Pearl EJ, Jarikji Z, Horb ME.

R01 DK077197-03 NIDDK NIH HHS , DK077197 NIDDK NIH HHS , R01 DK077197-05 NIDDK NIH HHS , R01 DK077197 NIDDK NIH HHS

	Fig. 1. Rfx6 expression in the endoderm and isolated gut tissue. (A) Sagittal bisection of an NF15 embryo showing an anteriorly localized stripe of rfx6 expression. (B) Sagittal bisection of an NF20 embryo showing rfx6 expression. (C) Transverse section through rfx6 expressing region of NF24 embryo showing broad expression in anterior endoderm. (D) NF28 embryo showing rfx6 expression restricted to a dorsal (arrow) and ventral (arrowhead) patch in the anterior foregut. (E) NF40 liver and pancreas showing rfx6 expression in the dorsal pancreas. (F) NF42 liver and pancreas showing rfx6 expression spreading to the ventral pancreas. (G) NF46 liver and pancreas showing rfx6 evenly expressed throughout the pancreas. (H) NF42 whole gut showing rfx6 expression in the dorsal pancreas, stomach and intestine. (A, B, D) Anterior is left. (A–D) Dorsal is up. (H) li, liver; p, pancreas; st, stomach. (I) RT-PCR of rfx6 in whole tadpoles at various stages throughout development.
	Fig. 2. Rfx6 LOF leads to decreased expression of foregut and pancreas markers early in pancreas development. (A) Western blot with anti-flag antibody of in vitro TnT assay of translation. This demonstrates that MO1 prevents translation of rfx6 mRNA, while the mis-match morpholino (MM) does not. Rfx6 mRNA, flag-tagged rfx6 mRNA plasmid; MM, mis-match morpholino. (B) Diagramatic representation of RT-PCR test of MO2 efficiency. (C) RT-PCR performed on wild-type (WT) and MO2-injected tadpoles. WT mRNA is processed leaving the reverse primer unable to bind resulting in no amplification in the Rfx6 panel. MO2 blocks mRNA processing at the intron 2 donor site (5' end of the intron) as demonstrated by the presence of PCR product in MO2 lane. Ef1α is a housekeeping gene used as a loading control. (D, E) Hnf6 expression is reduced in the morphant foregut endoderm at NF25 (24/28 reduced). (F, G) FoxA2 expression is reduced in the morphant foregut endoderm at NF25 (17/28). (H, I) Hnf6 expression is reduced in the morphant foregut at NF35 (10/13 reduced). (J, K) Pdx1 expression is reduced in the morphant (17/19 reduced). (L, M) Ptf1a expression is reduced in both the dorsal (arrow) and ventral (arrowhead) pancreatic buds of the morphant (n = 18). (N, O) Early insulin expression in the dorsal pancreatic bud (arrow) is lost in morphant tissue (56/62 reduced). Control tadpoles were injected with 25 ng mis-match morpholino.
	Fig. 3. Rfx6 is required for pancreas differentiation. Whole mount in situ hybridizations of NF42 whole guts from morphant (MO1) and mis-match (control) tadpoles. (A–D) Expression of pancreatic transcription factors pdx1 (n = 18) and ptf1a (n = 18) is almost completely abolished in morphant tissue. (E, F) Expression of acinar marker elastase is reduced (n = 16). (G, H) Schematic illustrating organs in the whole gut. Pink, liver; red, gall bladder; blue, pancreas; yellow, stomach, duodenum and intestine. (I, J) β cell marker insulin is reduced (72/75 reduced). (K–N) Glucagon and somatostatin, both markers of endocrine cells in the pancreas and stomach are reduced (glucagon n = 18, somatostatin 15/16 reduced). (O, P) NeuroD, developmental endocrine cell marker, is reduced (n = 17).
