Acinar Cell Apoptosis in Serpini2-Deficient Mice Models Pancreatic Insufficiency

Pancreatic insufficiency (PI) when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman–Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq), exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome transgenic expression or by pancreatic enzyme supplementation demonstrates that these phenotypes are secondary to malnutrition in pq/pq mice

A Sox10 Expression Screen Identifies an Amino Acid Essential for Erbb3 Function

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10 LacZ/+. Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1–4 (msp1–4), which show altered NC patterning. In homozygous msp1 embryos, Sox10 LacZ expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3 msp1, that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance o

A Sox10 expression screen identifies an amino acid essential for Erbb3 function.

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10(LacZ/+). Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1-4 (msp1-4), which show altered NC patterning. In homozygous msp1 embryos, Sox10(LacZ) expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3(msp1), that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance of the mutated residue for ERBB3 receptor function and activation. This study underscores the utility of using an ENU mutagenesis to identify genetic pathways regulating NC development and to dissect the roles of discrete protein domains, both of which contribute to a better understanding of gene function in a cellular and developmental setting

Pancreatic Enzyme Supplementation and BAC Transgenesis Rescue Lymphocyte Cells Numbers and Developmental Defects in <i>pequeño</i> Mice

<div><p>(A) <i>pequeño</i> mice with either enzyme supplementation (<i>pq/pq;</i> diet) or a BAC transgenic (<i>pq/pq;</i> BAC) display increased cellularity of the thymus, spleen, and bone marrow at day 28 of life (<i>n =</i> 4).</p><p>(B–D) Pancreatic enzyme supplementation and the BAC transgenic rescue the developmental defects in the <i>pequeño</i> mice. (B) CD4 and CD8 expression profiles of the thymus. (C) Expression profiles of B220 and IgM on total lymphoid cells in bone marrow (top row). The B220⁺IgM⁻ pro-/pre-B cell fraction (filled arrow) was gated to analyze the developmental stages using CD43 (middle row). The B220⁺IgM⁻CD43⁺ (less mature) fraction (open arrow) was then gated on to analyze CD24 expression (bottom row). (D) Splenic expression profile analysis of surface IgM/IgD expression on B220⁺ cells .</p></div

<i>pequeño</i> Malnutrition Is Rescued by BAC Transgenic Expressing <i>Serpini2</i> Gene or Pancreatic Enzyme Diet Supplementation

<div><p>(A–C) Treatment of <i>pq/pq</i> mice (blue) pharmacologically with daily pancreatic enzyme diet supplement (purple) or genetically with BAC 150E1 transgene (green), rescues (A) size, (B) weight, and (C) animal viability in comparison to wild-type mice (black). For (B) weight measurements are averaged from <i>n =</i> 6 males from each genotype.</p><p>(D–G) Hematoxylin and eosin staining of pancreas sections from 8-wk-old mice. Wild-type mice (D) have normal pancreatic acinar cells present and normal pancreatic morphology while in (E) <i>pq/pq</i> mice there is a loss of acinar cells. In the BAC+; <i>pq/pq</i> mice (F), pancreas acinar cells are present. The acinar cell loss in <i>pq/pq</i> mice treated with enzyme supplementation (G) is the same as untreated <i>pq/pq</i> mice (A–C), although diet supplementation is effective in combating the effects of malnutrition. Bar represents 25 μm.</p></div

Electron Microscopy Shows <i>pequeño</i> Acinar Cells Are Present but Undergoing Apoptosis at 1 Wk of Age

