High Resolution Melt Analysis (HRMA); a Viable Alternative to Agarose Gel Electrophoresis for Mouse Genotyping

Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.This work was supported by NHMRC (http://www.nhmrc.gov.au) grants 366476 and 1003648 (to R.M.A.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Overlapping and distinct expression domains of Zic2 and Zic3 during mouse gastrulation

The Zic genes are the vertebrate homologues of the Drosophila Odd-paired gene. Mutations in two of these genes are associated with human congenital genetic disorders. Mutation of human and mouse Zic2 is associated with holoprosencephaly which is caused by a defect of ventral forebrain development and mutation of human and mouse Zic3 is associated with a X-linked heterotaxy syndrome that results from a failure of left-right axis formation. The embryological role of the Zic genes in these disorders is not well understood. Here we show that both of these genes are expressed prior to and throughout gastrulation. The genes show some broad similarities in their expression domains. Both genes however are also uniquely expressed in some tissues and these unique domains correlate with regions that potentially play a role in the aetiology of the respective genetic disorders. During primitive streak stages Zic2 is expressed transiently and uniquely in the node and the head process mesendoderm. The head process is known to be required for the establishment or maintenance of the ventral forebrain, which is the region disrupted in holoprosencephaly. Zic3 is not expressed in the node during primitive streak stages but is expressed in and around the node beginning from the head fold stages of development. This expression of Zic3 correlates well with the first steps in the establishment of the left-right axis. We also examined the expression of the closely related gene, Zic1, and did not detect any transcripts in gastrulation stage embryos

Functional Analysis of Transforming Growth Factor-Beta Related Molecules During Early Mouse Development

Embryogenesis requires cell-cell communication in which individual cells send and receive signals that determine their final fate. The transforming growth factor-β (TGF-β) superfamily of molecules encodes secreted factors which are capable of regulating cellular growth and differentiation. Recently, members of the TGF-β superfamily have emerged as candidates for many postulated embryonic inductive interactions. As a step towards understanding such interactions, this thesis investigates the hypothesis that TGF-β superfamily molecules may act as embryonic inducing factors in the mouse. The embryonic expression of a number of TGF-β superfamily members and a putative inhibitor of one of these, follistatin, were investigated. While activin (a mesoderm inducing factor in Xenopus animal caps) was not expressed in the mouse embryo, follistatin, BMP-7, BMP-2 and Vgr-2 were expressed during gastrulation and early organogenesis. Strategies were designed to ectopically express two of these molecules, BMP-7 and Vgr-2 in embryogenesis. Firstly, embryonic stem (ES) cells were used as a vehicle to secrete Vgr-2 throughout the embryo. No effect on embryonic morphology was detected, although conditioned medium from the ES cells is capable of inducing mesoderm in Xenopus. Secondly, COS cells were used to deliver a localised source of BMP-7 to the cranial neural plate in cultured embryos. Such ectopic expression resulted in the induction of ectopic dorsal neural marker expression (Msx1 and AP-2), the diminished expression of a ventral marker (Shh), and a localised expansion of neurectodermal tissue. The localisation of endogenous BMP-7 mRNA during formation and patterning of the neural axis, together with the activities shown in this assay, suggest that BMP-7 may direct one or more aspects of early central nervous system formation in the mouse

Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.This research was supported in part by the Intramural Research Program of NHGRI, NIH, and the Wellcome Trust and by NHMRC Australia grant 366746. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Genome-Wide ENU Mutagenesis in Combination with High Density SNP Analysis and Exome Sequencing Provides Rapid Identification of Novel Mouse Models of Developmental Disease

BACKGROUND Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU). METHODOLOGY/PRINCIPAL FINDINGS ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. CONCLUSIONS/SIGNIFICANCE In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.This work was enabled by the Australian Phenomics Network and partly supported by funding from the Australian Government’s National Collaborative Research Infrastructure Strategy, a Strategic Grant from the Faculty of Medicine, Nursing and Health Sciences at Monash University, and the Victorian Government’s Operational Infrastructure Support Program. IS acknowledges support through the NH&MRC R. Douglas Wright and ARC Future Fellowship schemes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A murine Zic3 transcript with a premature termination codon evades nonsense-mediated decay during axis formation

The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-c

Systematized reporter assays reveal ZIC protein regulatory abilities are Subclass-specific and dependent upon transcription factor binding site context

The ZIC proteins are a family of transcription regulators with a well-defined zinc finger DNA-binding domain and there is evidence that they elicit functional DNA binding at a ZIC DNA binding site. Little is known, however, regarding domains within ZIC proteins that confer trans-activation or -repression. To address this question, a new cell-based trans-activation assay system suitable for ZIC proteins in HEK293T cells was constructed. This identified two previously unannotated evolutionarily conserved regions of ZIC3 that are necessary for trans-activation. These domains are found in all Subclass A ZIC proteins, but not in the Subclass B proteins. Additionally, the Subclass B proteins fail to elicit functional binding at a multimerised ZIC DNA binding site. All ZIC proteins, however, exhibit functional binding when the ZIC DNA binding site is embedded in a multiple transcription factor locus derived from ZIC target genes in the mouse genome. This ability is due to several domains, some of which are found in all ZIC proteins, that exhibit context dependent trans-activation or -repression activity. This knowledge is valuable for assessing the likely pathogenicity of variant ZIC proteins associated with human disorders and for determining factors that influence functional transcription factor binding

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.Grant-in-Aid for Scientific Research(B), Japan Society for the Promotion of Science (JSPS KAKENHI 17H03568) Fumihiro Sugiyama Grant-in-Aid for Scientific Research(S), Japan Society for the Promotion of Science (JSPS KAKENHI 26221004) Satoru Takahashi Grant-in-Aid for Scientific Research(C), Japan Society for the Promotion of Science (JSPS KAKENHI 17K07130) Hiroyoshi Iseki Grant-in-Aid for Young Scientists (B), Japan Society for the Promotion of Science (JSPS KAKENHI 19K16020) Tra Thi Huong Dinh Grant-in-Aid for Scientific Research(A), Japan Society for the Promotion of Science (JSPS KAKENHI 20H00444) Fumihiro Sugiyama The Cooperative Research Project Program of Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA Center), University of Tsukuba, Japan (182107) Fumihiro Sugiyam

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway

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