nucleus RNA-seq. We also infer completely rewired cell-to-cell communication patterns in FLC including signaling mediated by SPP1-CD44, MIF-ACKR3, GDF15-TGFBR2, and FGF7-FGFR. Finally, we validate findings with loss-of-function studies in several models including patient tissue slices, identifying vulnerabilities that merit further investigation as candidate therapeutic targets in FLC.

cancer biology↗

bioRxiv2024

Hormone-induced thrombosis is mediated through non-canonical fibrin(ogen) aggregation and a novel estrogen target in zebrafish

Venous thrombosis is a well-known complication of estrogen exposure, with nearly every woman at risk across her lifetime through contraception, pregnancy, or hormone therapy. Although estrogens alter expression of coagulation factors, the mechanisms that mediate estrogen-induced thrombosis are poorly understood, partially due to the absence of an animal model. Identification of these mediators is central to understanding of hormone-induced pathophysiology, could ascertain patients at higher risk for thrombosis, and pinpoint future therapeutic targets. The zebrafish is characterized by external development, high fecundity, optical transparency, and hemostasis is highly conserved with humans. Through a transgenic line that generates GFP-tagged fibrinogen, we show rapid onset of thrombosis after exposure to various estrogens, but not progestins or testosterone. Thrombi are localized to the venous system with evidence for clot contraction. Thrombosis is only partially impeded by anticoagulants, occurs in the absence of factor VII, factor X, and prothrombin, but is dependent on tissue factor and fibrin(ogen). Finally, targeting of all known estrogen receptors does not eliminate thrombosis. The inability to completely inhibit thrombosis through genetic/pharmacologic anticoagulation or estrogen receptor disruption suggests mechanisms different from canonical coagulation/thrombosis. These studies suggest that estrogen-induced thrombosis is a unique entity distinct from other forms of venous thrombosis.

pathology↗

bioRxiv2024

Bnip3lb-driven mitophagy sustains expansion of the embryonic hematopoietic stem cell pool

Embryonic hematopoietic stem and progenitor cells (HSPCs) have the unique ability to undergo rapid proliferation while maintaining multipotency, a clinically-valuable quality which currently cannot be replicated in vitro. Here, we show that embryonic HSPCs achieve this state by precise spatio-temporal regulation of reactive oxygen species (ROS) via Bnip3lb-associated developmentally-programmed mitophagy, a distinct autophagic regulatory mechanism from that of adult HSPCs. While ROS drives HSPC specification in the dorsal aorta, scRNAseq and live-imaging of Tg(ubi:mitoQC) zebrafish indicate that mitophagy initiates as HSPCs undergo endothelial-to-hematopoietic transition and colonize the caudal hematopoietic tissue (CHT). Knockdown of bnip3lb reduced mitophagy and HSPC numbers in the CHT by promoting myeloid-biased differentiation and apoptosis, which was rescued by anti-oxidant exposure. Conversely, induction of mitophagy enhanced both embryonic HSPC and lymphoid progenitor numbers. Significantly, mitophagy activation improved ex vivo functional capacity of hematopoietic progenitors derived from human-induced pluripotent stem cells (hiPSCs), enhancing serial-replating hematopoietic colony forming potential. HIGHLIGHTSO_LIROS promotes HSPC formation in the dorsal aorta but negatively affects maintenance thereafter. C_LIO_LIHSPCs colonizing secondary niches control ROS levels via Bnip3lb-directed mitophagy. C_LIO_LIMitophagy protects nascent HSPCs from ROS-associated apoptosis and maintains multipotency. C_LIO_LIInduction of mitophagy enhances long-term hematopoietic potential of iPSC-derived HSPCs. C_LI

cell biology↗

bioRxiv2024

Harmonizing terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) with multiplexed iterative immunofluorescence enriches spatial contextualization of cell death

Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) is an essential tool for the detection of cell death in tissues. Although TUNEL is not known to be compatible with multiplexed spatial proteomic methods, harmonizing TUNEL with such methods offers the opportunity to delineate cell-type specific cell death labeling and precise spatial contextualization of cell death in complex tissues. Here we evaluated variations of the TUNEL assay for their compatibility with a multiplexed immunofluorescence method, multiple iterative labeling by antibody neodeposition (MILAN), in two different tissues and injury models for cell death, acetaminophen-induced hepatocyte necrosis and dexamethasone-induced adrenocortical apoptosis. Using a commercial Click-iT-based assay as a standard, TUNEL signal could be reliably produced independent of antigen-retrieval method, with tissue-specific minor differences in signal-to-noise. In contrast, proteinase K treatment consistently reduced or even abrogated protein antigenicity, while pressure cooker treatment consistently enhanced protein antigenicity for the targets tested. Antibody-based TUNEL protocols using pressure-cooker antigen retrieval were MILAN erasure-compatible thus enabling harmonization of TUNEL with MILAN. As many as four staining cycles could be performed without loss of subsequent TUNEL signal, while first-round TUNEL did not influence protein antigenicity in subsequent rounds. We conclude this harmonized assay performs comparably to an established commercial assay, but preserves protein antigenicity, thus enabling versatile integration with multiplexed immunofluorescence using MILAN. We anticipate this harmonized protocol will enable broad and flexible integration of TUNEL into multiplexed spatial proteomic assays, thus vastly enhancing the spatial contextualization of cell death in complex tissues.

pathology↗

bioRxiv2023

Chronic metabolic stress drives developmental programs and loss of tissue functions in non-transformed liver that mirror tumor states and stratify survival

Under chronic stress, cells must balance competing demands between cellular survival and tissue function. In metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD/NASH), hepatocytes cooperate with structural and immune cells to perform crucial metabolic, synthetic, and detoxification functions despite nutrient imbalances. While prior work has emphasized stress-induced drivers of cell death, the dynamic adaptations of surviving cells and their functional repercussions remain unclear. Namely, we do not know which pathways and programs define cellular responses, what regulatory factors mediate (mal)adaptations, and how this aberrant activity connects to tissue-scale dysfunction and long-term disease outcomes. Here, by applying longitudinal single-cell multi-omics to a mouse model of chronic metabolic stress and extending to human cohorts, we show that stress drives survival-linked tradeoffs and metabolic rewiring, manifesting as shifts towards development-associated states in non-transformed hepatocytes with accompanying decreases in their professional functionality. Diet-induced adaptations occur significantly prior to tumorigenesis but parallel tumorigenesis-induced phenotypes and predict worsened human cancer survival. Through the development of a multi-omic computational gene regulatory inference framework and human in vitro and mouse in vivo genetic perturbations, we validate transcriptional (RELB, SOX4) and metabolic (HMGCS2) mediators that co-regulate and couple the balance between developmental state and hepatocyte functional identity programming. Our work defines cellular features of liver adaptation to chronic stress as well as their links to long-term disease outcomes and cancer hallmarks, unifying diverse axes of cellular dysfunction around core causal mechanisms.

systems biology↗

bioRxiv2022

A machine learning liver-on-a-chip system for safer drug formulation

Drug metabolism leads to biotransformations of pharmaceutical substances that alter drug efficacy, toxicity, as well as drug interactions. Modeling these processes ex vivo stands to greatly accelerate our capacity to develop safe and efficacious drugs and formulations. Recognizing the liver as the primary site of drug metabolism, here we report a novel whole-tissue ex vivo liver screening platform that enabled modeling of hepatic metabolism and tracking of hepatotoxic drug metabolites. We applied the system for the characterization of acetaminophen (APAP) metabolism and identified interactions that can mitigate the generation of toxic metabolites ex vivo. Combining our experimental platform with state-of-the-art machine learning, we validated two novel functional excipients that can prevent APAP hepatotoxicity in vivo in mice. To assess translational potential, we prototyped a novel solid dosage form with controlled release of both APAP and our functional excipients. Our this platform provides innovative potential access to actionable data on drug metabolism to support the development of new therapeutic approaches.

bioengineering↗

bioRxiv2020

osr1 maintains renal progenitors and regulates podocyte development by promoting wnt2ba through antagonism of hand2

Knowledge about the genetic pathways that control renal cell lineage development is essential to better understand the basis of congenital malformations of the kidney and design regenerative medicine therapies. The embryonic zebrafish kidney, or pronephros, contains two nephrons that are conserved with humans. Recently, the transcription factors Osr1 and Hand2 were found to exert antagonistic influences to balance kidney specification (Perens et al., 2016). Here, we performed a forward genetic screen in zebrafish to identify nephrogenesis regulators, where whole genome sequencing of the novel oceanside (ocn) mutant revealed a nonsense mutation in osr1. ocn mutants evince severe pronephros defects including abrogation of podocytes and proximal tubule cells. Our studies reveal that osr1 is not needed to specify renal progenitors, but rather required to maintain their survival. Additionally, osr1 is requisite for expression of the canonical Wnt ligand wnt2ba, where wnt2ba is expressed in the intermediate mesoderm (IM) and later restricts to podocytes. Deficiency of wnt2ba reduced podocyte progenitors, where overexpression of wnt2ba was sufficient to rescue the podocyte lineage as well as osr1 loss of function. Finally, we demonstrate that reciprocal antagonism between osr1 and hand2 mediates podocyte development specifically by controlling wnt2ba expression in the IM. Together, our data show that Osr1 is essential for a sequence of temporal functions that mediate the survival and lineage decisions of IM progenitors, and subsequently the maintenance of podocytes and proximal tubule epithelium in the embryonic nephron.

