The liver and exocrine pancreas share a common structure, with functioning units (hepatic plates and pancreatic acini) connected to the ductal tree. Here we show that Sox9 is expressed throughout the biliary and pancreatic ductal epithelia, which are connected to the intestinal stem-cell zone. Cre-based lineage tracing showed that adult intestinal cells, hepatocytes and pancreatic acinar cells are supplied physiologically from Sox9-expressing progenitors. Combination of lineage analysis and hepatic injury experiments showed involvement of Sox9-positive precursors in liver regeneration. Embryonic pancreatic Sox9-expressing cells differentiate into all types of mature cells, but their capacity for endocrine differentiation diminishes shortly after birth, when endocrine cells detach from the epithelial lining of the ducts and form the islets of Langerhans. We observed a developmental switch in the hepatic progenitor cell type from Sox9-negative to Sox9-positive progenitors as the biliary tree develops. These results suggest interdependence between the structure and homeostasis of endodermal organs, with Sox9 expression being linked to progenitor status.

We have used confocal microangiography to examine and describe the vascular anatomy of the developing zebrafish, Danio rerio. This method and the profound optical clarity of zebrafish embryos make it possible to view the entire developing vasculature with unprecedented resolution. A staged series of three-dimensional images of the vascular system were collected beginning shortly after the onset of circulation at 1 day postfertilization through early- to midlarval stages at approximately 7 days postfertilization. Blood vessels in every region of the animal were imaged at each stage, and detailed "wiring patterns" were derived describing the interconnections between every major vessel. We present an overview of these data here in this paper and in an accompanying Web site "The interactive atlas of zebrafish vascular anatomy" online at (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We find a highly dynamic but also highly stereotypic pattern of vascular connections, with different sets of primitive embryonic vessels severing connections and rewiring in new configurations according to a reproducible plan. We also find that despite variation in the details of the vascular anatomy, the basic vascular plan of the developing zebrafish shows strong similarity to that of other vertebrates. This atlas will provide an invaluable foundation for future genetic and experimental studies of vascular development in the zebrafish.

doi:10.1242/dev.00733 We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP) y1 Summary zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aortaderived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this networ

Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca channel Ca1.2 to trigger intracellular Ca oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca channel blockers (CCBs) or by knockdown of Ca1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Ca1.2 restored Ca oscillations and virus infection in Ca1.2-depleted cells, demonstrating the significance of Ca1.2 sialylation. Taken together, we identify Ca1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.

Ectopic pancreas is a developmental anomaly occasionally found in humans. Hes1, a main effector of Notch signaling, regulates the fate and differentiation of many cell types during development. To gain insights into the role of the Notch pathway in pancreatic fate determination, we combined the use of Hes1-knockout mice and lineage tracing employing the Cre/loxP system to specifically mark pancreatic precursor cells and their progeny in Ptf1a-cre and Rosa26 reporter mice. We show that inactivation of Hes1 induces misexpression of Ptf1a in discrete regions of the primitive stomach and duodenum and throughout the common bile duct. All ectopic Ptf1a-expressing cells were reprogrammed, or transcommitted, to multipotent pancreatic progenitor status and subsequently differentiated into mature pancreatic exocrine, endocrine, and duct cells. This process recapitulated normal pancreatogenesis in terms of morphological and genetic features. Furthermore, analysis of Hes1/Ptf1a double mutants revealed that ectopic Ptf1a-cre lineage-labeled cells adopted the fate of regionappropriate gut epithelium or endocrine cells similarly to Ptf1a-inactivated cells in the native pancreatic buds. Our data demonstrate that the Hes1-mediated Notch pathway is required for region-appropriate specification of pancreas in the developing foregut endoderm through regulation of Ptf1a expression, providing novel insight into the pathogenesis of ectopic pancreas development in a mouse model.

