Vertebrate Liver Research Articles

Identification and expression of a novel class of glutathione- S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver

Glutathione- S-transferases have been identified in all the living species examined so far, yet little is known to date about them in amphioxus, a model organism for insights into the origin and evolution of vertebrates. We have isolated a cDNA encoding an amphioxus ( Branchiostoma belcheri) glutathione- S-transferase with a predicted molecular mass of approximately 26 kDa, from the gut cDNA library. The glutathione- S-transferase had 43.7–51.8% identity to most glutathione- S-transferases identified from aquatic organisms including fish and green alga, but it was much less identical (<27%) to other cytosolic glutathione- S-transferase classes. The phylogenetic analysis revealed that the glutathione- S-transferase was grouped together with most piscine and algal glutathione- S-transferases, separating from other cytosolic glutathione- S-transferase classes. Moreover, the glutathione- S-transferase had an exon–intron organization typical of zebrafish putative GST, red sea bream GSTR1 and plaice GSTA1 genes. The recombinant glutathione- S-transferase has been successfully expressed and purified, which showed a relatively high catalytic activity (3.37 ± 0.1 unit/mg) toward 1-chloro-2, 4-dinitrobenzene and a moderate activity toward ethacrynic acid (0.41 ± 0.01 unit/mg), although it had no detectable activity toward 1, 2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 4-nitrobenzyl chloride and cumene hydroperoxide. In addition, we have revealed a tissue-specific expression pattern of the glutathione- S-transferase gene in B. belcheri, with the most abundant expression in the hepatic caecum. All these indicate that the amphioxus glutathione- S-transferase belongs to a novel rho-class of glutathione- S-transferases with a tissue-specific expression pattern. The relation between the glutathione- S-transferase expression in amphioxus hepatic caecum and the origin of vertebrate liver is also discussed.

PHENOTYPIC CHARACTERIZATION OF PEKIN, A NOVEL ZEBRAFISH MUTANT WITH DEFECTS IN BILE DUCT DEVELOPMENT AND PIGMENTATION

We use the zebrafish as a model to study liver development. Vertebrate liver and biliary development is well conserved across species, and zebrafish are well suited to study developmental genetics. We have been screening F3 larvae generated from fish mutagenized with ethylnitrosourea using the compound PED6. PED6 is a fluorescent lipid that is swallowed by the larva and is transported through the portal circulation to the liver, and then concentrates in the gallbladder. Defects along this pathway, including morphological defects of the intrahepatic bile ducts, result in decreased uptake by the gallbladder, which can be easily screened for in large numbers of F3 families. We have identified several novel mutants in which intrahepatic bile duct development is affected. One of these mutants, pekin (pkn), also exhibits abnormal pigmentation. We report here the initial phenotypic characterization of pkn.

Alanine Aminotransferase in Amphioxus: Presence, Localization and Up-regulation after Acute Lipopolysaccharide Exposure

Alanine aminotransferase (AAT) is mainly synthesized in the liver, and its level in mammalian serum is elevated after acute phase induction. Here we demonstrated that sheep anti-human AAT antibody cross-reacted with amphioxus humoral fluids as well as human serum; and the concentration of AAT in the humoral fluids in amphioxus increased after the acute challenge with lipopolysaccharide, while the level of total proteins remains unchanged. These suggest the presence of the same acute phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AAT was localized in the hepatic diverticulum, ovary and testis. It appears that the hepatic diverticulum in amphioxus is functionally homologous to the vertebrate liver in respect of AAT synthesis, supporting the hypothesis that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.

Identification of two cationic amino acid transporters required for nutritional signaling during mosquito reproduction

The defining characteristic of anautogenous mosquitoes is their requirement for a blood meal to initiate reproduction. The need for blood drives the association of vector and host, and is the primary reason why anautogenous mosquitoes are effective disease vectors. During mosquito vitellogenesis, a key process in reproduction, yolk protein precursor (YPP) gene expression is activated specifically in the fat body, the insect analogue of the vertebrate liver. We have demonstrated that blood meal derived amino acids (AAs) activate YPP genes via the target of rapamycin (TOR)-signal transduction pathway. Here we show, by stimulating fat bodies with balanced AA solutions lacking individual AAs, that specific cationic and branched AAs are essential for activation of the vitellogenin (vg) gene, the major YPP gene. Treatment of fat bodies with AA uptake inhibitors results in a strong inhibition of AA-induced vg gene expression proving that an active transport mechanism is necessary to transduce the AA signal. We identified two cationic AA transporters (CATs) in the fat body of Aedes aegypti females--Aa slimfast and iCAT2. RNAi knockdown of slimfast and iCAT2 results in a strong decrease in the response to AAs by the vg gene similar to that seen due to TOR inhibition. These data demonstrate that active uptake of specific AAs plays a key role in nutritional signaling during the onset of vitellogenic gene expression in mosquitoes and it is mediated by two cationic AA transporters.