developmental biology↗

bioRxiv2020

The cationic amino acid exporter Slc7a7 is vital for and induced in tissue macrophages with sustained efferocytic activity

Most tissues harbor a substantial population of resident macrophages. It is not quite known yet how their quite diverse phenotypes are shaped by the functions that they assume in each tissue. In this study, we elucidate a functional link between the Slc7a7 cationic amino acid transporter and tissue macrophages. We had identified a mutant zebrafish devoid of microglia due to a mutation in the slc7a7 gene. We found that in Slc7a7 deficient larvae, macrophages do enter the retina and brain to become microglia, but then die during the developmental wave of neuronal apoptosis, which triggers intense efferocytic work from them. A similar macrophage demise occurs at other tissues and stages whereby macrophages have to engulf many cell corpses, be it due to developmental or experimentally triggered cell death. We found that slc7a7 is by far the main cationic amino acid transporter gene expressed in macrophages of wild type zebrafish larvae, and that its expression is induced in tissue macrophages within 1-2 hrs upon efferocytosis. Our data altogether indicate that a high level of Slc7a7 is vital not only for microglia but also for any steadily efferocytic tissue macrophages, and that slc7a7 gene induction is one of the adaptive responses that allow them to cope with the catabolism of numerous dead cells without compromising their own viability.

developmental biology↗

bioRxiv2020

Live imaging defines the dynamics and molecular basis of in vivo mitophagy

Mitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia, and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive, fluorescent probes mito-Keima and mito-EGFP-mCherry and used quantitative intravital imaging to illuminate mitophagy during physiological stresses--embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress-response dynamics, and time-lapse imaging revealed mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor bnip3 prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes bnip3la (nix), fundc1, pink1 or prkn (Parkin) had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.

cell biology↗

bioRxiv2018

Tfap2a is a novel gatekeeper of differentiation in renal progenitors during kidney development

Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular populations with discrete physiological tasks. Knowledge about the terminal differentiation programs of each nephron segment has central importance for understanding kidney disease and to advance regenerative medicine, as mammalian nephrons grown in organoid cultures from pluripotent cells fail to terminally differentiate. Here, from a novel forward genetic screen using zebrafish we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that controls the progression of nephron distal segment progenitors into the differentiated state. Overexpression of tfap2a rescued differentiation in mutants and caused ectopic expression of distal segment markers in wild-type nephrons, indicating tfap2a is sufficient to instigate the distal segment differentiation program. tfap2a/2b deficiency exacerbated distal nephron segment differentiation defects, revealing functional redundancy where tfap2a has a dominant role upstream of its family member. With further genetic studies, we assembled a blueprint of the tfap2a gene regulatory network during nephrogenesis. We demonstrate that tfap2a acts downstream of Iroquois homeobox 3b, a conserved distal lineage transcription factor. tfap2a controls a circuit consisting of irx1a, tfap2b, and genes encoding solute transporters that dictate the specialized metabolic functions of the distal nephron segments, and we show for the first time that this regulatory node is distinct from the pathway circuits controlling aspects such as apical-basal polarity and ciliogenesis during the differentiation process. Thus, our studies reveal new insights into the genetic control of differentiation, where tfap2a regulates the suite of segment transporter traits. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to make functional units that can facilitate organoid applications such as drug discovery and regenerative therapies.\n\nSummary StatementHere, we report for the first time that transcription factor AP-2 alpha (tfap2a) controls the progression from nephron progenitor into the fully differentiated state. This fundamentally deepens our knowledge about the genetic control of kidney development.

developmental biology↗

bioRxiv2018

Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap-/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis: transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap-/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improved Yap-deficiency phenotypes, indicating functional importance of glucose-fuelled nucleotide biosynthesis. Yap-regulated Glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signalling in metabolic homeostasis.

developmental biology↗