Variations in dysfunction of sister chromatid cohesion in esco2 mutant zebrafish reflect the phenotypic diversity of Roberts syndrome

Mutations in ESCO2, one of two establishment of cohesion factors necessary for proper sister chromatid cohesion (SCC), cause a spectrum of developmental defects in the autosomal-recessive disorder Roberts syndrome (RBS), warranting in vivo analysis of the consequence of cohesion dysfunction. Through a genetic screen in zebrafish targeting embryonic-lethal mutants that have increased genomic instability, we have identified an esco2 mutant zebrafish. Utilizing the natural transparency of zebrafish embryos, we have developed a novel technique to observe chromosome dynamics within a single cell during mitosis in a live vertebrate embryo. Within esco2 mutant embryos, we observed premature chromatid separation, a unique chromosome scattering, prolonged mitotic delay, and genomic instability in the form of anaphase bridges and micronuclei formation. Cytogenetic studies indicated complete chromatid separation and high levels of aneuploidy within mutant embryos. Amongst aneuploid spreads, we predominantly observed decreases in chromosome number, suggesting that either cells with micronuclei or micronuclei themselves are eliminated. We also demonstrated that the genomic instability leads to p53-dependent neural tube apoptosis. Surprisingly, although many cells required Esco2 to establish cohesion, 10-20% of cells had only weakened cohesion in the absence of Esco2, suggesting that compensatory cohesion mechanisms exist in these cells that undergo a normal mitotic division. These studies provide a unique in vivo vertebrate view of the mitotic defects and consequences of cohesion establishment loss, and they provide a compensation-based model to explain the RBS phenotypes

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic

Genetic modeling of Li-Fraumeni syndrome in zebrafish

Li-Fraumeni syndrome (LFS) is a highly penetrant, autosomal dominant, human familial cancer predisposition. Although a key role for the tumor suppressor p53 has been implicated in LFS, the genetic and cellular mechanisms underpinning this disease remain unknown. Therefore, modeling LFS in a vertebrate system that is accessible to both large-scale genetic screens and in vivo cell biological studies will facilitate the in vivo dissection of disease mechanisms, help identify candidate genes, and spur the discovery of therapeutic compounds. Here, we describe a forward genetic screen in zebrafish embryos that was used to identify LFS candidate genes, which yielded a p53 mutant (p53I166T) that as an adult develops tumors, predominantly sarcomas, with 100% penetrance. As in humans with LFS, tumors arise in heterozygotes and display loss of heterozygosity (LOH). This report of LOH indicates that Knudson’s two-hit hypothesis, a hallmark of human autosomal dominant cancer syndromes, can be modeled in zebrafish. Furthermore, as with some LFS mutations, the zebrafish p53I166T allele is a loss-of-function allele with dominant-negative activity in vivo. Additionally, we demonstrate that the p53 regulatory pathway, including Mdm2 regulation, is evolutionarily conserved in zebrafish, providing a bona fide biological context in which to systematically uncover novel modifier genes and therapeutic agents for human LFS

Tissue-Specific Differences of p53 Inhibition by Mdm2 and Mdm4

The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

<div><p>With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.</p></div

HRM established genotype-phenotype correlation within Esco2 mutant embryos from a G0 intercross.

<p>A) wild-type and mutant phenotypes with Mendelian frequencies in embryos derived from heterozygous intercross of the Esco2 retroviral insertion mutant hi2865. Note the head necrosis in the mutant embryos. B) HRM genotyping of wild-type (grey and blue curves) and mutant (red curves) embryos display perfect genotype-phenotype correlation. C) wild-type and mutant phenotypes in embryos derived from intercross of G0 Esco2 CRISPR injected fish. Note the head necrosis in mutant embryos. D) Select HRM curves of 6 mutant and 6 wild-type embryos that were subsequently sequences to reveal specific alleles result in specific curves. All Wild-type animals (beyond the 6 displayed here) make up the green and grey curves; while 5 of 6 mutant animals make up the unique red and blue curves establishing a genotype phenotype correlation. E&G) are wild-type and mutant phenotypes and frequencies of G0#9 (E) or G0#10 (G) crossed to Esco2 hi2865 heterozygous animals. F&H) are HRM curves of mutant and wild-type embryos (from E&G) that are Esco2 hi2865 heterozygous. All heterozygous curves (red) are mutants, and all grey curves are normal phenotypically.</p

HRM established genotype-phenotype correlation within Esco2 mutant embryos from a G0 intercross.

<p>A) wild-type and mutant phenotypes with Mendelian frequencies in embryos derived from heterozygous intercross of the Esco2 retroviral insertion mutant hi2865. Note the head necrosis in the mutant embryos. B) HRM genotyping of wild-type (grey and blue curves) and mutant (red curves) embryos display perfect genotype-phenotype correlation. C) wild-type and mutant phenotypes in embryos derived from intercross of G0 Esco2 CRISPR injected fish. Note the head necrosis in mutant embryos. D) Select HRM curves of 6 mutant and 6 wild-type embryos that were subsequently sequences to reveal specific alleles result in specific curves. All Wild-type animals (beyond the 6 displayed here) make up the green and grey curves; while 5 of 6 mutant animals make up the unique red and blue curves establishing a genotype phenotype correlation. E&G) are wild-type and mutant phenotypes and frequencies of G0#9 (E) or G0#10 (G) crossed to Esco2 hi2865 heterozygous animals. F&H) are HRM curves of mutant and wild-type embryos (from E&G) that are Esco2 hi2865 heterozygous. All heterozygous curves (red) are mutants, and all grey curves are normal phenotypically.</p

HRM can efficiently detect low level chimeric mutations.

<p>A) Depicts different degrees of chimerism (amount red colored cells) that could occur due to custom nuclease injection. B) HRM curves of TA cloned 300 bp amplicons (100 ng/ul) or genomic DNA (100 ng/ul) of wild-type (grey curves) and either RBΔ2 or RBΔ13 mutations at different allele ratios with wild-type DNA.</p

HRM is highly efficient at detecting CRISPR derived mutations.

<p>HRM is highly efficient at detecting CRISPR derived mutations.</p

HRM analysis reveals low frequency of off-target cleavage by the CRISPR system.

<p>Using the CRISPR Design (<a href="http://crispr.mit.edu/" target="_blank">http://crispr.mit.edu/</a>) web tool we identified the most likely 4 off-target sites in the zebrafish genome for 5 different guides with different overall scores (in parenthesis next to gene name). The sequences are depicted with the PAM site in green and the nucleotide different from the guide sequence in red. To discern SNP's within the Oft amplicons we performed HRM analysis of genomic DNA from 24 wild-type adults. The HRM positive frequency within genomic DNA obtained from 24 G0 embryos injected with the guide/Cas9 RNAs is displayed. Also the HRM positive frequency within genomic DNA obtained from 12 F1 embryos derived from 4 different G0's is displayed. Select HRM curves that display unique features are displayed for G0 embryos as well as F1 embryos. For Ptgs1 Oft 1 is displayed to depict no off-target hits. For Esco2 Oft 3 is displayed to depict a common SNP derived from the wild-type AB. For Wapal1 Oft 1 is displayed to depict no off-target hits. For P107 Oft 2 is displayed to: 1) depict a SNP found in wild-type AB strain, G0 and F1 animals; and 2) novel CRISPR derived off-target hits in some of the F1 progeny (orange and green curves). For Bub1bb, Oft 1 is displayed to depict a low frequency off-target hit (red curve) in the F1 progeny. * in frequencies denotes that SNP curves were not considered a HRM positive.</p