	Fig. 4. Rfx6 mRNA rescues the morpholino knockdown phenotype. (A) Control whole gut stained for elastase expression (n = 9). (B, C) Whole guts from embryos injected with 40 ng MO2, elastase expression is reduced (n = 47, 6 moderate, 41 severe). (B) Moderate MO2 phenotype, (C) Severe MO2 phenotype. (D) Whole gut from embryo injected with 40 ng MO2 and 100 pg rfx6-GR mRNA. No dexamethosone was added. Elastase expression is reduced (n = 32, 8 normal, 15 moderate, 9 severe). (E) Whole gut from embryo injected with 40 ng MO2 and 100 pg rfx6-GR mRNA. 2 μg/mL dexamethosone was added at NF25 to activate Rfx6-GR protein. Elastase expression is restored (n = 36, 20 normal, 15 moderate, 1 severe). (F) Rescue results from all experimental whole guts. Severe phenotype refers to complete absence of elastase expression and gut malformation; moderate phenotype refers to reduced elastase expression and some gut malformation; normal refers to whole guts with normal levels of elastase expression and normal overall gut morphology.
	Fig. 5. Mutant rfx6 mRNA cannot rescue the morpholino knockdown phenotype. (A) Control whole gut stained for elastase expression (n = 17). (B) Whole gut from embryo injected with 40 ng MO2, elastase expression is reduced (n = 26, 4 normal, 8 moderate, 14 severe). (C) Whole gut from embryo injected with 40 ng MO2 and 100 pg rfx6R181Q-GR mRNA. No dexamethosone was added. Elastase expression is reduced (n = 12, 1 moderate, 11 severe). (D) Whole gut from embryo injected with 40 ng MO2 and 100 pg rfx6R181Q-GR mRNA. 2 μg/mL dexamethosone was added at NF25 to activate Rfx6R181Q-GR protein. Elastase expression remains reduced (n = 16, 3 normal, 4 moderate, 9 severe). (E) Rescue results for three rfx6 mutants: R181Q, S217P, and EX7 (Rfx6 is truncated after exon 7). Severe phenotype refers to complete absence of elastase expression and gut malformation; moderate phenotype refers to reduced elastase expression and some gut malformation; normal refers to whole guts with normal levels of elastase expression and normal overall gut morphology. S217P −dex n = 16, 2 normal, 2 moderate, 14 severe. S217P + dex n = 16, 2 moderate, 14 severe. EX7 −dex n = 13, 1 normal, 1 moderate, 11 severe. EX7 + dex n = 16, 1 normal, 1 moderate, 14 severe.
	Fig. 6. LOF microarray experiment schematic and RT-PCR verification of selected genes. (A) Schematic diagram of LOF microarray experiment. Eight-cell stage embryos were injected with 25 ng of either Rfx6 start site morpholino (MO) or mis-match control (MM). Samples were collected at NF30, NF40 and NF44, RNA was extracted and hybridized to Affymetrix Xenopus laevis GeneChip 2.0. (B–D) RT-PCR verification of selected differentially expressed genes from the LOF microarray analysis. (B) NF30. (C) NF40. (D) NF44. +, microarray experiment showed upregulation; −, microarray experiment showed downregulation, NA, this probe was not on the microarray but was shown to be downregulated in in situ experiments, NC, the microarray experiment detected no change. Differential expression in RT-PCR findings are consistent with results seen in the microarray experiment. MM, mis-match control; MO, MO1; -RT, control reaction without reverse transcriptase.
	Fig. 7. In situ hybridization confirms differential expression of organ marker genes for stomach, lung, heart and kidney in LOF microarray. (A, B) Isolated whole gut NF42 showing expression of stomach marker agr2 almost entirely abolished in morphant tissue (n = 23). (C, D) Expression of stomach marker frp5 was reduced (n = 18). (E–J) Whole tadpoles at NF42 with ventral skin removed for in situ hybridization. (E, F) Lung marker surfactant C (SurfC) is decreased in morphant tissue, closed arrows (10/14 reduced). (G, H) Heart marker nkx2.5 is decreased in morphant tissue, open arrow (8/14 reduced). (I, J) Kidney marker kcnJ1 is increased in morphant tissue, arrowhead (8/11 increased). (K,L) Expression of liver marker hex is unaffected by rfx6 knockdown (n = 32). (M, N) Expression of gall bladder and duodenum marker hnf6 is reduced in duodenum but not gall bladder (17/18). (O, P) Gall bladder marker sox17α was not reduced (18/20 showed normal expression).
	rfx6(regulatory factor X, 6) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Lateral view of bissected embryos: anterior left, dorsal up.
	rfx6(regulatory factor X, 6) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	rfx6(regulatory factor X, 6) gene expression in Xenopus laevis embryos, NF stage 42, as assayed by in situ hybridization. Lateral view of dissected foregut: anterior left, dorsal up.

