Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

A Novel System for Monitoring in vivo Cell Signaling Pathways Involved in Early Embryonic Patterning

<p>Early developmental events, such as the arrangement of the head-tail axis, are fundamentally driven by cell signalling cascades. Such incidents are regulated in a highly complex manner by promoters and inhibitors at many levels of the cascade. This complexity makes it difficult to understand where and when certain signalling occurs, and what effects additional factors have on the signalling system. Nodal signalling, executed by intracellular Smad2/3 signal propagation, is thought to induce the anterior-posterior and head-tail patterning of the early mouse embryo. Target gene outputs of this signalling are fine-tuned by a vast array of modulators; TGBβ co-receptors, extracellular ligand and receptor inhibitors, DNA binding cofactors, and intracellular enhancers and inhibitors. The endogenous target genes of this system cannot be used as a measure of signalling as they themselves feedback on the original system and others, creating diverse signals. In this body of work, we have distilled the Nodal signalling cascade to a single variable by creating a fluorescent genetic reporter to semi-quantitatively measure Smad signalling during early embryonic development. Reporter constructs contain Smad binding elements, a minimal promoter and fluorescent protein elements. Various sensitivity Smad binding elements were created to respond to different thresholds of signalling. Fluorescent microscopy and flow cytometry were used to verify responsiveness of reporter constructs, tested first in a mouse embryonic fibroblast line and subsequently in transgenic embryos. This study will provide an understanding of how extracellular cues dictate gene expression during early embryonic formation. The knowledge acquired from this work may have implications in dairy cattle and human fertility.</p>

A Novel System for Monitoring in vivo Cell Signaling Pathways Involved in Early Embryonic Patterning

<p>Early developmental events, such as the arrangement of the head-tail axis, are fundamentally driven by cell signalling cascades. Such incidents are regulated in a highly complex manner by promoters and inhibitors at many levels of the cascade. This complexity makes it difficult to understand where and when certain signalling occurs, and what effects additional factors have on the signalling system. Nodal signalling, executed by intracellular Smad2/3 signal propagation, is thought to induce the anterior-posterior and head-tail patterning of the early mouse embryo. Target gene outputs of this signalling are fine-tuned by a vast array of modulators; TGBβ co-receptors, extracellular ligand and receptor inhibitors, DNA binding cofactors, and intracellular enhancers and inhibitors. The endogenous target genes of this system cannot be used as a measure of signalling as they themselves feedback on the original system and others, creating diverse signals. In this body of work, we have distilled the Nodal signalling cascade to a single variable by creating a fluorescent genetic reporter to semi-quantitatively measure Smad signalling during early embryonic development. Reporter constructs contain Smad binding elements, a minimal promoter and fluorescent protein elements. Various sensitivity Smad binding elements were created to respond to different thresholds of signalling. Fluorescent microscopy and flow cytometry were used to verify responsiveness of reporter constructs, tested first in a mouse embryonic fibroblast line and subsequently in transgenic embryos. This study will provide an understanding of how extracellular cues dictate gene expression during early embryonic formation. The knowledge acquired from this work may have implications in dairy cattle and human fertility.</p>