Immunohistochemical localization of vitellogenin in the hepatic diverticulum of the amphioxus Branchiostoma belcheri tsingtauense, with implications for the origin of the liver

Abstract. The localization of vitellogenin (Vg) remains untested in amphioxus. Western blotting analysis showed that mouse anti‐amphioxus Vg sera cross‐reacted with the crude extracts of the hepatic diverticulum and ovary, and humoral fluids, including blood and coelomic fluid, from specimens of Branchiostoma belcheri. Similarly, immunohistochemical staining also revealed that Vg is localized in the hepatic diverticulum, sub‐intestinal vessels, and ovary in amphioxus. The hepatic diverticulum of amphioxus, like the vertebrate liver, synthesizes Vg, suggesting that amphioxus hepatic diverticulum is functionally equivalent to the vertebrate liver in respect of the synthesis of this compound. This agrees with the idea that amphioxus hepatic diverticulum is the precursor of vertebrate liver.

Demonstration of plasminogen‐like protein in amphioxus with implications for the origin of vertebrate liver

AbstractPlasminogen, the proenzyme of serine protease plasmin, is a plasma glycoprotein synthesized primarily in the liver, and its evolutionary origin in chordates remains unclear. We demonstrated here that the humoral fluid in amphioxus is capable of cross‐reacting with anti‐human or anti‐mouse plasminogen antibodies, and the hepatic diverticulum in amphioxus is the site of plasminogen‐like protein synthesis. The presence of plasminogen‐like protein in amphioxus pushes the origin of plasminogen to before the last common ancestor of vertebrates. In addition, the localization of plasminogen‐like protein in the hepatic diverticulum suggests that the diverticulum in amphioxus is functionally homologous to the vertebrate liver in respect of plasminogen synthesis, supporting the hypothesis that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus‐like ancestor during early chordate evolution.

The Influence of Chloride on Glucose Export in Marine Crabs: Sensitivity of Glucose-6-Phosphatase to Chloride Ion

Abstract The export of cellular glucose depends on the endoplasmic reticulum bound enzyme glucose-6-phosphatase (G-6-Pase), which catalyzes the hydrolysis of glucose-6-phosphate to glucose-6, the final step required for the release of new glucose into the blood or hemolymph. The current study examined the effect of chloride on G-6-Pase activity in two marine crustaceans: the ion-regulating crab Menippe mercenaria, and the ion-conforming crab Libinia dubia. Elevated chloride (from 0 to 250 mmol L−1 Cl−) stimulated hepatopancreas G-6-Pase activity in both crabs (2 times in M. mercenaria and up to 1.6 times in L. dubia). Elevated chloride also increased glucose export from isolated M. mercenaria hepatopancreas. These results are in stark contrast to G-6-Pase chloride inhibition previously reported in vertebrate liver. The sensitivity of G-6-Pase to changes in environmental chloride was determined in crabs acclimated to low (20‰) and high (45‰) salinity. M. mercenaria acclimated to high salinity became less s...

Vitellogenin detection in Caiman latirostris (Crocodylia: Alligatoridae): a tool to assess environmental estrogen exposure in wildlife

Environmental pollution with endocrine disrupting compounds (EDCs) has adverse effects on the ecosystem's health. Caiman latirostris are widely distributed in South American aquatic ecosystems. Caimans have physiological and ecological characteristics that make them particularly vulnerable to EDCs exposure and suitable candidate as a sentinel species. Vitellogenin (Vtg) is a yolk pre-cursor protein synthesized by the liver of non-mammalian vertebrates and induced in response to estrogen. Purified plasma Vtg from caimans injected with estradiol-17beta (E2) was used to generate a polyclonal anti-body. Anti-body specificity was assessed using Western blot. The antiserum was also effective in detecting turtle Vtg, exhibiting high cross-reactivity with Vtg from Phrynops hilarii and Trachemys scripta dorbigni. We developed a specific and highly sensitive ELISA for caiman Vtg. This method has a detection limit of 0.1 ng/ml of plasma. The ELISA did not detect Vtg in plasma of non-induced male caimans. Induction of Vtg in male caimans was evaluated in response to one or two (7 days apart) doses of E2. Due to its high sensitivity, ELISA allows to measure the small increases in plasma Vtg after exposure to exogenous estrogen. A priming effect was observed following the second E2 dose, with a tenfold increase in circulating Vtg. Hepatic synthesis was confirmed by immunohistochemistry. The results presented herein suggest that detection of plasma Vtg in male caimans might become a valuable tool in biomonitoring xenoestrogen exposure in a polluted environment.

