ß-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.

Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.

Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration.

Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here, we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery.

Hematopoietic stem cell development is dependent on blood flow.

During vertebrate embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonads-mesonephros (AGM) region. We report here that blood flow is a conserved regulator of HSC formation. In zebrafish, chemical blood flow modulators regulated HSC development, and silent heart (sih) embryos, lacking a heartbeat and blood circulation, exhibited severely reduced HSCs. Flow-modifying compounds primarily affected HSC induction after the onset of heartbeat; however, nitric oxide (NO) donors regulated HSC number even when treatment occurred before the initiation of circulation, and rescued HSCs in sih mutants. Morpholino knockdown of nos1 (nnos/enos) blocked HSC development, and its requirement was shown to be cell autonomous. In the mouse, Nos3 (eNos) was expressed in HSCs in the AGM. Intrauterine Nos inhibition or embryonic Nos3 deficiency resulted in a reduction of hematopoietic clusters and transplantable murine HSCs. This work links blood flow to AGM hematopoiesis and identifies NO as a conserved downstream regulator of HSC development.

Quantitative intravital imaging in zebrafish reveals in vivo dynamics of physiological-stress-induced 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, namely, embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress response dynamics, and time-lapse imaging revealed that 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.

Phase Ib and Expansion Study of Gemcitabine, Nab-Paclitaxel, and Ficlatuzumab in Patients With Metastatic Pancreatic Cancer.

BACKGROUND: In preclinical pancreatic ductal adenocarcinoma (PDAC) models, inhibition of hepatocyte growth factor (HGF) signaling using ficlatuzumab, a recombinant humanized anti-HGF antibody, and gemcitabine reduced tumor burden. METHODS: Patients with previously untreated metastatic PDAC enrolled in a phase Ib dose escalation study with 3 + 3 design of 2 dose cohorts of ficlatuzumab 10 and 20 mg/kg administered intravenously every other week with gemcitabine 1000 mg/m2 and albumin-bound paclitaxel 125 mg/m2 given 3 weeks on and 1 week off. This was followed by an expansion phase at the maximally tolerated dose of the combination. RESULTS: Twenty-six patients (sex, 12 male:14 female; median age, 68 years [range, 49-83 years]) were enrolled, 22 patients were evaluable. No dose-limiting toxicities were identified (N = 7 pts) and ficlatuzumab at 20 mg/kg was chosen as the maximum tolerated dose. Among the 21 patients treated at the MTD, best response by RECISTv1.1: 6 (29%) partial response, 12 (57%) stable disease, 1 (5%) progressive disease, and 2 (9%) not evaluable. Median progression-free survival and overall survival times were 11.0 months (95% CI, 7.6-11.4 months) and 16.2 months (95% CI, 9.1 months to not reached), respectively. Toxicities attributed to ficlatuzumab included hypoalbuminemia (grade 3, 16%; any grade, 52%) and edema (grade 3, 8%; any grade, 48%). Immunohistochemistry for c-Met pathway activation demonstrated higher tumor cell p-Met levels in patients who experienced response to therapy. CONCLUSION: In this phase Ib trial, ficlatuzumab, gemcitabine, and albumin-bound paclitaxel were associated with durable treatment responses and increased rates of hypoalbuminemia and edema.

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 improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled 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 signaling in glucose metabolic homeostasis.

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

Most tissues harbor a substantial population of resident macrophages. Here, we elucidate a functional link between the Slc7a7 cationic amino acid transporter and tissue macrophages. We 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 in other tissues, at stages where macrophages have to engulf many cell corpses, whether due to developmental or experimentally triggered cell death. We found that Slc7a7 is the main cationic amino acid transporter expressed in macrophages of zebrafish larvae, and that its expression is induced in tissue macrophages within 1-2 h upon efferocytosis. Our data indicate that 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.

Tfap2a is a novel gatekeeper of nephron differentiation during kidney development.

Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular compartments with discrete physiological identities. Here, from a forward genetic screen using zebrafish, we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that activates the terminal differentiation program of distal segments in the pronephros. We found that tfap2a acts downstream of Iroquois homeobox 3b (irx3b), a distal lineage transcription factor, to operate a circuit consisting of tfap2b, irx1a and genes encoding solute transporters that dictate the specialized metabolic functions of distal nephron segments. Interestingly, this regulatory node is distinct from other checkpoints of differentiation, such as polarity establishment and ciliogenesis. Thus, our studies reveal insights into the genetic control of differentiation, where tfap2a is essential for regulating a suite of segment transporter traits at the final tier of zebrafish pronephros ontogeny. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to facilitate drug discovery and regenerative therapies.

Hepatic Nervous System in Development, Regeneration, and Disease.

The liver is innervated by autonomic and sensory fibers of the sympathetic and parasympathetic nervous systems that regulate liver function, regeneration, and disease. Although the importance of the hepatic nervous system in maintaining and restoring liver homeostasis is increasingly appreciated, much remains unknown about the specific mechanisms by which hepatic nerves both influence and are influenced by liver diseases. While recent work has begun to illuminate the developmental mechanisms underlying recruitment of nerves to the liver, evolutionary differences contributing to species-specific patterns of hepatic innervation remain elusive. In this review, we summarize current knowledge on the development of the hepatic nervous system and its role in liver regeneration and disease. We also highlight areas in which further investigation would greatly enhance our understanding of the evolution and function of liver innervation.

Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

Estrogen Acts Through Estrogen Receptor 2b to Regulate Hepatobiliary Fate During Vertebrate Development.

BACKGROUND AND AIMS: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions. APPROACH AND RESULTS: Exposure of zebrafish embryos to 17ß-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b-/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development. CONCLUSIONS: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy.

Clinicopathological findings in patients with COVID-19-associated ischaemic enterocolitis.

AIMS: Coronavirus disease 2019 (COVID-19) has been recognised as a predominantly respiratory tract infection, but some patients manifest severe systemic symptoms/coagulation abnormalities. The aim of this study was to evaluate the impact of severe COVID-19 infection on the gastrointestinal tract. METHODS AND RESULTS: We examined clinicopathological findings in 28 resected ischaemic bowels from 22 patients with severe COVID-19. Most patients required intubation preoperatively and presented with acute decompensation shortly before surgery. D-dimer levels were markedly elevated in all measured cases (mean, 5394 ng/ml). Histologically, 25 cases (19 patients) showed evidence of acute ischaemia with necrosis. In this group, the most characteristic finding was the presence of small vessel fibrin thrombi (24 of 25 cases, 96%), which were numerous in 64% of cases. Patients with COVID-19 were significantly more likely than a control cohort of 35 non-COVID-19-associated acute ischaemic bowels to show isolated small intestine involvement (32% versus 6%, P < 0.001), small vessel fibrin thrombi (100% versus 43%, P < 0.001), submucosal vessels with fibrinous degeneration and perivascular neutrophils (90% versus 54%, P < 0.001), fibrin strands within submucosal vessels (58% versus 20%, P = 0.007), and histological evidence of pneumatosis (74% versus 34%, P = 0.010). Three cases in this cohort had histopathological findings normally seen in the setting of chronic ischaemia, notably prominent fibroblastic proliferation affecting the outer layer of the muscularis propria. CONCLUSIONS: Herein, we describe the histopathological findings in COVID-19-associated ischaemic bowels and postulate a relationship with the hypercoagulable state seen in patients with severe COVID-19 infection. Additional experience with these cases may further elucidate specific features or mechanisms of COVID-19-associated ischaemic enterocolitis.

Hepatobiliary Differentiation: Principles from Embryonic Liver Development.

Hepatocytes and biliary epithelial cells (BECs), the two endodermal cell types of the liver, originate from progenitor cells called hepatoblasts. Based principally on in vitro data, hepatoblasts are thought to be bipotent stem cells with the potential to produce both hepatocytes and BECs. However, robust in vivo evidence for this model has only recently emerged. We examine the molecular mechanisms that stimulate hepatoblast differentiation into hepatocytes or BECs. In the absence of extrinsic cues, the default fate of hepatoblasts is hepatocyte differentiation. Inductive cues from the hepatic portal vein, however, initiate transcription factor expression in hepatoblasts, driving biliary specification. Defining the mechanisms of hepatobiliary differentiation provides important insights into congenital disorders, such as Alagille syndrome, and may help to better characterize the poorly understood hepatic lineage relationships observed during regeneration from liver injury.

