Anterior Endoderm Research Articles

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.

Early Activation of FGF and Nodal Pathways Mediates Cardiac Specification Independently of Wnt/β-Catenin Signaling

BackgroundCardiac induction, the first step in heart development in vertebrate embryos, is thought to be initiated by anterior endoderm during gastrulation, but what the signals are and how they act is unknown. Several signaling pathways, including FGF, Nodal, BMP and Wnt have been implicated in cardiac specification, in both gain- and loss-of-function experiments. However, as these pathways regulate germ layer formation and patterning, their specific roles in cardiac induction have been difficult to define.Methodology/Principal FindingsTo investigate the mechanisms of cardiac induction directly we devised an assay based on conjugates of anterior endoderm from early gastrula stage Xenopus embryos as the inducing tissue and pluripotent ectodermal explants as the responding tissue. We show that the anterior endoderm produces a specific signal, as skeletal muscle is not induced. Cardiac inducing signal needs up to two hours of interaction with the responding tissue to produce an effect. While we found that the BMP pathway was not necessary, our results demonstrate that the FGF and Nodal pathways are essential for cardiogenesis. They were required only during the first hour of cardiogenesis, while sustained activation of ERK was required for at least four hours. Our results also show that transient early activation of the Wnt/β-catenin pathway has no effect on cardiogenesis, while later activation of the pathway antagonizes cardiac differentiation.Conclusions/SignificanceWe have described an assay for investigating the mechanisms of cardiac induction by anterior endoderm. The assay was used to provide evidence for a direct, early and transient requirement of FGF and Nodal pathways. In addition, we demonstrate that Wnt/β-catenin pathway plays no direct role in vertebrate cardiac specification, but needs to be suppressed just prior to differentiation.

The expression of a novel cxcr4 gene in Xenopus embryo.

The aim of the present work was to identify a homologue of zebrafish cxcr4b in Xenopus, which could be involved in primordial germ cell (PGC) guidance migration. Following a BLAST analysis, the clone gi 27519681, homologous to the zebrafish gene z-cxcr4b, was identified, inserted into pCMV-SPORT6 plasmid and cloned in Escherichia coli. Embryonic expression of x-cxcr4b was analyzed by RT-PCR. X-cxcr4b was weakly expressed maternally but sharply increased after the mid-blastula transition (MBT), declining significantly at stage 45 when PGCs migration is complete. In contrast, RT-PCR of isolated presumptive PGCs showed strong maternal expression at stage 8, which decreased by stage 10 post-MBT and was not detected at stage 14. Whole mount in situ hybridization of x-cxcr4b mRNA showed that this gene is expressed in neural and haematopoietic tissues, and should be linked to important processes during embryonic development of these organs. Although weak staining could be seen in some samples within the anterior endoderm, expression of x-cxcr4b was never coincident with that of Xpat mRNA, which labels PGCs restricted to the posterior endoderm. Therefore, maternal x-cxcr4b is specifically downregulated within PGCs at pre-migratory stages while it is expressed in other tissues.

14-P005 The role of fatty acid binding proteins and polyunsaturated fatty acids in hippocampal neurogenesis

nature of Hex+Cxcr4+ (H+C+) ES cell derived ADE. Global expression analysis of ES cell derived ADE lead to the identification of an H+C+ gene expression cluster that contains well know anterior endoderm markers alongside a novel set of new sequence tags. Based on multiple pairwise comparisons between the H+C+ fraction and both differentiated and undifferentiated ES cell fractions, 66 genes were selected for further analysis. Based on cytokine response and the kinetics of gene expression in differentiation, this list was reduced to 47. In vivo expression analysis of these genes during mouse development (6.0–9.0 dpc) alongside a quantitative PCR analysis of anterior restriction, has produced an early ADE signature of five genes in addition to a number of endoderm markers that are expressed in both the anterior and posterior of the embryo. Analysis of gene ontology and domain structures in these novel genes suggests this list includes proteins involved in cell signalling, and one novel transcription factor.

Differentiation of embryonic stem cells into anterior definitive endoderm.

Anterior definitive endoderm (ADE) is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other visceral organs. Here we describe a method for the differentiation of mouse embryonic stem (ES) cells to endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (aggregation or embryoid body) culture and in a serum-free adherent monolayer culture. Purified ES cell-derived ADE cells appear committed to endodermal fates and can undergo further differentiation in vitro towards liver and pancreas with enhanced efficiency.

Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis.

BackgroundEndoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer.Methodology/Principal FindingsRA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA + cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity.Conclusions/SignificanceOur work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.

Cyp26 enzymes function in endoderm to regulate pancreatic field size

The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates are specified by retinoic acid (RA), and proper size and localization of the pancreatic field are dependent on tight control of RA signaling. Here we show that the RA-degrading Cyp26 enzymes play a critical role in defining the normal anterior limit of the pancreatic field. Disruption of Cyp26 function causes a dramatic expansion of pancreatic cell types toward the anterior of the embryo. The cyp26a1 gene is expressed in the anterior trunk endoderm at developmental stages when RA is signaling to specify pancreas, and analysis of cyp26a1/giraffe (gir) mutant zebrafish embryos confirms that cyp26a1 plays the primary role in setting the anterior limit of the pancreas. Analysis of the gir mutants further reveals that cyp26b1 and cyp26c1 function redundantly to partially compensate for loss of Cyp26a1 function. We used cell transplantation to determine that Cyp26a1 functions directly in endoderm to modulate RA signaling and limit the pancreatic field. Taken together with our finding that endodermal expression of cyp26 genes is subject to positive regulation by RA, our data reveal a feedback loop within the endoderm. Such feedback can maintain consistent levels of RA signaling, despite environmental fluctuations in RA concentration, thus ensuring a consistent size and location of the pancreatic field.

Genetic dissection of cardiac progenitor migration

The formation of heart is regulated by coordinated complex processes: cardiac progenitors migrate from both sides of the anterior lateral plate mesoderm toward the midline and differentiate into the heart tube including atrium and ventricle. Forward genetic analysis using zebrafish has identified several mutants defective in myocardial migration, resulting in two hearts known as cardia bifida. Identification of genes responsible for the cardia bifida mutations has revealed key players that regulate the migration and assembly of cardiac progenitors. Both anterior endoderm and the extraembryonic tissue yolk syncytial layer (YSL) provide for this process, thereby controlling the coordinated movement of cardiac progenitors. Further, the signaling pathway mediated by sphingosine 1-phosphate (S1P) is essential for the migration of cardiac progenitors in zebrafish.

Expression of Xenopus tropicalis HNF6/Onecut-1

Onecut genes belong to a family of transcription factors that are known to be important in embryonic development. In the present study, we analyzed the pattern of expression of Onecut-1/HNF6 in Xenopus tropicalis using RT-PCR and whole mount in situ hybridization. Expression of the Xenopus tropicalis Onecut-1/HNF6 gene was found to be conserved in the neural tube, the sensory placodes and in the anterior ventral endoderm in a domain consistent with the developing liver primordium.

Expression of Islet1 in thyroid development related to budding, migration, and fusion of primordia

The LIM homeodomain transcription factor Isl1 was investigated in mouse thyroid organogenesis. All progenitor cells of the midline thyroid diverticulum and lateral primordia (ultimobranchial bodies) expressed Isl1. This pattern persisted until the growing anlagen fused at embryonic day (E) 13.5. In Isl1 null mutants thyroid progenitors expressing Nkx2.1 and Pax8 were readily specified in the anterior endoderm but the size of the thyroid rudiment was reduced. In late development, only immature C-cells expressed Isl1. In the adult gland the number of Isl1+ cells was small compared with cells expressing calcitonin. Analysis of microarray profiles indicated a higher level of Isl1 expression in medullary thyroid carcinomas than in tumors derived from follicular cells. Together, these findings suggest that Isl1 may be a novel regulator of thyroid development before terminal differentiation of the endocrine cell types. Isl1 is an embryonic C-cell precursor marker that may be relevant also in cancer developed from the mature C-cell.

Anterior Definitive Endoderm from ESCs Reveals a Role for FGF Signaling

The use of embryonic stem cell (ESC) differentiation to generate functional hepatic or pancreatic progenitors and as a tool for developmental biology is limited by an inability to isolate in vitro equivalents of regionally specified anterior definitive endoderm (ADE). To address this, we devised a strategy using a fluorescent reporter gene under the transcriptional control of the anterior endoderm marker Hex alongside the definitive mesendoderm marker Cxcr4. Isolation of Hex(+)Cxcr4(+) differentiating ESCs yielded a population expressing ADE markers that both can be expanded and is competent to undergo differentiation toward liver and pancreatic fates. Hex reporter ESCs were also used to define conditions for ADE specification in serum-free adherent culture and revealed an unexpected role for FGF signaling in the generation of ADE. Our findings in defined monolayer differentiation suggest FGF signaling is an important regulator of early anterior mesendoderm differentiation rather than merely a mediator of morphogenetic movement.

