Positional Cloning of “Lisch-like”, a Candidate Modifier of Susceptibility to Type 2 Diabetes in Mice

In 404 Lepob/ob F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lepob. The phenotypes of B6.DBA congenic mice include reduced β-cell replication rates accompanied by reduced β-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated “Lisch-like” (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646–amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes

Analysis of beta cell proliferation dynamics in zebrafish

Among the different mechanisms invoked to explain the beta cell mass expansion during postnatal stages and adulthood, self-replication is being considered the major cellular event occurring both under physiological conditions and in regenerating pancreas after partial pancreactomy. Neogenesis, i.e. differentiation from pancreatic progenitors, has been demonstrated to act concurrently with beta cell replication during pancreatic regeneration. Both phenomena have been largely elucidated in higher vertebrates (mouse, rat and guinea pig), but an extensive description of beta cell dynamics in other animal models is currently lacking. We, therefore, explored in zebrafish the cellular origins of new beta cells in both adult and larval stages. By integrating the results from in vivo time lapse analysis and immunostaining, we provide a detailed reconstruction of the major processes involved in fish beta cell genesis and maintenance. Moreover, by establishing the selective ablation of proliferating beta cells, through the ganciclovir-HSVTK system, we could show that in larval stages self-replication is the main mechanism of beta cells expansion. Since the same mechanism of proliferation has been observed to occur during early and late life stages, we suggest that zebrafish larvae can be used as an alternative tool for an in vivo exploration and screening of new potential mitogens specifically targeting beta cells

Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish

The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of Delta and Jagged/Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different delta and jagged genes. Specifically, deltaA knock-down embryos lack alpha cells, similarly to mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely, jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state

Optimal Experimental Design for Systems and Synthetic Biology Using AMIGO2

19 pages, 6 figuresDynamic modeling in systems and synthetic biology is still quite a challenge—the complex nature of the interactions results in nonlinear models, which include unknown parameters (or functions). Ideally, time-series data support the estimation of model unknowns through data fitting. Goodness-of-fit measures would lead to the best model among a set of candidates. However, even when state-of-the-art measuring techniques allow for an unprecedented amount of data, not all data suit dynamic modeling. Model-based optimal experimental design (OED) is intended to improve model predictive capabilities. OED can be used to define the set of experiments that would (a) identify the best model or (b) improve the identifiability of unknown parameters. In this chapter, we present a detailed practical procedure to compute optimal experiments using the AMIGO2 toolboxThe authors acknowledge financial support from the Spanish Ministry of Science, Innovation and Universities and the European Union FEDER (project grant RTI2018-093744-B-C33). This work was also supported by a Royal Society of Edinburgh-MoST grant, EPSRC grant EP/R035350/1 and EP/S001921/1 to Dr. Menolascina, and the EPSRC grant EP/P017134/1-CONDSYC to Dr. BandieraN