MLL/AF9 Expression Causes Leukemia in Zebrafish

Background Acute Leukaemia (AL) is a genetically heterogeneous disorder caused by somatic mutations and acquired chromosomal translocations. Translocations can lead to the formation of fusion genes such as the MLL/AF9 fusion, which results from the t(9;11)(p22;q23). A better understanding the molecular pathophysiology of AML, of the mechanisms of treatment resistance, disease relapse can be achieved by developing animal models. The MLL/AF9 fusion is frequently used to model AML in mice. However, to date, no MLL/AF9 leukemia models in zebrafish have been reported. Aim Our aim was to establish a transgenic zebrafish leukemia model using the human MLL/AF9 fusion gene. Methods To generate transgenic fish, two constructs (pTol2-Runx1+23: MLL-AF9-IRES-EGFP-cmlc-GFP and pTol2-Runx1+23: MLL-AF9-IRES-mCherry-cmlc-GFP) were injected together with Tol2 transposase mRNA into one-cell stage zebrafish embryos. We used the murine Runx1+23 enhancer to direct MLL/AF9 expression to hematopoietic stem cells and EGFP or mCherry as fluorescent markers. We selected transgenic embryos 24 hours post-fertilization based on the heart marker expression (cmlc). Results 29% (100 of 340 embryos) of the transgenics reached adulthood (6 weeks). After 6 to 24 months, 77% (77) of them developed signs of sickness. They became less active with protruding eyes and hump formation on the nose. Some started bleeding from the gills and/or showed tumor formation around the abdomen and head. Sick fish were euthanized and dissected. The autopsies showed pale and dysmorphic kidneys, pale and enlarged spleens, and in some cases white granular spots on the spleen. Histological sections revealed increased kidney, spleen, and liver cellularity with massive cellular infiltration of cells in these organs. In flow cytometry, kidney marrow cells from the transgenic fish showed a different forward scatter (FSC) and side scatter (SSC) profile compared to that of the normal zebrafish kidney marrow cells. The transgenic F 0 zebrafish showed increased numbers of lymphoid cells (12 fish), precursor cells (9 fish), or myeloid cells (6 fish). Peripheral blood smears showed many intermediate-sized mononuclear cells with an increased nuclear-cytoplasmic ratio, with nuclei containing dipsersed chromatin and inconspicuous nucleoli resembling blasts. There were occasional myelocytes and no mature granulocytes. These data are consistent with the development of acute leukemia in our MLL/AF9 transgenic fish. Whole-mount in situ hybridization (WISH) was performed on F 1 embryos. RNA probes for early hematopoietic markers (gata1, scl, runx1, ikaros, cmyb, mpx, and lyz) were hybridized to 24 and 48 hpf F1 transgenic embryos. There were expression changes of these markers compared to age-matched wild-type larvae, including low expression of gata1, scl, cmyb and high expression of lyz, mpx and ikaros in the caudal hematopoietic organ. We also performed transplantation experiments with the kidney marrow cells from diseased fish to test whether the disease was transplantable. The disease was serially transplantable into secondary and tertiary recipient fish. Transplanted fish had a significantly shorter latency to disease development of only 2 to 6 weeks. The morphological evidence and the serial transplantability of the disease proves that we have in fact succeeded in establishing an MLL/AF9-driven acute leukemia model in zebrafish. The long latency and incomplete penetrance observed in our F 0 MA9 zebrafish, along with a shorter latency in the transplanted fish, suggests that additional somatic mutations are required for leukemogenesis in this model. We performed whole exome sequencing to find cooperating somatic mutations and RNASeq to identify differentially expressed genes in MA9 leukemia fish. Whole exome sequencing on six samples identified putative somatic mutations in genes such as Stat5, Cyp2j20, Ms4a17a.3, Tapbp.1 and Herc5.3, which have been reported to be mutated in human cancer. RNA-seq analysis on seven samples showed 67 differentially expressed genes with a q value &lt; 0.05 (e.g., cxcl32b.1, myof1, ctdsp2, egr3, il2rb) and nine enriched pathways with a P-value of &lt; 0.054 (e.g.: KRAS, TP53, MEK) in our transgenic leukemic MLL/AF9 fish. Conclusion Our MLL/AF9 zebrafish acute leukemia model will be a helpful tool to understand leukemia biology and enable testing of new therapeutic strategies. Disclosures Browett: AbbVie: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

FOXO1 represses Sprouty2 and Sprouty4 expression in endothelial cells to promote arterial specification and vascular remodeling in the mouse yolk sac

The establishment of a functional circulatory system is required for post-implantation development during mouse embryogenesis. Previous studies in null mouse models have reported that FOXO1, a Forkhead family transcription factor, is essential for yolk sac vascular remodeling and survival beyond embryonic day (E) 11. Here, we show that loss of FoxO1 in E8.25 endothelial cells results in increased Sprouty2 and Sprouty4 gene expression, reduced expression of arterial genes, and reduced Flk1/Vegfr2 expression without affecting overall endothelial cell identity, survival or proliferation. Using Dll4-BAC-nlacZ reporter, we found that one of the earliest expressed arterial genes, Dll4, is significantly reduced in the yolk sac of FoxO1 mutants without being substantially affected in the embryo. We show that in the yolk sac, FOXO1 not only binds directly to a subset of previously identified activating Sprouty2 regulatory regions and newly identified conserved Sprouty4 regulatory regions, but can also repress their expression. Additionally, over expression of Sprouty4 in E8.25 transient transgenic embryos largely recapitulates reduced expression of arterial genes seen in endothelial FoxO1 mutants. These data reveal a novel role for FOXO1 as a key transcriptional repressor in early, pre-flow arterial specification and subsequent vessel remodeling within the murine yolk sac.

