HNF-6-independent differentiation of mouse embryonic stem cells into insulin-producing cells.

AIMS/HYPOTHESIS: Embryonic stem cells, when grown as embryoid bodies, spontaneously generate insulin-producing cells which could be used in therapy of diabetes mellitus, provided that their selection and differentiation are optimized. To achieve such optimization, one needs to know whether the differentiation of cells in embryoid bodies mimicks that of pancreatic beta cells in embryos. To address this question we verified if the differentiation of the insulin-producing cells in embryoid bodies requires Hepatocyte Nuclear Factor-6 (HNF-6), a transcription factor known to control pancreatic endocrine differentiation in embryos. METHODS: We generated mouse Hnf6-/- embryonic stem cells and grew them as embryoid bodies. The expression of HNF-6, insulin, and transcription factors that are regulated by HNF-6 in developing pancreas was compared in wild-type and Hnf6-/- embryoid bodies. RESULTS: No difference was observed in the expression of insulin between wild-type and Hnf6-/-embryoid bodies. In both cases insulin was expressed in the outer layer of cells, which is similar to the visceral endoderm. In wild-type embryoid bodies HNF-6 was transiently expressed in the outer layer of cells, but was not co-expressed with insulin. The expression of genes that are targets of HNF-6 in developing pancreas was unaffected in Hnf6-/-embryoid bodies. CONCLUSION/INTERPRETATION: In contrast to the development of pancreatic beta cells, the differentiation of insulin-producing cells in embryoid bodies did not require HNF-6. Thus, the differentiation mechanism of insulin-producing cells in embryoid bodies differs from that of the beta cells and it is likely to resemble that of insulin-producing cells in the visceral endoderm.

Liver glucokinase gene expression is controlled by the onecut transcription factor hepatocyte nuclear factor-6.

AIMS/HYPOTHESIS: Glucokinase plays a key role in glucose homeostasis and the expression of its gene is differentially regulated in pancreatic beta cells and in the liver through distinct promoters. The factors that determine the tissue-specific expression of the glucokinase gene are not known. Putative binding sites for hepatocyte nuclear factor (HNF)-6, the prototype of the ONECUT family of transcription factors, are present in the hepatic promoter of the glucokinase gene and hnf6 knockout mice are diabetic [corrected]. We hypothesized that HNF-6 controls the activity of the hepatic glucokinase promoter. METHODS: We tested the binding of recombinant HNF-6 to DNA sequences from the mouse hepatic glucokinase promoter in vitro and the effect of HNF-6 on promoter activity in transfected cells. We investigated in vivo the role of HNF-6 in mice by examining the effect of inactivating the hnf6 gene on glucokinase gene-specific deoxyribonuclease I hypersensitive sites in liver chromatin and on liver glucokinase mRNA concentration. RESULTS: HNF-6 bound to the hepatic promoter of the glucokinase gene and stimulated its activity. Inactivation of the hnf6 gene did not modify the pattern of deoxyribonuclease I hypersensitive sites but was associated with a decrease of liver glucokinase mRNA to half the control value. CONCLUSIONS/INTERPRETATION: Although HNF-6 is not required to open chromatin of the hepatic promoter of the glucokinase gene, it stimulates transcription of the glucokinase gene in the liver. This could partly explain the diabetes observed in hnf6 knockout mice.

The transcription factor onecut-2 controls the microphthalmia-associated transcription factor gene.

Microphthalmia-associated transcription factor (MITF) is essential for melanocyte differentiation. MITF mutations are associated with some cases of Waardenburg syndrome (WS) type 2. WS is a dominantly inherited disease characterized by auditory-pigmentary defects that result from the absence of melanocytes. The lack of mutation in MITF coding sequences in some WS2 patients suggests that unidentified factors controlling MITF expression might be involved. We show here that the cut-homeodomain transcription factor Onecut-2 (OC-2) is expressed in melanocytes and binds to the MITF gene promoter. Overexpression of OC-2 in transfected cells stimulates MITF promoter activity. Mutations that prevent OC-2 binding decrease MITF promoter activity by 75%. Based on these results, we searched in 56 WS2 patients for mutations in the OC2 gene or in OC-2 binding sites in the MITF promoter, but none was found. These results show that OC-2 stimulates MITF expression and that OC2 is a candidate gene, but not a common cause, of WS.

Control of gene expression by growth hormone in liver: key role of a network of transcription factors.

Growth hormone (GH) controls gene expression in liver. Recent work suggests that this can result in part from the stimulation by GH of the synthesis of liver-specific transcription factors, one of which is HNF-6. The liver-specific factors HNF-4 and C/EBP alpha respectively stimulate and inhibit transcription of the hnf 6 gene. Upon GH stimulation, the affinity of HNF-4 for the hnf 6 promoter is increased and the binding of C/EBP alpha is decreased. GH therefore controls hnf 6 by a combination of stimulatory and derepressive mechanisms. On the other hand, HNF-6 stimulates transcription of the hnf 3beta and hnf 4 genes, the stimulation of hnf 4 resulting most likely from the GH-induced increase in HNF-6 concentration. We conclude that in liver GH is likely to control the synthesis of a whole set of proteins whose genes are regulated by a GH-sensitive network of transcription factors, which regulate each other by feed-back and autoregulatory loops.

Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3.

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class of cut homeodomain transcription factors. During mouse development, HNF-6 is expressed in the epithelial cells that are precursors of the exocrine and endocrine pancreatic cells. We have investigated the role of HNF-6 in pancreas differentiation by inactivating its gene in the mouse. In hnf6(-/-) embryos, the exocrine pancreas appeared to be normal but endocrine cell differentiation was impaired. The expression of neurogenin 3 (Ngn-3), a transcription factor that is essential for determination of endocrine cell precursors, was almost abolished. Consistent with this, we demonstrated that HNF-6 binds to and stimulates the ngn3 gene promoter. At birth, only a few endocrine cells were found and the islets of Langerhans were missing. Later, the number of endocrine cells increased and islets appeared. However, the architecture of the islets was perturbed, and their beta cells were deficient in glucose transporter 2 expression. Adult hnf6(-/-) mice were diabetic. Taken together, our data demonstrate that HNF-6 controls pancreatic endocrine differentiation at the precursor stage and identify HNF-6 as the first positive regulator of the proendocrine gene ngn3 in the pancreas. They also suggest that HNF-6 is a candidate gene for diabetes mellitus in humans.

CCAAT/enhancer-binding protein-alpha is a component of the growth hormone-regulated network of liver transcription factors.

GH regulates gene expression by modulating the concentration or activity of transcription factors. To identify transcription factors that mediate the effects of GH in liver we analyzed the promoter of the gene coding for hepatocyte nuclear factor-6 (HNF-6), whose expression in liver is stimulated by GH. In protein-DNA interaction studies and in transfection experiments, we found that the liver-enriched transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) binds to the hnf6 gene and inhibits its expression. This inhibitory effect involved an N-terminal subdomain of C/EBPalpha and two sites in the hnf6 gene promoter. Using liver nuclear extracts from GH-treated hypophysectomized rats, we found that GH induces a rapid, transient decrease in the amount of C/EBPalpha protein. This GH-induced change is concomitant with the transient stimulatory effect of GH on the hnf6 gene. Stimulation of the hnf6 gene by GH therefore involves lifting of the repression exerted by C/EBPalpha in addition to the known GH-induced stimulatory effects of STAT5 (signal transducer and activator of transcription-5) and HNF-4 on that gene. Our data provide further evidence that GH controls a network of liver transcription factors and show that C/EBPalpha participates in this process.

Transcriptional stimulation by hepatocyte nuclear factor-6. Target-specific recruitment of either CREB-binding protein (CBP) or p300/CBP-associated factor (p/CAF).

Transcription factors of the ONECUT class, whose prototype is HNF-6, contain a single cut domain and a divergent homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. The cut domain is required for DNA binding. The homeodomain is required either for DNA binding or for transcriptional stimulation, depending on the target gene. Transcriptional stimulation by the homeodomain involves the F48M50 dyad. We investigate here how HNF-6 stimulates transcription. We identify transcriptionally active domains of HNF-6 that are conserved among members of the ONECUT class and show that the cut domain of HNF-6 participates to DNA binding and, via a LXXLL motif, to transcriptional stimulation. We also demonstrate that, on a target gene to which HNF-6 binds without requirement for the homeodomain, transcriptional stimulation involves an interaction of HNF-6 with the coactivator CREB-binding protein (CBP). This interaction depends both on the LXXLL motif of the cut domain and on the F48M50 dyad of the homeodomain. On a target gene for which the homeodomain is required for DNA binding, but not for transcriptional stimulation, HNF-6 interacts with the coactivator p300/CBP-associated factor but not with CBP. These data show that a transcription factor can act via different, sequence-specific, mechanisms that combine distinct modes of DNA binding with the use of different coactivators.

Involvement of STAT5 (signal transducer and activator of transcription 5) and HNF-4 (hepatocyte nuclear factor 4) in the transcriptional control of the hnf6 gene by growth hormone.

HNF-6 is a tissue-restricted transcription factor that participates in the regulation of several genes in liver. We reported earlier that in adult rats, HNF-6 mRNA concentration in liver drops to almost undetectable levels after hypophysectomy and returns to normal after 1 week of GH treatment. We now show that this results from a rapid effect of GH, and we characterize its molecular mechanism. In hypophysectomized rats, HNF-6 mRNAs increased within 1 h after a single injection of GH. The same GH-dependent induction was reproduced on isolated hepatocytes. To determine whether GH regulates hnf6 expression at the gene level, we studied its promoter. DNA binding experiments showed that 1) the transcription factors STAT5 (signal transducer and activator of transcription 5) and HNF-4 (hepatocyte nuclear factor 4) bind to sites located around -110 and -650, respectively; and 2) STAT5 binding is induced and HNF-4 binding affinity is increased in liver within 1 h after GH injection to hypophysectomized rats. Using transfection experiments and site-directed mutagenesis, we found that STAT5 and HNF-4 stimulated transcription of an hnf6 gene promoter-reporter construct. Furthermore, GH stimulated transcription of this construct in cells that express GH receptors. Consistent with our earlier finding that HNF-6 stimulates the hnf4 and hnf3beta gene promoters, GH treatment of hypophysectomized rats increased the liver concentration of HNF-4 and HNF-3beta mRNAs. Together, these data demonstrate that GH stimulates transcription of the hnf6 gene by a mechanism involving STAT5 and HNF-4. They show that HNF-6 participates not only as an effector, but also as a target, to the regulatory network of liver transcription factors, and that several members of this network are GH regulated.

Hepatocyte nuclear factor-6: associations between genetic variability and type II diabetes and between genetic variability and estimates of insulin secretion.

AIMS/HYPOTHESIS: The transcription factor hepatocyte nuclear factor (HNF)-6 is an upstream regulator of several genes involved in the pathogenesis of maturity-onset diabetes of the young. We therefore tested the hypothesis that variability in the HNF-6 gene is associated with subsets of Type II (non-insulin-dependent) diabetes mellitus and estimates of insulin secretion in glucose tolerant subjects. METHODS: We cloned the coding region as well as the intron-exon boundaries of the HNF-6 gene. We then examined them on genomic DNA in six MODY probands without mutations in the MODY1, MODY3 and MODY4 genes and in 54 patients with late-onset Type II diabetes by combined single strand conformational polymorphism-heteroduplex analysis followed by direct sequencing of identified variants. An identified missense variant was examined in association studies and genotype-phenotype studies. RESULTS: We identified two silent and one missense (Pro75 Ala) variant. In an association study the allelic frequency of the Pro75Ala polymorphism was 3.2% (95% confidence interval, 1.9-4.5) in 330 patients with Type II diabetes mellitus compared with 4.2% (2.4-6.0) in 238 age-matched glucose tolerant control subjects. Moreover, in studies of 238 middle-aged glucose tolerant subjects, of 226 glucose tolerant offspring of Type II diabetic patients and of 367 young healthy subjects, the carriers of the polymorphism did not differ from non-carriers in glucose induced serum insulin or C-peptide responses. CONCLUSION/INTERPRETATION: Mutations in the coding region of the HNF-6 gene are not associated with Type II diabetes or with changes in insulin responses to glucose among the Caucasians examined.

Antiglucocorticoid activity of hepatocyte nuclear factor-6.

Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity. In vitro and in vivo experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.

Insulin inhibition of glucocorticoid-stimulated gene transcription: requirement for an insulin response element?

The glucocorticoid hormone receptor binds to regulatory elements of target genes and activates transcription through interactions with coactivators. For a subset of genes, glucocorticoid receptor activity is inhibited by insulin. The present paper analyzes recent data on the molecular mechanisms whereby insulin exerts this antiglucocorticoid effect. Two models are proposed. In the first model insulin controls the activity of an insulin-responsive factor bound to an insulin-responsive DNA element. In a second model, insulin targets a non-DNA bound coactivator of the glucocorticoid receptor. Here, the gene-specificity of the effect of insulin is conferred by the combined action of the glucocorticoid receptor, of DNA-bound transcription factors and of coactivators, which form a higher order structure that binds to a DNA sequence called glucocorticoid/insulin responsive unit.

OC-2, a novel mammalian member of the ONECUT class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6.

Transcription factors of the ONECUT class, whose prototype is hepatocyte nuclear factor (HNF)-6, are characterized by the presence of a single cut domain and by a peculiar homeodomain (Lannoy, V. J., Bürglin, T. R., Rousseau, G. G., and Lemaigre, F. P. (1998) J. Biol. Chem. 273, 13552-13562). We report here the identification and characterization of human OC-2, the second mammalian member of this class. The OC-2 gene is located on human chromosome 18. The distribution of OC-2 mRNA in humans is tissue-restricted, the strongest expression being detected in the liver and skin. The amino acid sequence of OC-2 contains several regions of high similarity to HNF-6. The recognition properties of OC-2 for binding sites present in regulatory regions of liver-expressed genes differ from, but overlap with, those of HNF-6. Like HNF-6, OC-2 stimulates transcription of the hnf-3beta gene in transient transfection experiments, suggesting that OC-2 participates in the network of transcription factors required for liver differentiation and metabolism.

Inhibition by insulin of glucocorticoid-induced gene transcription: involvement of the ligand-binding domain of the glucocorticoid receptor and independence from the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways.

Insulin can inhibit the stimulatory effect of glucocorticoid hormones on the transcription of genes coding for enzymes involved in glucose metabolism. We reported earlier that insulin inhibits the glucocorticoid-stimulated transcription of the gene coding for liver 6-phosphofructo-2-kinase (PFK-2). To elucidate the mechanism of these hormonal effects, we have studied the regulatory regions of the PFK-2 gene in transfection experiments. We found that both glucocorticoids and insulin act via the glucocorticoid response unit (GRU) located in the first intron. Footprinting experiments showed that the GRU binds not only the glucocorticoid receptor (GR), but also ubiquitous [nuclear factor I (NF-I)] and liver-enriched [hepatocyte nuclear factor (HNF)-3, HNF-6, CAAT/enhancer binding protein (C/EBP)] transcription factors. Site-directed mutational analysis of the GRU revealed that these factors modulate glucocorticoid action but that none of them seems to be individually involved in the inhibitory effect of insulin. We did not find an insulin response element in the GRU, but we showed that insulin targets the GR. Insulin-induced inhibition of the glucocorticoid stimulation required the ligand-binding domain of the GR. Finally, the insulin-signaling cascade involved was independent of the phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways. Together, these results suggest that insulin acts on the PFK-2 gene via another pathway and targets either the GR in its ligand-binding domain or a cofactor interacting with this domain.

Hepatocyte nuclear factor 6: organization and chromosomal assignment of the rat gene and characterization of its promoter.

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a family of tissue-specific transcription factors characterized by a bipartite DNA-binding domain consisting of a single cut domain and a novel type of homeodomain. We have previously cloned rat cDNA species coding for two isoforms, HNF-6alpha (465 residues) and beta (491 residues), which differ only by the length of the spacer between the two DNA-binding domains. We have now localized the rat Hnf6 gene to chromosome 8q24-q31 by Southern blotting of DNA from somatic cell hybrids and by fluorescence in situ hybridization. Cloning and sequencing of the rat gene showed that the two HNF-6 isoforms are generated by alternative splicing of three exons that are more than 10 kb apart from each other. Exon 1 codes for the N-terminal part and the cut domain, exon 2 codes for the 26 HNF-6beta-specific amino acids, and exon 3 codes for the homeodomain and the C-terminal amino acids. The transcription initiation site was mapped by ribonuclease protection and 5' rapid amplification of cDNA ends. Transfection experiments showed that promoter activity was contained within 0.75 kb upstream of the transcription initiation site. This activity was detected by the transfection of liver-derived HepG2 cells, but not of Rat-1 fibroblasts, suggesting that the promoter is sufficient to confer liver-specific expression.

Isoforms of hepatocyte nuclear factor-6 differ in DNA-binding properties, contain a bifunctional homeodomain, and define the new ONECUT class of homeodomain proteins.