Expandable Endodermal Progenitors: New Tools to Explore Endoderm and Its Derivatives

In the two recent issues of Cell Stem Cell, Rossant and colleagues (Seguin et al., 2008) produced expandable endoderm from hESCs by constitutive expression of Sox transcription factors, while Brickman and associates (Morrison et al., 2008) used a reporter gene strategy to isolate replicating anterior definitive endoderm from mESCs.

A differential screen for genes expressed in the extraembryonic endodermal layer of pre-primitive streak stage chick embryos reveals expression of Apolipoprotein A1 in hypoblast, endoblast and endoderm

The lower layer of the pre-gastrulating chick embryo is an extra-embryonic tissue made up of two different cell populations, the hypoblast and the endoblast. The hypoblast is characterized by the expression of inhibitory signalling molecules (e.g. Cerberus, Dickkopf1, Crescent) and others (e.g. Otx2, goosecoid, Hex, Hesx1/RPX, FGF8). However, no genes expressed in the endoblast have yet been found. We designed a differential screen to identify markers differentially expressed in these two cell populations. This only revealed one novel gene, Apolipoprotein A1 ( APO A1) with restricted endodermal layer expression. Expression of APO A1 begins very early throughout the lower layer (both hypoblast and endoblast). At later stages it is also expressed in the endoderm and its derivatives, the anterior intestinal portal endoderm and the growing liver bud.

Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo

This fate-mapping study reveals that the progenitors of all major parts of the embryonic gut are already present in endoderm of the early-head-fold to early-somite stage (1–9 somites) mouse embryo. The anterior endoderm contributes primarily to the anterior intestinal portal of the early-organogenesis stage (16–19 somites) embryo. Endoderm cells around and lateral to the node are allocated to the open “midgut” region of the embryonic gut. The posterior (post-nodal) endoderm contributes not only to the posterior intestinal portal but also the open “midgut”. Descendants of the posterior endoderm span a length of the gut from the level of the 3rd–5th somites to the posterior end of the embryonic gut. The formation of the anterior and posterior intestinal portals is accompanied by similar repertoires of morphogenetic tissue movement. We also discovered that cells on contralateral sides of the anterior endoderm are distributed asymmetrically to the dorsal and ventral sides of the anterior intestinal portal, heralding the acquisition of laterality by the embryonic foregut.

Expression of the chick Sizzled gene in progenitors of the cardiac outflow tract

The secreted Frizzled receptor related proteins (Sfrp’s) belong to a protein family that comprises antagonists and modifiers of Wnt and BMP signalling. Here we report the isolation and expression pattern of the Sfrp gene “Sizzled” in the chick. Sizzled genes, as well as the closely related crescent genes, exist in the genomes of fishes, frogs and chicks, but not of mammals. The chicken Sizzled gene (Szl) is initially expressed in the anterior endoderm of gastrulating and early head fold embryos. An additional, separate expression domain develops at the posterior end of the embryo from the head process stage onwards. Szl transcripts are then detected in precardial mesodermal cells, are transiently transcribed in the straight heart tube, and later prominently in the splanchnic mesoderm surrounding the arterial pole of the developing heart, the anterior heart field. These cells are subsequently recruited to form the cardiac outflow tract. cSzl expression is downregulated when the septation of the outflow tract by neural crest derived cells begins.

Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of theCionaembryo

Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube.

Smad2 and smad3 Are Required for Mesendoderm Induction by Transforming Growth Factor-β/Nodal Signals in Zebrafish

The transforming growth factor-beta ligands Nodal, activin, and Vg1 play important roles in mesendoderm induction and patterning during vertebrate embryogenesis. These ligands are believed to transduce the signal through the receptor-activated transcription factors Smad2 and Smad3. However, the roles of smad2/3 genes in development of zebrafish embryos are largely unknown because the presence of multiple smad2/3 genes and their maternal expression have hampered the investigation of their developmental roles. We generated potent and specific dominant-negative forms of zebrafish Smad2, Smad3a, and Smad3b by mutating multiple amino acids. Overexpression of these mutants abolished mesendoderm induction by ectopic Nodal signaling in zebrafish embryos. Expression of dominant-negative smad2/3 abrogated Smad2/3 activities in wild-type embryos and caused various mesendodermal defects similar to those in Nodal-deficient embryos. Smad2/3-deficient cells transplanted into the blastodermal margin of wild-type hosts preferentially differentiated into ectodermal tissues rather than mesendodermal tissues, supporting the idea that response of cells to mesendoderm inducers requires Smad2/3 activities. Interference with Smad2/3 activities in Zoep, Moep, and MZoep mutant embryos resulted in more severe mesendodermal defects. Thus, our data reveal that Nodal signaling and mesendoderm induction depend on Smad2/3 and suggest that transforming growth factor-beta signals other than Nodal also contribute to Smad2/3 signaling and embryonic patterning.

Towards Regenerative Therapy for Thymic Insufficiency after Hematopoietic Stem Cell Transplantation: Generation of MTS24 Positive Definitive Endoderm from Murine Embryonic Stem Cells.

Following hematopoietic stem cell transplantation (HSCT), longterm T-cell reconstitution should be established by thymus-dependent de-novo generation of naïve T-cells (thymopoiesis), which is especially important for generating a naïve T-cell pool with a broad T-cell receptor (TCR) repertoire. However, while erythroid and myeloid hematopoietic cell lineages recover rapidly following HSCT, T-cell development may severely lag behind due to thymic insufficiency. Recent studies in fetal mice have identified common thymic epithelial progenitor cells (TEPC) that were capable to re-establish a thymus in-vivo upon transplantation into a-thymic nude mice. These TEPC are characterized by expression of the transcription factor Foxn1 and by cell surface expression of MTS24. These TEPC arise exclusively from progenitors originating from the anterior foregut endoderm during embryogenesis. Therefore, we hypothesized that common TEPC may be generated in-vitro from embryonic stem (ES) cells that have differentiated towards definitive endoderm. Currently, the mechanisms underlying commitment of definitive endoderm towards a thymic fate are unknown. In order to differentiate murine ES cells towards definitive endoderm and TEPC and to identify the factors involved in the commitment of endoderm towards a thymic fate we investigated the expression of MTS24 and of genes associated with thymic differentiation in ES-cell derived endoderm using a Gcs–GFP/Sox17–huCD25 reporter ES cell line. Culture of these GscgfpSox17huCD25 ES cells in the presence of Activin A resulted in a rapid induction of mesendodermal differentiation. After 6 days of culture the majority of cells differentiated towards mesoderm (Gsc+Sox17−, 60%) and definitive endoderm (GSC+Sox17+, 35%). Apart from the addition of Activin A, the use of low passage number ES-cells and a seeding density between 200–300 cells/cm2 were the most important factors determining efficient differentiation towards definitive endoderm. Addition of insulin or WNT-3a had no significant effect on differentiation, while usage of a high passage number of ES-cells and/or a high seeding density mainly promoted development of visceral endoderm. Real-time quantitative PCR of the definitive endoderm fraction of these cultures not only showed expression of genes associated with definitive endoderm and gut tube formation (i.e. Sox17, Foxa2, Hnf4a and TCF2) but also of genes associated with anterior foregut endoderm (i.e. Hhex, Pax9) and a low, but significant, expression of Foxn1. Analysis of MTS24 expression within these cultures showed the presence of this antigen on all three cell types. The percentage of cells expressing MTS24 was highest in visceral endoderm (30–50%) and lowest in mesoderm (5–10%). The expression was approximately 12% in definitive endoderm. We conclude that murine ES cells cultured in the presence of Activin A can efficiently differentiate towards gut-tube like endoderm, including anterior forgut endoderm, and that a fraction of the generated endoderm also expresses the surface marker MTS24, suggesting the generation of epithelial progenitors with phenotypic characteristics of TEPC.

Highlights in DD

“Highlights” is a new feature that calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section will spotlight significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Quieting the Noise (Dev Dyn 236:1416–1421) Without a conductor, musicians drift out of synch, their song fragmenting into a disorganized clatter of notes. Similarly, adjacent cells in the posterior presomitic mesoderm (PSM) rely on oscillators to coordinate the rhythmic cycling of segmentation gene transcription, translation, and transcriptional repression. One challenge the segmentation clock faces is that 10–15% of PSM cells are in M-phase, during which time transcription and translation are quiescent, meaning initiation of their segmentation cycles are staggered. In this review, the authors propose a model for how synchrony is achieved. PSM cells have an intracellular oscillator led by hairy, which in a direct autoinhibitory feedback loop represses its own expression, and also represses Delta expression. Delta, in turn, is an intercellular oscillator that binds the Notch receptor on adjacent cells, and activates hairy expression. In vivo and mathematical modeling experiments support their model that Delta reduces biological noise by coupling oscillator cycling in neighboring cells. Profiling Endoderm (Dev Dyn 236:1633–1649) If you research molecular mechanisms of endoderm development, then this paper is a must-see. Endoderm, which gives rise to lung, stomach, esophagus, pancreas, liver, duodenum, gallbladder, and intestine, is arguably the least studied of the three primary germ layers. In search of genes that regulate development of these organs, Han and colleagues performed microarray analysis on Xenopus endoderm at three crucial stages: the onset of regional specification, organogenesis, and differentiation of organ-specific cell types. Anterior and posterior endoderm were compared at each stage to enrich for organspecific genes. Their painstaking approach yielded 915 candidates that are differentially expressed by at least threefold. Of these, 104 were selected for in situ hybridization. A table detailing gene expression in individual organs reveals some intriguing patterns. Interested in the liver? You may want to investigate transcription factors Tbx2 and MafB. Curious about the lung? Look into signaling molecules Semaphorin6D and Frizzled-7. The information is there for the taking. Recipe for Mature Chondrocytes (Dev Dyn 236:1954–1962) Science is a lot like cooking. Mix the ingredients, incubate, and voila! Your product is ready. Presented here is a recipe for making mature chondrocytes. The transcription factor Runx2 drives the chondrocyte maturation step, chondrocyte hypertrophy, and regulates osteoblast development. Surprisingly, retroviral overexpression of Runx2 in presomitic mesoderm (PSM) explants fails to activate expression of the chondrocyte hypertrophic marker Collagen X. However, if PSM explants are first treated with Shh then BMP4, signals surrounding developing axial chondrocytes in vivo, then ectopic Runx2 can activate chondrocyte hypertrophy markers. Forced expression of Sox9 or Nkx3.2, transcription factors expressed in immature chondrocytes, can substitute for Shh. In other conditions, Runx2 overexpressing explants instead activate osteopontin, an osteoblast marker, suggesting that Runx2 only activates Collagen X when in the right cellular milieu. In agreement, overexpression of Runx2 in combination with a transcription factor that transduces bone morphogenetic protein signals, Smad1, activates a Collagen X reporter in hypertrophic chondrocytes, but not in fibroblasts or immature chondrocyte primary cultures. What is the permissive factor necessary for chondrocyte maturation? Now that contexts in which it functions are known, it will not be long until the secret ingredient is revealed.

An Amphioxus Gli Gene Reveals Conservation of Midline Patterning and the Evolution of Hedgehog Signalling Diversity in Chordates

BackgroundHedgehog signalling, interpreted in receiving cells by Gli transcription factors, plays a central role in the development of vertebrate and Drosophila embryos. Many aspects of the signalling pathway are conserved between these lineages, however vertebrates have diverged in at least one key aspect: they have evolved multiple Gli genes encoding functionally-distinct proteins, increasing the complexity of the hedgehog-dependent transcriptional response. Amphioxus is one of the closest living relatives of the vertebrates, having split from the vertebrate lineage prior to the widespread gene duplication prominent in early vertebrate evolution.Principal FindingsWe show that amphioxus has a single Gli gene, which is deployed in tissues adjacent to sources of hedgehog signalling derived from the midline and anterior endoderm. This shows the duplication and divergence of the Gli gene family, and hence the origin of vertebrate Gli functional diversity, was specific to the vertebrate lineage. However we also show that the single amphioxus Gli gene produces two distinct transcripts encoding different proteins. We utilise three tests of Gli function to examine the transcription regulatory capacities of these different proteins, demonstrating one has activating activity similar to Gli2, while the other acts as a weak repressor, similar to Gli3.ConclusionsThese data show that vertebrates and amphioxus have evolved functionally-similar repertoires of Gli proteins using parallel molecular routes; vertebrates via gene duplication and divergence, and amphioxus via alternate splicing of a single gene. Our results demonstrate that similar functional complexity of intercellular signalling can be achieved via different evolutionary pathways.