Transgenic Plasmodium berghei sporozoites expressing β-galactosidase for quantification of sporozoite transmission

Malaria transmission occurs during a blood-meal of an infected Anopheles mosquito. Visualization and quantification of sporozoites along the journey from the mosquito midgut, where they develop, to the vertebrate liver, their final target organ, is important for understanding many aspects of sporozoite biology. Here we describe the generation of Plasmodium berghei parasites that express the reporter gene lacZ as a stable transgene, under the control of the sporozoite-specific CSP promoter. Transgenic sporozoites expressing β-galactosidase can be simply visualized and quantified in an enzymatic assay. In addition, these sporozoites can be used to quantify sporozoites deposited in subcutaneous tissue during natural infection.

Presence and localization of antithrombin and its regulation after acute lipopolysaccharide exposure in amphioxus, with implications for the origin of vertebrate liver

Antithrombin (AT), which is mainly synthesized in the liver, is an acute-phase plasma protein in mammalian species. Here, we demonstrated that sheep anti-human AT antibody cross-reacted with the humoral fluids in amphioxus Branchiostoma belcheri tsingtauense as well as human serum. The concentration of AT in the humoral fluids in amphioxus decreased slightly at first and then increased after the acute challenge with lipopolysaccharide, while the level of total proteins remained unchanged. These suggest the presence of the same acute-phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AT was localized in the hepatic diverticulum. It is clear that the hepatic diverticulum in amphioxus is homologous to the vertebrate liver with respect to AT synthesis. This lends support to the hypothesis originally suggested by Müller that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.

Antagonistic Actions of Ecdysone and Insulins Determine Final Size in Drosophila

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. We show that 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. We also demonstrate that the larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size.

A mosquito-specific protein family includes candidate receptors for malaria sporozoite invasion of salivary glands

We describe a previously unrecognized protein family from Aedes and Anopheles mosquitoes, here named SGS proteins. There are no SGS homologues in Drosophila or other eukaryotes, but SGS presence in two mosquito genera suggests that the protein family is widespread among mosquitoes. Ae. aegypti aaSGS1 mRNA and protein are salivary gland specific, and protein is localized in the basal lamina covering the anatomical regions that are preferentially invaded by malaria sporozoites. Anti-aaSGS1 antibodies inhibited sporozoite invasion into the salivary glands in vivo, confirming aaSGS1 as a candidate sporozoite receptor. By homology to aaSGS1 we identified the complete complement of four SGS genes in An. gambiae, which were not recognized in the genome annotation. Two An. gambiae SGS genes display salivary gland specific expression like aaSGS1. Bioinformatic analysis predicts that SGS proteins possess heparin-binding domains, and have among the highest density of tyrosine sulphation sites of all An. gambiae proteins. The major sporozoite surface proteins (CS and TRAP) also bind heparin, and interact with sulphoconjugates during liver cell invasion. Thus, we speculate that sporozoite invasion of mosquito salivary glands and subsequently the vertebrate liver may share similar mechanisms based on sulphation. Phylogenomic analysis suggests that an SGS ancestor was involved in a lateral gene transfer.

Biochemical and physiological responses after exposure to microcystins in the crab Chasmagnathus granulatus (Decapoda, Brachyura)

Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 μg kg −1) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 μg kg −1 of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 μg kg −1. LPO levels increased in doses equal to or higher than 34 μg kg −1 after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 μg kg −1), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 μg kg −1. After 12 h of exposure to 860 μg kg −1 of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.17±0.56 μg kg −1. In crabs exposed to doses higher than 172 μg kg −1 during 72 h this value raised to 32.14±4.12 μg kg −1. The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances.