Estrogen Activation of G-Protein-Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes.

BACKGROUND & AIMS: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. METHODS: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein-coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry. RESULTS: Exposure to 17ß-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. CONCLUSIONS: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. TRANSCRIPT PROFILING: The accession number in the Gene Expression Omnibus is GSE92544.

A phase 2 clinical trial of the heat shock protein 90 (HSP 90) inhibitor ganetespib in patients with refractory advanced esophagogastric cancer.

Subsets of esophagogastric (EG) cancers harbor genetic abnormalities, including amplification of HER2, MET, or FGFR2 or mutations in PIK3CA, EGFR, or BRAF. Ganetespib which is a novel triazolone heterocyclic inhibitor of HSP90, is a potentially biologically rational treatment strategy for advanced EG cancers with these gene amplification. This multicenter, single-arm phase 2 trial enrolled patients with histologically confirmed advanced EG cancer with progression on at least one line of systemic therapy. Patients received Ganetespib 200 mg/m2 IV on Days 1, 8, and 15 of a 28-day cycle. The primary endpoint was overall response rate (ORR). Secondary endpoints included: Progression Free Survival (PFS); to correlate the presence of HSP clients with ORR and PFS; evaluating the safety, tolerability and adverse events profile. In this study 26 eligible patients mainly: male 77%, median age 64 years were enrolled. The most common drug-related adverse events were diarrhea (77%), fatigue (65%), elevated ALKP (42%), and elevated AST (38%). The most common grade 3/4 AEs included: leucopenia (12%), fatigue (12%), diarrhea (8%), and elevated ALKP (8%). The ORR of 4% reflects the single patient of 26 who had a complete response and stayed on treatment for more than seventy (70) months. Median PFS and OS was 61 days (2.0 months), 94 days (3.1 months) respectively. Ganetespib showed manageable toxicity. While the study was terminated early due to insufficient evidence of single-agent activity, the durable CR and 2 minor responses suggest that there may be a subset of EG patients who could benefit from this drug.

Cannabinoid receptor signaling regulates liver development and metabolism.

Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methionine-dependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.

Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis.

Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

The zebrafish kidney mutant zeppelin reveals that brca2/fancd1 is essential for pronephros development.

The zebrafish kidney is conserved with other vertebrates, making it an excellent genetic model to study renal development. The kidney collects metabolic waste using a blood filter with specialized epithelial cells known as podocytes. Podocyte formation is poorly understood but relevant to many kidney diseases, as podocyte injury leads to progressive scarring and organ failure. zeppelin (zep) was isolated in a forward screen for kidney mutants and identified as a homozygous recessive lethal allele that causes reduced podocyte numbers, deficient filtration, and fluid imbalance. Interestingly, zep mutants had a larger interrenal gland, the teleostean counterpart of the mammalian adrenal gland, which suggested a fate switch with the related podocyte lineage since cell proliferation and cell death were unchanged within the shared progenitor field from which these two identities arise. Cloning of zep by whole genome sequencing (WGS) identified a splicing mutation in breast cancer 2, early onset (brca2)/fancd1, which was confirmed by sequencing of individual fish. Several independent brca2 morpholinos (MOs) phenocopied zep, causing edema, reduced podocyte number, and increased interrenal cell number. Complementation analysis between zep and brca2ZM_00057434 -/- zebrafish, which have an insertional mutation, revealed that the interrenal lineage was expanded. Importantly, overexpression of brca2 rescued podocyte formation in zep mutants, providing critical evidence that the brca2 lesion encoded by zep specifically disrupts the balance of nephrogenesis. Taken together, these data suggest for the first time that brca2/fancd1 is essential for vertebrate kidney ontogeny. Thus, our findings impart novel insights into the genetic components that impact renal development, and because BRCA2/FANCD1 mutations in humans cause Fanconi anemia and several common cancers, this work has identified a new zebrafish model to further study brca2/fancd1 in disease.

Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development.

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease.