Endoderm Development Research Articles

Functional analysis of Rfx6 and mutant variants associated with neonatal diabetes

Mutations in rfx6 were recently associated with Mitchell–Riley syndrome, which involves neonatal diabetes, and other digestive system defects. To better define the function of Rfx6 in early endoderm development we cloned the Xenopus homologue. Expression of rfx6 begins early, showing broad expression throughout the anterior endoderm; at later stages rfx6 expression becomes restricted to the endocrine cells of the gut and pancreas. Morpholino knockdown of rfx6 caused a loss of pancreas marker expression, as well as other abnormalities. Co-injection of exogenous wild-type rfx6 rescued the morpholino phenotype in Xenopus tadpoles, whereas attempts to rescue the loss-of-function phenotype using mutant rfx6 based on Mitchell–Riley patients were unsuccessful. To better define the pleiotropic effects, we performed microarray analyses of gene expression in knockdown foregut tissue. In addition to pancreatic defects, the microarray analyses revealed downregulation of lung, stomach and heart markers and an upregulation of kidney markers. We verified these results using RT-PCR and in situ hybridization. Based on the different rfx6 expression patterns and our functional analyses, we propose that rfx6 has both early and late functions. In early development Rfx6 plays a broad role, being essential for development of most anterior endodermal organs. At later stages however, Rfx6 function is restricted to endocrine cells.

Endodermal progenitor cells isolated from mouse pancreas

In this study we have isolated and identified a progenitor cell population located in murine pancreatic tissue. This cell population has specific properties that make it different from previously described candidate pancreatic progenytor cell populations. It is located in the periductal region outside the islet architecture yet the cells have features consistent with endodermal development and endocrine functions within the islet. Cells are proliferative and capable of differentiation into pancreatic lineages expressing pancreatic associated transcription factors and proteins and maintaining the ability to produce insulin.

Patterning and plasticity in development of the respiratory lineage

The mammalian respiratory lineage, consisting of the trachea and lung, originates from the ventral foregut in an early embryo. Reciprocal signaling interactions between the foregut epithelium and its associated mesenchyme guide development of the respiratory endoderm, from a naive sheet of cells to multiple cell types that line a functional organ. This review synthesizes current understanding of the early events in respiratory system development, focusing on three main topics: (1) specification of the respiratory system as a distinct organ of the endoderm, (2) patterning and differentiation of the nascent respiratory epithelium along its proximal-distal axis, and (3) plasticity of the respiratory cells during the process of development. This review also highlights areas in need of further study, including determining how early endoderm cells rapidly switch their responses to the same signaling cues during development, and how the general proximal-distal pattern of the lung is converted to fine-scale organization of multiple cell types along this axis.

Identification of region-specific genes in the early chicken endoderm

In vertebrates, the endoderm gives rise to the epithelial lining of the digestive tract, respiratory system and endocrine organs. After gastrulation, the newly formed endoderm gradually becomes regionalized and differentiates into specific organs. To understand the molecular basis of early endoderm regionalization, which is largely unknown, it is necessary to identify novel region-specific genes as candidates potentially involved in this process. Applying an Affymetrix Array based approach we aimed for the identification of genes specifically upregulated in the foregut or mid-/hindgut endoderm at the onset of regionalization. Several genes exhibiting spatial and temporal restricted expression patterns in the developing early endoderm were identified and their expression was validated via RT-PCR and whole mount in situ hybridization. We report here the detailed gene expression patterns of two novel genes specifically associated with foregut endoderm and of eight novel genes specifically expressed in the mid-/hindgut endoderm at HH stages 10–11. Future functional analysis of these genes may help to elucidate the mechanisms involved in endoderm development and regionalization.

FGF2 Promotes Primitive Endoderm Development in Bovine Blastocysts.

Cell fate specification begins in preimplantation embryo with the emergence of trophectoderm (TE). Primitive endoderm (PE) is the second extraembryonic lineage to form from cells within the inner cell mass (ICM) during embryogenesis. Ruminant conceptuses elongate dramatically prior to uterine attachment and extensive migration and proliferation of TE and PE is crucial for normal conceptus development and elongation. Work described here provides evidence that FGF2 and potentially other FGFs impact PE development during bovine embryonic development. Bovine blastocyst outgrowths were generated from in vitro-produced embryos using a feeder-layer free culture system containing a Matrigel-coated surface. Individually cultured bovine blastocysts were supplemented with FGF2 at the beginning of culture (day 8 post-IVF) and identification of PE-like cells was evaluated microscopically on days 13 and 15. In controls, only a single blastocyst outgrowth contained what appeared morphologically as PE cells (1.3% of all outgrowths). By contrast, 10.5±6.7%, 19.7±0.28% and 22.4±0.26% of outgrowths contained PE colonies on day 15 when embryos were exposed to 0.5, 5 and 50ng/ml FGF2, respectively. A subset of outgrowths (n=6 TE only and 6 PE-containing) were selected and cultured to day 20, then lineage-specific markers were evaluated using qRT-PCR. Transcripts for IFNT and CDX2 (TE markers) were present in TE outgrowths. Trace amounts of IFNT and CDX2 mRNA were detected in PE colonies (P<0.05) likely because residual TE cells still existed in some of these outgrowths. PE-containing outgrowths contained ample quantities of GATA4 and GATA6 mRNA (putative PE markers) whereas TE outgrowths contained no GATA4 and low amounts of GATA6 mRNA. None of the outgrowths contained NANOG mRNA whereas all TE and PE outgrowths contained OCT4 mRNA. Western blot analysis determined PE-containing outgrowths produced transferrin, a PE product. Moreover, immunoreactive GATA4 localized to nuclei of PE cells but was not detected in TE cells. By contrast, CDX2 localized to TE nuclei but was not detected in PE cells. Continued cultivation and selection for TE and PE yielded pure cell populations (n=6 of each). Differences in relative abundance of FGF receptors (FGFRs) and selective alternatively spliced receptor isoforms were evident between the cell types. Specifically, transcripts for FGFR2b and FGFR3 predominated in TE cells while FGFR1b represented the primary transcript in PE cells. TE and PE cells responded differently to FGF2 treatment. FGF2 supplementation did not affect the mitotic index of TE cells whereas the mitotic index of PE cells increased (P<0.05) following exposure to 5 or 50ng/ml FGF2 (35.1±0.09% and 34.7±0.11%, respectively versus 19.9±0.06% for controls). These observations indicate that the non-TE cells formed with blastocyst exposure to FGF2 are PE, and these cells respond differently to FGF2 than TE. Collectively, this work provides new insight into another vital activity for FGF2 and potentially other FGFs during early embryonic development in bovine. This project was supported by NRICGP number 2008-35203-19106 from the USDA-CSREES. (platform)

Klf5 regulates lineage formation in the pre-implantation mouse embryo

Kruppel-like transcription factors (Klfs) are essential for the induction and maintenance of pluripotency of embryonic stem cells (ESCs), yet little is known about their roles in establishing the three lineages of the pre-implantation embryo. Here, we show that Klf5 is required for the formation of the trophectoderm (TE) and the inner cell mass (ICM), and for repressing primitive endoderm (PE) development. Although cell polarity appeared normal, Klf5 mutant embryos arrested at the blastocyst stage and failed to hatch due to defective TE development. Klf5 acted cell-autonomously in the TE, downstream of Fgf4 and upstream of Cdx2, Eomes and Krt8. In the ICM, loss of Klf5 resulted in reduced expression of pluripotency markers Oct4 and Nanog, but led to increased Sox17 expression in the PE, suggesting that Klf5 suppresses the PE lineage. Consistent with this, overexpression of Klf5 in transgenic embryos was sufficient to suppress the Sox17(+) PE lineage in the ICM. Klf5 overexpression led to a dose-dependent decrease in Sox17 promoter activity in reporter assays in cultured cells. Moreover, in chimeric embryos, Klf5(-/-) cells preferentially contributed to the Sox17(+) PE lineage and Cdx2 expression was not rescued in Klf5(-/-) outer cells. Finally, outgrowths from Klf5(-/-) embryos failed to form an ICM/pluripotent colony, had very few Oct4(+) or Cdx2(+) cells, but showed an increase in the percentage of Sox17(+) PE cells. These findings demonstrate that Klf5 is a dynamic regulator of all three lineages in the pre-implantation embryo by promoting the TE and epiblast lineages while suppressing the PE lineage.

Intrinsic properties and external factors determine the differentiation bias of human embryonic stem cell lines

A major goal of human embryonic stem cell (hESC) research is to regulate differentiation through external means to generate specific cell types with high purity for regenerative medicine applications. Although all hESC lines express pluripotency-associated genes, their differentiation ability to various lineages differs considerably. We have compared spontaneous differentiation propensity of the two hESC lines, RelicellhES1 and BG01. Spontaneous differentiation of hESC lines grown in different media conditions was followed by differentiation using two methods. Kinetic data generated by real-time gene expression studies for differentiated cell types were analyzed, and confirmed at protein levels. Both cell lines showed upregulation of genes associated with the 3 germ layers, although stark contrast was evident in the magnitude of upregulation of lineage specific genes. A distinct difference was also found in the rate at which the pluripoteny factors, Oct-4 and Nanog, were downregulated during differentiation. Once differentiation was initiated, both Oct-4 and Nanog gene expression was drastically reduced in RelicellhES1, whereas a gradual decrease was observed in BG01. A clear trend is seen in RelicellhES1 to differentiate into neuroectodermal and mesenchymal lineages, whereas BG01 cells are more prone to mesoderm and endoderm development. In addition, suspension versus plated methods of cell culture significantly influenced the outcome of differentiation of certain types of cells. Results obtained by spontaneous differentiation of hESCs were also amplified by induced differentiation. Thus, differential rates of downregulation of pluripotency markers along with culture conditions seem to play an important role in determining the developmental bias of human ES cell lines.

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells*

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.

Endoderm development in Caenorhabditis elegans: The synergistic action of ELT-2 and -7 mediates the specification→differentiation transition

The transition from specification of cell identity to the differentiation of cells into an appropriate and enduring state is critical to the development of embryos. Transcriptional profiling in Caenorhabditis elegans has revealed a large number of genes that are expressed in the fully differentiated intestine; however, no regulatory factor has been found to be essential to initiate their expression once the endoderm has been specified. These gut-expressed genes possess a preponderance of GATA factor binding sites and one GATA factor, ELT-2, fulfills the expected characteristics of a key regulator of these genes based on its persistent expression exclusively in the developing and differentiated intestine and its ability to bind these regulatory sites. However, a striking characteristic of elt-2(0) knockout mutants is that while they die shortly after hatching owing to an obstructed gut passage, they nevertheless contain a gut that has undergone complete morphological differentiation. We have discovered a second gut-specific GATA factor, ELT-7, that profoundly synergizes with ELT-2 to create a transcriptional switch essential for gut cell differentiation. ELT-7 is first expressed in the early endoderm lineage and, when expressed ectopically, is sufficient to activate gut differentiation in nonendodermal progenitors. elt-7 is transcriptionally activated by the redundant endoderm-specifying factors END-1 and -3, and its product in turn activates both its own expression and that of elt-2, constituting an apparent positive feedback system. While elt-7 loss-of-function mutants lack a discernible phenotype, simultaneous loss of both elt-7 and elt-2 results in a striking all-or-none block to morphological differentiation of groups of gut cells with a region-specific bias, as well as reduced or abolished gut-specific expression of a number of terminal differentiation genes. ELT-2 and -7 synergize not only in activation of gene expression but also in repression of a gene that is normally expressed in the valve cells, which immediately flank the termini of the gut tube. Our results point to a developmental strategy whereby positive feedback and cross-regulatory interactions between two synergistically acting regulatory factors promote a decisive and persistent transition of specified endoderm progenitors into the program of intestinal differentiation.

Repression of Wnt signaling by a Fer-type nonreceptor tyrosine kinase

The Wnt signaling pathway must be properly modulated to ensure an appropriate output: pathological conditions result from either insufficient or excessive levels of Wnt signal. For example, hyperactivation of the Wnt pathway is associated with various cancers and subnormal Wnt signaling can lead to increased invasiveness of tumor cells. We found that the Caenorhabditis elegans ortholog of the Fer nonreceptor tyrosine kinase, FRK-1, limits Wnt signaling by preventing the adhesion complex-associated β-catenin, HMP-2, from participating in Wnt-dependent specification of the endoderm during embryogenesis. Removal of FRK-1 function results in relocalization of HMP-2 to the nucleus of epidermal cells, and allows it to substitute for WRM-1, the nuclear β-catenin that normally transduces the Wnt signal during endoderm development. APR-1, the C. elegans APC ortholog, is similarly required to prevent HMP-2 relocalization and keeps it from participating in Wnt signal transduction; this finding partially explains the paradoxical observation that APR-1 acts either negatively or positively in Wnt signaling, depending on context. The apparent hyperactivation of the Wnt response in the absence of FRK-1 leads to hyperproliferation in the endoderm, as is also seen when WRM-1 is overexpressed in wild-type embryos. The specification and proliferation activities of Wnt signaling are separable: although the Tcf/Lef factor POP-1 acts in Wnt-dependent endoderm specification, it is not apparently required for hyperproliferation resulting from excessive Wnt signaling. These findings highlight a role for a Fer-type kinase in setting the proper levels of Wnt signaling and demonstrate the importance of this modulation in ensuring appropriate cell division.

Direct transcriptional regulation of Gata4 during early endoderm specification is controlled by FoxA2 binding to an intronic enhancer

The embryonic endoderm is a multipotent progenitor cell population that gives rise to the epithelia of the digestive and respiratory tracts, the liver and the pancreas. Among the transcription factors that have been shown to be important for endoderm development and gut morphogenesis is GATA4. Despite the important role of GATA4 in endoderm development, its transcriptional regulation is not well understood. In this study, we identified an intronic enhancer from the mouse Gata4 gene that directs expression to the definitive endoderm in the early embryo. The activity of this enhancer is initially broad in all endodermal progenitors, as demonstrated by fate mapping analysis using the Cre/loxP system, but becomes restricted to the dorsal foregut and midgut, and associated organs such as dorsal pancreas and stomach. The function of the intronic Gata4 enhancer is dependent upon a conserved Forkhead transcription factor-binding site, which is bound by recombinant FoxA2 in vitro. These studies identify Gata4 as a direct transcriptional target of FoxA2 in the hierarchy of the transcriptional regulatory network that controls the development of the definitive endoderm.

Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)

Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. Mct8 (monocarboxylate transporter 8), expressed predominantly in the brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells toward neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All-trans-RA (tRA) and other RA receptor (RAR) agonists dramatically (>300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1- and 4.3-fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and 5'-rapid amplification of cDNA ends PCR analysis in F9 cells identified 11 transcription start sites and a proximal Sp1 site but no TATA box. tRA significantly enhanced Mct8 promoter activity through a consensus RA-responsive element located 6.6 kilobases upstream of the coding region. A chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid X receptor to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates cross-talk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.

Regulation of endoderm development by zebrafish Nipbl

Program/Abstract # 270 Regulation of endoderm development by zebrafish Nipbl Akihiko Muto a , Thomas F. Schilling b , Anne L. Calof b , Arthur D. Lander a a Dev & Cell Biol., Univ. of California, Irvine, CA, USA b Anatomy & Neurobiol., Univ. of California, Irvine, CA, USA Heterozygous loss of the Nipped-B-like gene (Nipbl) is the most common cause of Cornelia de Lange Syndrome (CdLS). Although Nipbl is a cohesin-associated protein conserved among all eukaryotes, recent studies suggest that it regulates gene expression through mechanisms independent of cohesin's established role in chromatid cohesion. There are 2 Nipbl genes in zebrafish, zNipbl-1 and zNipbl-2, and embryos injected with morpholinos targeting either gene (zNipbl-morphants) show defects in formation of the gut/visceral organs and heart with a range of severity from looping defects to organs duplication. Such morphants show significant reductions in sox32, sox17 and foxa2 expressions in endoderm cells, and we found that combined sox17/foxa2-double morphants exhibited gut bifurca- tions similar to zNipbl-morphants. Interestingly, the degree of sox17 and foxa2 suppression in zNipbl-morphants was greater than could be explained by the reduction in sox32 (a known positive regulator of these genes), and the response of these genes to ectopic sox32 was significantly blunted. These data suggest that zNipbls may influence sox17 and foxa2 transcription directly. In zNipbl-morphants, we also observed abnormal spatial expression of the left–right patterning genes, lefty2 and southpaw, although Kuppfer's vesicle (KV), a critical organ for left–right patterning that depends on sox32 function, formed normally, implying that zNipbls act downstream of KV formation. These results support the idea that zNipbls regulate embryonic development through modulating gene expression at multiple levels. Supported by NIH P01-HD052860. doi:10.1016/j.ydbio.2010.05.433

BrunoL1 regulates endoderm proliferation through translational enhancement of cyclin A2 mRNA

Developmental control of proliferation relies on tight regulation of protein expression. Although this has been well studied in early embryogenesis, how the cell cycle is regulated during organogenesis is not well understood. Bruno-Like RNA binding proteins bind to consensus sequences in the 3′UTR of specific mRNAs and repress protein translation, but much of this functional information is derived from studies on mainly two members, Drosophila Bruno and vertebrate BrunoL2 (CUGBP1). There are however, six vertebrate and three Drosophila Bruno family members, but less is known about these other family members, and none have been shown to function in the endoderm. We recently identified BrunoL1 as a dorsal pancreas enriched gene, and in this paper we define BrunoL1 function in Xenopus endoderm development. We find that, in contrast to other Bruno-Like proteins, BrunoL1 acts to enhance rather than repress translation. We demonstrate that BrunoL1 regulates proliferation of endoderm cells through translational control of cyclin A2 mRNA. Specifically BrunoL1 enhanced translation of cyclin A2 through binding consensus Bruno Response Elements (BREs) in its 3′UTR. We compared the ability of other Bruno-Like proteins, both vertebrate and invertebrate, to stimulate translation via the cyclin A2 3′UTR and found that only Drosophila Bru-3 had similar activity. In addition, we also found that both BrunoL1 and Bru-3 enhanced translation of mRNAs containing the 3′UTRs of Drosophila oskar or cyclin A, which have been well characterized to mediate repression. Lastly, we show that it is the Linker region of BrunoL1 that is both necessary and sufficient for this activity. These results are the first example of BRE-dependent translational enhancement and are the first demonstration in vertebrates of Bruno-Like proteins regulating translation through BREs.

ESCs Require PRC2 to Direct the Successful Reprogramming of Differentiated Cells toward Pluripotency

Embryonic stem cells (ESCs) are pluripotent, self-renewing, and have the ability to reprogram differentiated cell types to pluripotency upon cellular fusion. Polycomb-group (PcG) proteins are important for restraining the inappropriate expression of lineage-specifying factors in ESCs. To investigate whether PcG proteins are required for establishing, rather than maintaining, the pluripotent state, we compared the ability of wild-type, PRC1-, and PRC2-depleted ESCs to reprogram human lymphocytes. We show that ESCs lacking either PRC1 or PRC2 are unable to successfully reprogram B cells toward pluripotency. This defect is a direct consequence of the lack of PcG activity because it could be efficiently rescued by reconstituting PRC2 activity in PRC2-deficient ESCs. Surprisingly, the failure of PRC2-deficient ESCs to reprogram somatic cells is functionally dominant, demonstrating a critical requirement for PcG proteins in the chromatin-remodeling events required for the direct conversion of differentiated cells toward pluripotency.

Klf6/copeb is required for hepatic outgrowth in zebrafish and for hepatocyte specification in mouse ES cells

Krüppel-like factor 6 (Klf6; copeb in zebrafish) is a zinc-finger transcription factor and tumor suppressor gene. Klf6−/− mice have defects in hematopoiesis and angiogenesis and do not form a liver. However, the vascular abnormalities in Klf6−/− mice obfuscate its role in liver development since these two processes are linked in mammals. We utilized zebrafish and mouse ES cells to investigate the role of copeb in endoderm specification and hepatogenesis separate from its function in angiogenesis. During zebrafish development, copeb expression is enriched in digestive organs. Morpholino knockdown of copeb blocks expansion of the liver, pancreas and intestine, but does not affect their specification, differentiation or the vascularization of the liver. Decreased hepatocyte proliferation in copeb morphants is accompanied by upregulation of the cell cycle inhibitor, cdkn1a, a Copeb transcriptional target. A cell autonomous role for Klf6 in endoderm and hepatic development was investigated by manipulating Klf6 expression in mouse ES cells driven to differentiate along the hepatic lineage. Expression of the endoderm markers Hnf3β, Gata4, Sox17, and CxCr4 is not induced in Klf6−/− cells but is upregulated in ES cells over-expressing Klf6. Collectively, these findings indicate that copeb/Klf6 is essential for the development of endoderm-derived organs.

Prep1.2 and aldh1a2 participate to a positive loop required for branchial arches development in zebrafish

Segmentation is a key step in embryonic development. Acting in all germ layers, it is responsible for the generation of antero-posterior asymmetries. Hox genes, with their diverse expression in individual segments, are fundamental players in the determination of different segmental fates. In vertebrates, Hox gene products gain specificity for DNA sequences by interacting with Pbx, Prep and Meis homeodomain transcription factors. In this work we cloned and analysed prep1.2 in zebrafish. In-situ hybridization experiments show that prep1.2 is maternally and ubiquitously expressed up to early somitogenesis when its expression pattern becomes more restricted to the head and trunk mesenchyme. Experiments of loss of function with prep1.2 morpholinos change the shape of the hyoid and third pharyngeal cartilages while arches 4–7 and pectoral fins are absent, a phenotype strikingly similar to that caused by loss of retinoic acid (RA). In fact, we show that prep1.2 is positively regulated by RA and required for the normal expression of aldh1a2 at later stages, particularly in tissues involved in the development of the branchial arches and pectoral fins. Thus, prep1.2 and aldh1a2 are members of an indirect positive feedback loop required for pharyngeal endoderm and posterior branchial arches development. As the paralogue gene prep1.1 is more important in hindbrain patterning and neural crest chondrogenesis, we provide evidence of a functional specialization of prep genes in zebrafish head segmentation and morphogenesis.

Wnt/beta‐catenin signaling in patterning and differentiation of the vertebrate endoderm

The endoderm gives rise to organs as different in anatomy and function as the lungs, colon and liver, and we want to understand the signaling processes controlling this diversification. Our primary interest is in the pancreas, and our work indicates multiple roles for the Wnt/beta (β)‐catenin signaling pathway in its development. Early, Wnt signaling inhibits pancreas specification within the foregut; later, it promotes expansion of multipotent progenitor cells and their differentiation into exocrine acinar tissue. Until recently, our studies have focused on response to Wnt ligands, mediated by the non‐redundant functions of β‐catenin. To better understand the role of Wnt ligand production, while bypassing redundancy between the ligand‐encoding genes, we have targeted the mouse Porcn gene, homologous to Drosophila porcupine. Porcupine is required for wingless secretion and bioactivity, and we find that mouse cells lacking Porcn cannot produce any of several Wnt ligands tested, but retain normal responsiveness to exogenous Wnts. The Porcn mutant defect thus closely resembles that of fly porcupine, indicating a conserved and non‐redundant role for this gene in Wnt production. Wnt production by Porcn mutant cells is rescued by wildtype human PORCN, but not a PORCN mutant recently identified in a pleiotropic congenital syndrome, Focal Dermal Hypoplasia. Mice carrying a conditional Porcn allele are viable, and will provide a novel genetic tool to understand Wnt function in endoderm development and beyond.Supported by the NIH (R01‐DK075072) and Searle Scholars Program (06‐B‐116).

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

Conditional control of the differentiation competence of pancreatic endocrine and ductal cells by Fgf10

Fgf10 is a critical component of mesenchymal-to-epithelial signaling during endodermal development. In the Fgf10 null pancreas, the embryonic progenitor population fails to expand, while ectopic Fgf10 expression forces progenitor arrest and organ hyperplasia. Using a conditional Fgf10 gain-of-function model, we observed that the timing of Fgf10 expression affected the cellular competence of the arrested pancreatic progenitors. We present evidence that the Fgf10-arrested progenitor state is reversible and that terminal differentiation resumes upon cessation of Fgf10 production. However, competence towards the individual pancreatic cell lineages depended upon the gestational time of when Fgf10 expression was attenuated. This revealed a competence window of endocrine and ductal cell formation that coincided with the pancreatic secondary transition between E13.5 and E15.5. We demonstrate that maintaining the Fgf10-arrested state during this period leads to permanent loss of competence for the endocrine and ductal cell fates. However, competence of the arrested progenitors towards the exocrine cell fate was retained throughout the secondary transition. Sustained Fgf10 expression caused irreversible loss of Ngn3 expression, which may underlie the loss of endocrine competence. Maintenance of exocrine competence may be attributable to continuous Ptf1a expression in the Fgf10-arrested progenitors. This may explain the rapid induction of Bhlhb8, a normally distalized cell intrinsic marker, following loss of ectopic Fgf10 expression. We conclude that the window for endocrine and ductal cell competence ceases during the secondary transition in pancreatic development.