ENU-induced phenovariance in mice: inferences from 587 mutations.

BACKGROUND: We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. FINDINGS: In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. CONCLUSIONS: Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Rapid identification of a disease allele in mouse through whole genome sequencing and bulk segregation analysis.

In a pedigree of C57BL/6J mice homozygous for germline mutations induced by the mutagen N-ethyl-N-nitrosourea (ENU), numerous animals died under specific pathogen-free (SPF) conditions between 6 and 7 months of age. Death was caused by nephritic syndrome, which progressed to renal failure associated with focal segmental glomerulosclerosis. To identify the mutation responsible for renal disease, we sequenced genomic DNA from an affected animal using the Applied Biosystems SOLiD sequencing platform. Approximately 74% of the nucleotides comprising coding sequences and splice junctions in the mouse genome were covered at least three times. Within this portion of the genome, 64 discrepancies were flagged as potential homozygous mutations and 82 were flagged as potential heterozygous mutations. A total of 10 of these calls, all homozygous, were validated by capillary sequencing. One of the validated mutations disrupted splicing of the Col4a4 transcript. Genetic mapping by bulk segregation analysis excluded all mutations but this one as the cause of renal disease in Aoba mice. Col4a4 has not been targeted in the mouse, and this strain, named Aoba, represents the first functionally null allele in this species. Our study demonstrates the speed and utility of whole genome sequencing coupled with low resolution meiotic mapping as a means of identifying causative mutations induced by ENU.

Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells.

Despite their low frequency, plasmacytoid dendritic cells (pDCs) produce most of the type I IFN that is detectable in the blood following viral infection. The endosomal Toll-like receptors (TLRs) TLR7 and TLR9 are required for pDCs, as well as other cell types, to sense viral nucleic acids, but the mechanism by which signaling through these shared receptors results in the prodigious production of type I IFN by pDCs is not understood. We designed a genetic screen to identify proteins required for the development and specialized function of pDCs. One phenovariant, which we named feeble, showed abrogation of both TLR-induced type I IFN and proinflammatory cytokine production by pDCs, while leaving TLR responses intact in other cells. The feeble phenotype was mapped to a mutation in Slc15a4, which encodes the peptide/histidine transporter 1 (PHT1) and has not previously been implicated in pDC function. The identification of the feeble mutation led to our subsequent observations that AP-3, as well as the BLOC-1 and BLOC-2 Hermansky-Pudlak syndrome proteins are essential for pDC signaling through TLR7 and TLR9. These proteins are not necessary for TLR7 or TLR9 signaling in conventional DCs and thus comprise a membrane trafficking pathway uniquely required for endosomal TLR signaling in pDCs.

A mutation of Ikbkg causes immune deficiency without impairing degradation of IkappaB alpha.

Null alleles of the gene encoding NEMO (NF-kappaB essential modulator) are lethal in hemizygous mice and men, whereas hypomorphic alleles typically cause a syndrome of immune deficiency and ectodermal dysplasia. Here we describe an allele of Ikbkg in mice that impaired Toll-like receptor signaling, lymph node formation, development of memory and regulatory T cells, and Ig production, but did not cause ectodermal dysplasia. Degradation of IkappaB alpha, which is considered a primary requirement for NEMO-mediated immune signaling, occurred normally in response to Toll-like receptor stimulation, yet ERK phosphorylation and NF-kappaB p65 nuclear translocation were severely impaired. This selective loss of function highlights the immunological importance of NEMO-regulated pathways beyond IkappaB alpha degradation, and offers a biochemical explanation for rare immune deficiencies in man.

The Tpl2 mutation Sluggish impairs type I IFN production and increases susceptibility to group B streptococcal disease.

Sluggish was identified in a population of third generation mice descended from N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes exhibited impaired TNF-alpha production in response to all TLR ligands tested and displayed impaired type I IFN production in response to TLR7 and TLR9 stimulations. The phenotype was confined to a critical region on mouse chromosome 18 and then ascribed to a T to A transversion in the acceptor splice site of intron 4 at position 13346 of the Map3k8 gene, resulting in defective splicing. The Map3k8(Sluggish) mutation does not result in susceptibility to viral infections, but Sluggish mice displayed high susceptibility to group B streptococcus infection, with impaired TNF-alpha and type I IFN production in infected macrophages. Our data demonstrate that the encoded protein kinase Tpl2 plays an essential role in cell signaling in the immune response to certain pathogens.

Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function.

The classical recessive coat color mutation misty (m) arose spontaneously on the DBA/J background and causes generalized hypopigmentation and localized white-spotting in mice, with a lack of pigment on the belly, tail tip, and paws. Here we describe moonlight (mnlt), a second hypopigmentation and white-spotting mutation identified on the C57BL/6J background, which yields a phenotypic copy of m/m coat color traits. We demonstrate that the 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result from mutations that truncate the dedicator of cytokinesis 7 protein (DOCK7), a widely expressed Rho family guanine nucleotide exchange factor. Although Dock7 is transcribed at high levels in the developing brain and has been implicated in both axon development and myelination by in vitro studies, we find no requirement for DOCK7 in neurobehavioral function in vivo. However, DOCK7 has non-redundant role(s) related to the distribution and function of dermal and follicular melanocytes.

Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.

Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human beta-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implanted mice, human insulin and C-peptide are detected in sera at levels similar to those of mice transplanted with approximately 3,000 human islets. Moreover, the insulin-expressing cells generated after engraftment exhibit many properties of functional beta-cells, including expression of critical beta-cell transcription factors, appropriate processing of proinsulin and the presence of mature endocrine secretory granules. Finally, in a test of therapeutic potential, we demonstrate that implantation of hES cell-derived pancreatic endoderm protects against streptozotocin-induced hyperglycemia. Together, these data provide definitive evidence that hES cells are competent to generate glucose-responsive, insulin-secreting cells.

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus.

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.

GDF11 modulates NGN3+ islet progenitor cell number and promotes beta-cell differentiation in pancreas development.

Identification of endogenous signals that regulate expansion and maturation of organ-specific progenitor cells is a major goal in studies of organ development. Here we provide evidence that growth differentiation factor 11 (GDF11), a member of the TGF-beta ligand family, governs the number and maturation of islet progenitor cells in mouse pancreas development. Gdf11 is expressed in embryonic pancreatic epithelium during formation of islet progenitor cells that express neurogenin 3. Mice deficient for Gdf11 harbor increased numbers of NGN3+ cells, revealing that GDF11 negatively regulates production of islet progenitor cells. Despite a marked expansion of these NGN3+ islet progenitors, mice lacking Gdf11 have reduced beta-cell numbers and evidence of arrested beta-cell development, indicating that GDF11 is also required for beta-cell maturation. Similar precursor and islet cell phenotypes are observed in mice deficient for SMAD2, an intracellular signaling factor activated by TGF-beta signals. Our data suggest that Gdf11 and Smad2 regulate islet cell differentiation in parallel to the Notch pathway, which previously has been shown to control development of NGN3+ cells. Thus, our studies reveal mechanisms by which GDF11 regulates the production and maturation of islet progenitor cells in pancreas development.