The initiation of liver development is dependent on Foxa transcription factors

The specification of the vertebrate liver is thought to occur in a two-step process, beginning with the establishment of competence within the foregut endoderm for responding to organ-specific signals, followed by the induction of liver-specific genes. On the basis of expression and in vitro studies, it has been proposed that the Foxa transcription factors establish competence by opening compacted chromatin structures within liver-specific target genes. Here we show that Foxa1 and Foxa2 (forkhead box proteins A1 and A2) are required in concert for hepatic specification in mouse. In embryos deficient for both genes in the foregut endoderm, no liver bud is evident and expression of the hepatoblast marker alpha-fetoprotein (Afp) is lost. Furthermore, Foxa1/Foxa2-deficient endoderm cultured in the presence of exogenous fibroblast growth factor 2 (FGF2) fails to initiate expression of the liver markers albumin and transthyretin. Thus, Foxa1 and Foxa2 are required for the establishment of competence within the foregut endoderm and the onset of hepatogenesis.

Microsomal Triglyceride Transfer Protein Promotes the Secretion of Xenopus laevis Vitellogenin A1

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

Assessment of Estrogenic Endocrine-Disrupting Chemical Actions in the Brain Using in Vivo Somatic Gene Transfer

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element–thymidine kinase–luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17β-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 μg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy

The Alagille Syndrome (AGS) is a heritable disorder affecting the liver and other organs. Causative dominant mutations in human Jagged 1 have been identified in most AGS patients. Related organ defects occur in mice that carry jagged 1 and notch 2 mutations. Multiple jagged and notch genes are expressed in the developing zebrafish liver. Compound jagged and notch gene knockdowns alter zebrafish biliary, kidney, pancreatic, cardiac and craniofacial development in a manner compatible with an AGS phenocopy. These data confirm an evolutionarily conserved role for Notch signaling in vertebrate liver development, and support the zebrafish as a model system for diseases of the human biliary system.

Identification of HAX-1 as a Protein That Binds Bile Salt Export Protein and Regulates Its Abundance in the Apical Membrane of Madin-Darby Canine Kidney Cells

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters are targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport of bile acids, drugs, amphipathic cations, phospholipids, and conjugated organic anions, respectively. Changes in activity and abundance of transporters in the canalicular membrane regulate bile flow; however, little is known regarding cellular proteins that bind ABC transporters and regulate their trafficking. A yeast two-hybrid screen identified HAX-1 as a binding partner for BSEP, MDR1, and MDR2. The interactions were validated biochemically by glutathione S-transferase pull-down and co-immunoprecipitation assays. BSEP and HAX-1 were over-represented in rat liver subcellular fractions enriched for canalicular membrane vesicles, microsomes, and clathrin-coated vesicles. HAX-1 was bound to BSEP, MDR1, and MDR2 in canalicular membrane vesicles and co-localized with BSEP and MDR1 in the apical membrane of Madin-Darby canine kidney (MDCK) cells. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. Pulse-chase studies indicated that HAX-1 depletion did not affect BSEP translation, post-translational modification, delivery to the plasma membrane, or half-life. HAX-1 depletion resulted in an increased peak of metabolically labeled apical membrane BSEP at 4 h and enhanced retention at 6 and 9 h. HAX-1 also interacts with cortactin. Expression of dominant negative cortactin increased steady state levels of BSEP 2-fold in the apical membrane of MDCK cells, as did expression of dominant negative EPS15. These findings suggest that HAX-1 and cortactin participate in BSEP internalization from the apical membrane.

Target of rapamycin-mediated amino acid signaling in mosquito anautogeny.

Mosquitoes generate an enormous burden on human health worldwide. Disease-transmitting species use a reproductive strategy, termed anautogeny, that requires a blood meal to initiate egg maturation. Whereas this strategy is important for driving disease transmission, the molecular mechanisms underlying this phenomenon are still poorly understood. The production of yolk protein precursors (YPPs), a central event in egg maturation, is called vitellogenesis. YPPs are synthesized in the fat body, the insect analogue of the vertebrate liver. Mosquito vitellogenesis is regulated by the steroid hormone 20 hydroxyecdysone (20E). However, 20E alone is not capable of activating vitellogenesis in vivo. Here, we report that amino acid signaling through the nutrient-sensitive target of rapamycin (TOR) pathway is essential for the activation of YPP gene expression. An increase in extracellular amino acid levels, similar to the increase observed after a blood meal, is critical for 20E stimulation of YPP gene expression. Treatment with the TOR kinase inhibitor rapamycin significantly inhibits YPP expression. We used RNA interference to knockdown the expression of two key proteins of the TOR signaling pathway, TOR, and tuberous sclerosis complex 2. Knockdown of TOR inhibited amino acid stimulation while knockdown of tuberous sclerosis complex 2, a negative regulator of TOR signaling, resulted in enhanced YPP expression. Thus, amino acid-based TOR signaling regulates the activation of egg development after a blood meal, an adaptation to the unique life style of mosquitoes.

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.