Highly Efficient Knockin in Human iPS Cells and Rat Embryos by CRISPR/Cas9 Molecular Optimization

The CRISPR/Cas9 system is now the gold standard for the generation of genetically modified cell and animal models but knockin is a bottleneck. One reason could be that there is no consensus regarding the concentrations of its components to be used. Here, we defined optimal Cas9 protein, guide RNA and short donor DNA concentrations on a GFP to BFP conversion model of human induced pluripotent stem cells and point mutations on rat transgenic embryos. With a molecular rational approach of the CRISPR/Cas9 system and study of ribonucleoprotein complex formation by nanodifferential scanning fluorimetry, we defined that Cas9/guide RNA 1/1 molar ratio with 0.2µM and 0.4µM of Cas9, coupled with 2µM of ssODN are sufficient for optimal and high knockin frequencies in rat embryos and human induced pluripotent stem cells, respectively. These optimal conditions use lower concentrations of CRISPR reagents to form the RNP complex than most conditions published while achieving 50% of knockin. This study allowed us to reduce costs and toxicity while improving editing and knockin efficacy on two particularly key models to mimic human diseases.

The blood flow-klf6a-tagln2 axis drives vessel pruning in zebrafish by regulating endothelial cell rearrangement and actin cytoskeleton dynamics

Recent studies have focused on capillary pruning in various organs and species. However, the way in which large-diameter vessels are pruned remains unclear. Here we show that pruning of the zebrafish caudal vein (CV) from ventral capillaries of the CV plexus in different transgenic embryos is driven by endothelial cell (EC) rearrangement, which involves EC nucleus migration, junction remodeling, and actin cytoskeleton remodeling. Further observation reveals a growing difference in blood flow velocity between the two vessels in CV pruning in zebrafish embryos. With this model, we identify the critical role of Kruppel-like factor 6a (klf6a) in CV pruning. Disruption of klf6a functioning impairs CV pruning in zebrafish. klf6a is required for EC nucleus migration, junction remodeling, and actin cytoskeleton dynamics in zebrafish embryos. Moreover, actin-related protein transgelin 2 (tagln2) is a direct downstream target of klf6a in CV pruning in zebrafish embryos. Together these results demonstrate that the klf6a-tagln2 axis regulates CV pruning by promoting EC rearrangement.

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant’s phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.

Overdosage of HNF1B Gene Associated With Annular Pancreas Detected in Neonate Patients With 17q12 Duplication

The annular pancreas (AP) is a congenital anomaly of the pancreas that can cause acute abdominal pain and vomiting after birth. However, the genetic cause of AP is still unknown, and no study has reported AP in patients with 17q12 duplication. This study retrospectively analyzed the next-generation sequencing (NGS) data of individuals from January 2016 to June 2020 for 17q12 duplication. To identify the function of the key gene of HNF1B in the 17q12 duplication region, human HNF1B mRNA was microinjected into LiPan zebrafish transgenic embryos. A total of 19 cases of 17q12 duplication were confirmed. AP was diagnosed during exploratory laparotomy in four patients (21.1%). The other common features of 17q12 duplication included intellectual disability (50%), gross motor delay (50%), and seizures/epilepsy (31.58%). The ratio of the abnormal pancreas in zebrafish was significantly higher in the HNF1B overexpression models. In conclusion, we first reported AP in patients with duplication of the 17q12 region, resulting in the phenotype of 17q12 duplication syndrome. Furthermore, our zebrafish studies verified the role of the HNF1B gene in pancreatic development.

Epigenetic Events and Germ Line Transmission in Rag1 knock-out Chimeric Mice Produced by CRISPR/Cas9 Technology

Considering the effects of epigenetic changes on the embryonic development through altering the gene expression of pluripotency, trophectoderm, and imprinting genes, we determined the pattern of epigenetic alterations in transgenic embryos produced by injecting transgenic mESCs into the morula. To aim this, RAG1 knock-out mESCs were produced using CRISPR-Cas9 editing system and then microinjected into morulas to develop chimeric embryos. Afterward, the expression of pluripotency, trophectoderm genes, and imprinting genes in these embryos were evaluated using real-time PCR. Immunohistochemical analysis was carried out to determine the methylation rates of H3K9me3 and H3K4me3. In the following, since epigenetic alterations can be at least one of the possible reasons of male infertility and the loss of germ-line transmission capacity, the germ-line transmission ability in chimeric mice was also evaluated by breeding them and subsequent backcrosses to wild type strains. Our findings showed that the methylation rates of H3K4me3 and H3K9me3 were significantly lower and higher respectively in the RAG1 knock-out embryos compared others groups. Moreover, the chimeric embryos exhibited the decreased expression of NANOG, OCT4, CDX2, TEAD4, and H19 genes. Following the breeding of chimeric males with normal female mouse, 40% of chimeras had no germ-line transmission (GLT), and 60% were infertile.We showed that the manipulation of mESC by CRISPR-Cas9 approach remarkably changed the methylation status of H3K9me3 and H3K4me3, resulting in impaired development of embryos through dysregulation of genes involved embryonic development and then, may be one of the reasons of infertility and lack of GLT.