Activating transcription factor (ATF)/cAMP-response element (CRE)-binding (CREB) proteins induce the CRE-mediated gene transcription depending on the cAMP stimulation. cAMPdependent signaling oscillates in a circadian manner, which in turn also sustains core oscillation machinery of the circadian clock. Here, we show that among the ATF/CREB family proteins, ATF4 is essential for the circadian expression of the Period2 (Per2) gene, a key component of the circadian clock. Transcription of the Atf4 gene was regulated by core components of the circadian clock, and its expression exhibited circadian oscillation in mouse tissues as well as embryonic fibroblasts. ATF4 bound to the CRE of the Per2 promoter in a circadian time-dependent manner and periodically activated the transcription of the Per2 gene. Consequently, the oscillation of the Per2 expression was attenuated in embryonic cells prepared from Atf4-null mice. Furthermore, the loss of ATF4 also disrupted the rhythms in the expression of other clock genes. These results suggest that ATF4 is a component responsible for sustaining circadian oscillation of CRE-mediated gene expression and also constitute a molecular link connecting cAMP-dependent signaling to the circadian clock.Genetic and molecular approaches have identified a basic mechanism of the circadian oscillator that is governed by interconnected transcriptional and translational feedback loops (1, 2). Gene products of Clock and Bmal1 (also known as Arntl) form a heterodimer that activates the transcription of Period (Per) and Cryptochrome (Cry) genes. Once PER and CRY proteins have reached a critical concentration, they attenuate CLOCK/BMAL1-mediated transactivation, thus generating circadian oscillation in their own transcription. Rev-erb␣ (known as Nrd1d1) is also activated by CLOCK/BMAL1 and transrepressed by PER and CRY, resulting in circadian oscillation in the expression of Rev-erb␣. In turn, REV-ERB␣ periodically represses Bmal1 transcription, thereby interconnecting the positive and negative loops (3). Like the mechanism of Reverb␣ transcription, clock genes comprising the core oscillation loop transduce downstream events by regulating the expression of clock-controlled output genes (4). cAMP-dependent signaling is involved in the circadian output pathways, but the cAMP signaling subsequently sustains the core oscillation loops in the suprachiasmatic nucleus (SCN), 3 the center of the mammalian circadian clock (5, 6). However, the regulation mechanism of cAMP to sustain the circadian oscillator remains to be elucidated.The intracellular accumulation of cAMP induces CRE-mediated gene expression via ATF/CREB protein activation (7). ATF/CREB proteins belong to the bZIP transcription factor superfamily, and they are characterized by a conserved domain including highly charged basic amino acids that are required for DNA recognition at the TGACGT(C/A)(G/A) sequence (8). Although the phosphorylated states of CREB in the SCN vary in a circadian manner (5), the functional importance of the transcriptional...

The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common β subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.

OBJECTIVE-Most pancreatic endocrine cells derive fromPtf1a-expressing progenitor cells. In humans, nonsense mutations in Ptf1a have recently been identified as a cause of permanent neonatal diabetes associated with pancreatic agenesis. The death of Ptf1a-null mice soon after birth has not allowed further insight into the pathogenesis of the disease; it is therefore unclear how much pancreatic endocrine function is dependent on Ptf1a in mammals. This study aims to investigate gene-dosage effects of Ptf1a on pancreas development and function in mice.RESEARCH DESIGN AND METHODS-Combining hypomorphic and null alleles of Ptf1a and Cre-mediated lineage tracing, we followed the cell fate of reduced Ptf1a-expressing progenitors and analyzed pancreas development and function in mice. RESULTS-ReducedPtf1a dosage resulted in pancreatic hypoplasia and glucose intolerance with insufficient insulin secretion in a dosage-dependent manner. In hypomorphic mutant mice, pancreatic bud size was small and substantial proportions of pancreatic progenitors were misspecified to the common bile duct and duodenal cells. Growth with branching morphogenesis and subsequent exocrine cytodifferentiation was reduced and delayed. Total ␤-cell number was decreased, proportion of non-␤ islet cells was increased, and ␣-cells were abnormally intermingled with ␤-cells. Interestingly, Pdx1 expression was decreased in early pancreatic progenitors but elevated to normal level at the mid-to-late stages of pancreatogenesis.