Aftab, Identification and characterization of novel human tissue-specific RFX transcription factors. 2008, Pubmed

Aftab, Identification and characterization of novel human tissue-specific RFX transcription factors. 2008, Pubmed
Aguilar-Bryan, Neonatal diabetes mellitus. 2008, Pubmed
Ait-Lounis, The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression. 2010, Pubmed
Ait-Lounis, Novel function of the ciliogenic transcription factor RFX3 in development of the endocrine pancreas. 2007, Pubmed
Arima, A conserved imprinting control region at the HYMAI/ZAC domain is implicated in transient neonatal diabetes mellitus. 2001, Pubmed
Ashique, The Rfx4 transcription factor modulates Shh signaling by regional control of ciliogenesis. 2009, Pubmed
Babenko, Activating mutations in the ABCC8 gene in neonatal diabetes mellitus. 2006, Pubmed
Craig, An in-frame deletion in Kir6.2 (KCNJ11) causing neonatal diabetes reveals a site of interaction between Kir6.2 and SUR1. 2009, Pubmed , Xenbase
Delépine, EIF2AK3, encoding translation initiation factor 2-alpha kinase 3, is mutated in patients with Wolcott-Rallison syndrome. 2000, Pubmed
Edgar, Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. 2002, Pubmed
Edghill, Insulin mutation screening in 1,044 patients with diabetes: mutations in the INS gene are a common cause of neonatal diabetes but a rare cause of diabetes diagnosed in childhood or adulthood. 2008, Pubmed
Ellard, Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects. 2007, Pubmed
El Zein, RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies. 2009, Pubmed
Gajiwala, Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. 2000, Pubmed
Gardner, An imprinted locus associated with transient neonatal diabetes mellitus. 2000, Pubmed
Garvie, Characterization of the RFX complex and the RFX5(L66A) mutant: implications for the regulation of MHC class II gene expression. 2007, Pubmed
Gloyn, Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. 2004, Pubmed , Xenbase
Gradwohl, neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. 2000, Pubmed , Xenbase
Guilbault, Distinct pattern of lung gene expression in the Cftr-KO mice developing spontaneous lung disease compared with their littermate controls. 2006, Pubmed
Hamilton-Shield, Overview of neonatal diabetes. 2007, Pubmed
Horb, Experimental conversion of liver to pancreas. 2003, Pubmed , Xenbase
Horb, Expression of amylase and other pancreatic genes in Xenopus. 2002, Pubmed , Xenbase
Horb, Xenopus insm1 is essential for gastrointestinal and pancreatic endocrine cell development. 2009, Pubmed , Xenbase
Horb, BrunoL1 regulates endoderm proliferation through translational enhancement of cyclin A2 mRNA. 2010, Pubmed , Xenbase
Horvath, RFX2 is a candidate downstream amplifier of A-MYB regulation in mouse spermatogenesis. 2009, Pubmed
Horvath, RFX2 is a potential transcriptional regulatory factor for histone H1t and other genes expressed during the meiotic phase of spermatogenesis. 2004, Pubmed
Jabrane-Ferrat, Major histocompatibility complex class II transcriptional platform: assembly of nuclear factor Y and regulatory factor X (RFX) on DNA requires RFX5 dimers. 2002, Pubmed
Jacquemin, Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3. 2000, Pubmed
Jacquemin, The Onecut transcription factor HNF-6 (OC-1) is required for timely specification of the pancreas and acts upstream of Pdx-1 in the specification cascade. 2003, Pubmed
Jarikji, Differential ability of Ptf1a and Ptf1a-VP16 to convert stomach, duodenum and liver to pancreas. 2007, Pubmed , Xenbase
Jarikji, The tetraspanin Tm4sf3 is localized to the ventral pancreas and regulates fusion of the dorsal and ventral pancreatic buds. 2009, Pubmed , Xenbase
Kamiya, The cell cycle control gene ZAC/PLAGL1 is imprinted--a strong candidate gene for transient neonatal diabetes. 2000, Pubmed
Kistler, Differential expression of Rfx1-4 during mouse spermatogenesis. 2009, Pubmed
Krawczyk, New functions of the major histocompatibility complex class II-specific transcription factor RFXANK revealed by a high-resolution mutagenesis study. 2005, Pubmed
Mitchell, Neonatal diabetes, with hypoplastic pancreas, intestinal atresia and gall bladder hypoplasia: search for the aetiology of a new autosomal recessive syndrome. 2004, Pubmed
Nekrep, Mutation in a winged-helix DNA-binding motif causes atypical bare lymphocyte syndrome. 2002, Pubmed
Nicolino, A novel hypomorphic PDX1 mutation responsible for permanent neonatal diabetes with subclinical exocrine deficiency. 2010, Pubmed
Niesen, Technological advances in the study of HLA-DRA promoter regulation: extending the functions of CIITA, Oct-1, Rb, and RFX. 2009, Pubmed
Njølstad, Permanent neonatal diabetes caused by glucokinase deficiency: inborn error of the glucose-insulin signaling pathway. 2003, Pubmed
Njølstad, Neonatal diabetes mellitus due to complete glucokinase deficiency. 2001, Pubmed
Novak, Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution. 2006, Pubmed
Novak, Variation in fiberoptic bead-based oligonucleotide microarrays: dispersion characteristics among hybridization and biological replicate samples. 2006, Pubmed
Novak, Characterization of variability in large-scale gene expression data: implications for study design. 2002, Pubmed
Pearl, Xenopus pancreas development. 2009, Pubmed , Xenbase
Polak, Neonatal and very-early-onset diabetes mellitus. 2004, Pubmed
Polak, Neonatal diabetes mellitus: a disease linked to multiple mechanisms. 2007, Pubmed
Polak, Heterozygous missense mutations in the insulin gene are linked to permanent diabetes appearing in the neonatal period or in early infancy: a report from the French ND (Neonatal Diabetes) Study Group. 2008, Pubmed
Porter, Permanent neonatal diabetes in an Asian infant. 2005, Pubmed
Purvis, Transcriptional regulation of the Alström syndrome gene ALMS1 by members of the RFX family and Sp1. 2010, Pubmed
Rousseau, In vivo, RFX5 binds differently to the human leucocyte antigen-E, -F, and -G gene promoters and participates in HLA class I protein expression in a cell type-dependent manner. 2004, Pubmed
Rubio-Cabezas, Clinical heterogeneity in patients with FOXP3 mutations presenting with permanent neonatal diabetes. 2009, Pubmed
Rubio-Cabezas, Wolcott-Rallison syndrome is the most common genetic cause of permanent neonatal diabetes in consanguineous families. 2009, Pubmed
Schwitzgebel, Agenesis of human pancreas due to decreased half-life of insulin promoter factor 1. 2003, Pubmed
Seguín-Estévez, The transcription factor RFX protects MHC class II genes against epigenetic silencing by DNA methylation. 2009, Pubmed
Sellick, Mutations in PTF1A cause pancreatic and cerebellar agenesis. 2004, Pubmed
Senée, Wolcott-Rallison Syndrome: clinical, genetic, and functional study of EIF2AK3 mutations and suggestion of genetic heterogeneity. 2004, Pubmed
Senée, Mutations in GLIS3 are responsible for a rare syndrome with neonatal diabetes mellitus and congenital hypothyroidism. 2006, Pubmed
Shimomura, The first clinical case of a mutation at residue K185 of Kir6.2 (KCNJ11): a major ATP-binding residue. 2010, Pubmed , Xenbase
Smith, Rfx6 directs islet formation and insulin production in mice and humans. 2010, Pubmed
Soyer, Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development. 2010, Pubmed
Stoffers, Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. 1997, Pubmed
Støy, Insulin gene mutations as a cause of permanent neonatal diabetes. 2007, Pubmed
von Mühlendahl, Long-term course of neonatal diabetes. 1995, Pubmed
Wildin, X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. 2001, Pubmed
Wolfe, Transcription factor RFX2 is abundant in rat testis and enriched in nuclei of primary spermatocytes where it appears to be required for transcription of the testis-specific histone H1t gene. 2006, Pubmed
Wolfe, Transcription factor RFX4 binding to the testis-specific histone H1t promoter in spermatocytes may be important for regulation of H1t gene transcription during spermatogenesis. 2008, Pubmed
Yorifuji, Neonatal diabetes mellitus and neonatal polycystic, dysplastic kidneys: Phenotypically discordant recurrence of a mutation in the hepatocyte nuclear factor-1beta gene due to germline mosaicism. 2004, Pubmed