Hepatocyte nuclear factor-6 (HNF-6) contains a single cut domain and a homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. We describe here two isoforms of HNF-6 which differ by the linker that separates these domains. Both isoforms stimulated transcription. The affinity of HNF-6alpha and HNF-6beta for DNA differed, depending on the target sequence. Binding of HNF-6 to DNA involved the cut domain and the homeodomain, but the latter was not required for binding to a subset of sites. Mutations of the F48M50 dyad that did not affect DNA binding reduced the transcriptional stimulation of constructs that do not require the homeodomain for DNA binding, but did not affect the stimulation of constructs that do require the homeodomain. Comparative trees of mammalian, Drosophila, and Caenorhabditis elegans proteins showed that HNF-6 defines a new class, which we call ONECUT, of homeodomain proteins. C. elegans proteins of this class bound to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which hinge on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain.

Type I protein C deficiency caused by disruption of a hepatocyte nuclear factor (HNF)-6/HNF-1 binding site in the human protein C gene promoter.

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by proteolytic inactivation of factors Va and VIIIa. Individuals affected by protein C deficiency are at risk for venous thrombosis. One such affected individual was shown earlier to carry a -14 T --> C mutation in the promoter region of the protein C gene. It is shown here that the region around this mutation corresponds to a binding site for the transcription factor hepatocyte nuclear factor (HNF)-6 and that this site completely overlaps an HNF-1 binding site. HNF-6 and HNF-1 bound in a mutually exclusive manner. The -14 T --> C mutation reduced HNF-6 binding. In transient transfection experiments, HNF-6 transactivated the wild-type protein C promoter and introduction of the mutation abolished transactivation by HNF-6. Similar experiments showed that wild-type protein C promoter activity was reduced by cotransfection of an HNF-1 expression vector. This inhibiting effect of HNF-1 was reversed to a stimulatory effect when promoter sequences either upstream or downstream of the HNF-6/HNF-1 site were deleted. It is concluded that HNF-6 is a major determinant of protein C gene activity. Moreover, this is the first report describing the putative involvement of HNF-6 and of an HNF-6 binding site in human pathology.

Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth hormone.

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

In vivo protein-DNA interactions on a glucocorticoid response unit of a liver-specific gene: hormone-induced transcription factor binding to constitutively open chromatin.

Transcription from the liver promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) gene depends on the presence of glucocorticoids that act via a glucocorticoid response unit (GRU) located in the first intron. The promoter and the GRU are in a constitutively open chromatin configuration. To determine how glucocorticoids would affect factor binding to the GRU in absence of chromatin remodeling, we have used a combination of in vitro DNA-binding assays and in vivo genomic footprinting in rat hepatocytes and hepatoma cells. We found that, in the absence of glucocorticoids, the GRU binds nuclear factor-I (NF-I). Glucocorticoid treatment modified factor binding to the NF-I site and induced the binding of hepatocyte nuclear factor-3 (HNF-3). Transfection assays showed that HNF-3 cooperates with the glucocorticoid receptor in stimulating transcription. In contrast with the lack of effect of glucocorticoids on factor binding to constitutively open GRUs of other genes, HNF-3 binding to the open PFK-2 GRU was hormone-dependent. Therefore, the PFK-2 GRU behaves as a novel type of GRU.

HNF-6 is expressed in endoderm derivatives and nervous system of the mouse embryo and participates to the cross-regulatory network of liver-enriched transcription factors.

Hepatocyte nuclear factor-6 (HNF-6) is a liver-enriched transcription factor that contains a single cut domain and a novel type of homeodomain. Here we have studied the developmental expression pattern of HNF-6 in the mouse. In situ hybridization experiments showed that HNF-6 mRNA is detected in the liver at embryonic day (E) 9, at the onset of liver differentiation. HNF-6 mRNA disappeared transiently from the liver between E12.5 and E15. In transfection experiments HNF-6 stimulated the expression of HNF-4 and of HNF-3 beta, two transcription factors known to be involved in liver development and differentiation. HNF-6 was detected in the pancreas from E10.5 onward, where it was restricted to the exocrine cells. HNF-6 was also detected in the developing nervous system. Both the brain and the spinal cord started to express HNF-6 at E9-9.5 in postmitotic neuroblasts. Later on, HNF-6 was restricted to brain nuclei, to the retina, to the ventral horn of the spinal cord, and to dorsal root ganglia. Our observations that HNF-6 contributes to the control of the expression of transcription factors and is expressed at early stages of liver, pancreas, and neuronal differentiation suggest that HNF-6 regulates several developmental programs.