<div><p>(A) Wild-type acinar cells have normal cellular morphology and contained abundant numbers of ZGs (seen as black circular vesicles containing electron-dense material), and electron-dense material from ZGs is present in the ductal lumen (white arrow).</p><p>(B–D) <i>pq/pq</i> mice have fewer and smaller ZGs and no dense material in the ductal lumen (L) when compared to wild-type animals. A significant number of acinar cells are undergoing apoptosis, as indicated by the presence of cellular phagocytosis (C and D), rough endoplasmic reticulum whirling bodies (B and C) (black arrows), and nuclear condensation and fragmentation (D) (asterisk).</p><p>(E and F) TUNEL staining of 3-wk-old pancreas tissue from wild-type (E) and <i>pq/pq</i> (F) mice indicates an increase in TUNEL-positive, red-fushcin-staining cells in <i>pq/pq</i> mice as compared to wild-type.</p><p>L, lumen; N, nucleus; RER, rough endoplasmic reticulum. For (A–D) bar represents 5 μm. For (E and F) bar represents 50 μm.</p></div

Decreased Lymphocyte Numbers and Impaired B cell Development in <i>pequeño</i> Mice

<div><p>(A) <i>pequeño</i> mice exhibit a striking reduction in the cellularity of the thymus, spleen and bone marrow at days 21 and 28 of life (<i>n =</i> 3).</p><p>(B) <i>pequeño</i> mice show decreased early B cell progenitors in the bone marrow. Expression profiles of B220 and IgM on total lymphoid cells in bone marrow are shown for wild-type (top row, percentage of B220⁺IgM⁻ pro-/pre-B cell fraction are indicated). The B220⁺IgM⁻ pro-/pre-B cell fraction (filled arrow) was gated to analyze the developmental stages using CD43 (middle row). The B220⁺IgM⁻CD43⁺ (less mature) fraction (open arrow) was then gated on to analyze CD24 expression (bottom row). <i>pq/pq</i> mice exhibit an increase in the proportion of CD43⁺CD24⁻ B cells, indicating a block in the pre-/pro-B to early pro-B transition.</p><p>(C) Flow cytometric analysis of surface IgM⁺/IgD⁻ expression on B220⁺ cells in the spleen indicates a decreased percentage of immature (IgM⁺/IgD⁻) B cells in the <i>pequeño</i> mice.</p></div

Acinar Cell Loss in <i>pequeño</i> Mice

<div><p>(A) Size of <i>pq/pq</i> mice in relation to wild-type and heterozygous littermates at 8 wk of age.</p><p>(B) Average weight of <i>n =</i> 6 males for each genotype over a 32-wk period.</p><p>(C–H) Pancreas hematoxylin and eosin histological analyses were performed on 8-wk-old (C, D, and E), 3-wk-old (F), and 1-wk-old (G and H) mice. Wild-type (<i>+,+</i>) acinar cells (C) and heterozygous (<i>pq/+</i>) acinar cells (D) at 8 wk of age were morphologically normal. This is in contrast to homozygous <i>(pq/pq)</i> animals at 8 wk of age (E) and 3 wk of age (F), where there is a severe loss of exocrine pancreatic acinar cells while duct cells and islet cells (arrow) are normal. Acinar cells are present in pancreas samples from 1-wk-old <i>pq/pq</i> mice (H), but the cells are smaller and show reduced cytosolic volume when compared to wild-type littermates (G). Bar represents 25 μm.</p></div

Pequeño Phenotype Is the Result of a 210-kb Genomic Deletion

<div><p>(A) FISH to interphase <i>pq/+</i> chromosome spreads with a p3pTVA transgene probe (red) locates the transgene insertion on mouse Chromosome 3 between BACs RP23-338I15 (yellow) and RP23-296I12 (green).</p><p>(B) The genomic region defined by BAC FISH localization contained six known or predicted genes, one of which, <i>Serpini2,</i> is highly expressed in pancreas and adipose tissue. PCR primer sets A–P (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010038#pgen-0010038-st001" target="_blank">Table S1</a>) were used to characterize and define the identified ~210-kb deletion, indicated by red dashed line. PCR primer sets that correspond to known and predicted exon sequences are indicated by black vertical dashed lines. The genomic deletion encompassed the entire <i>Serpini2</i> gene locus and exons 4 to 17 of the predicted gene E-62199.1/E-38943.1.</p></div