Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes.

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce 'leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.

Presence of Rigid Red Blood Cells in Blood Flow Interferes with the Vascular Wall Adhesion of Leukocytes.

The symptoms of many blood diseases can often be attributed to irregularities in cellular dynamics produced by abnormalities in blood cells, particularly red blood cells (RBCs). Contingent on the disease and its severity, RBCs can be afflicted with increased membrane rigidity as seen in malaria and sickle cell disease. Despite this understanding, little experimental work has been conducted toward understanding the effect of RBC rigidity on cellular dynamics in physiologic blood flow. Though many have computationally modeled complex blood flow to postulate how RBC rigidity may disrupt normal hemodynamics, to date, there lacks a clear understanding of how rigid RBCs affect the blood cell segregation behavior in blood flow, known as margination, and the resulting change in the adhesion of white blood cells (WBCs). In this work, we utilized an in vitro blood flow model to examine how different RBC rigidities and volume fractions of rigid RBCs impact cell margination and the downstream effect on white blood cell (WBC) adhesion in blood flow. Healthy RBC membranes were rigidified and reconstituted into whole blood and then perfused over activated endothelial cells under physiologically relevant shear conditions. Rigid RBCs were shown to reduce WBC adhesion by up to 80%, contingent on the RBC rigidity and the fraction of treated RBCs present in blood flow. Furthermore, the RBC core was found to be slightly expanded with the presence of rigid RBCs, by up to ∼30% in size fully composed of rigid RBCs. Overall, the obtained results demonstrate an impact of RBC rigidity on cellular dynamics and WBC adhesion, which possibly contributes to the pathological understanding of diseases characterized by significant RBC rigidity.

Genome-wide view of TGFß/Foxh1 regulation of the early mesendoderm program.

Nodal/TGFß signaling regulates diverse biological responses. By combining RNA-seq on Foxh1 and Nodal signaling loss-of-function embryos with ChIP-seq of Foxh1 and Smad2/3, we report a comprehensive genome-wide interaction between Foxh1 and Smad2/3 in mediating Nodal signaling during vertebrate mesendoderm development. This study significantly increases the total number of Nodal target genes regulated by Foxh1 and Smad2/3, and reinforces the notion that Foxh1-Smad2/3-mediated Nodal signaling directly coordinates the expression of a cohort of genes involved in the control of gene transcription, signaling pathway modulation and tissue morphogenesis during gastrulation. We also show that Foxh1 may function independently of Nodal signaling, in addition to its role as a transcription factor mediating Nodal signaling via Smad2/3. Finally, we propose an evolutionarily conserved interaction between Foxh1 and PouV, a mechanism observed in Pou5f1-mediated regulation of pluripotency in human embryonic stem and epiblast cells.

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.

The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.

Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.

Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus.

We have developed a method of injecting bacterial artificial chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.

Uncovering the mesendoderm gene regulatory network through multi-omic data integration.

Mesendodermal specification is one of the earliest events in embryogenesis, where cells first acquire distinct identities. Cell differentiation is a highly regulated process that involves the function of numerous transcription factors (TFs) and signaling molecules, which can be described with gene regulatory networks (GRNs). Cell differentiation GRNs are difficult to build because existing mechanistic methods are low throughput, and high-throughput methods tend to be non-mechanistic. Additionally, integrating highly dimensional data composed of more than two data types is challenging. Here, we use linked self-organizing maps to combine chromatin immunoprecipitation sequencing (ChIP-seq)/ATAC-seq with temporal, spatial, and perturbation RNA sequencing (RNA-seq) data from Xenopus tropicalis mesendoderm development to build a high-resolution genome scale mechanistic GRN. We recover both known and previously unsuspected TF-DNA/TF-TF interactions validated through reporter assays. Our analysis provides insights into transcriptional regulation of early cell fate decisions and provides a general approach to building GRNs using highly dimensional multi-omic datasets.

Deformable microparticles for shuttling nanoparticles to the vascular wall.

Vascular-targeted drug carriers must localize to the wall (i.e., marginate) and adhere to a diseased endothelium to achieve clinical utility. The particle size has been reported as a critical physical property prescribing particle margination in vitro and in vivo blood flows. Different transport process steps yield conflicting requirements-microparticles are optimal for margination, but nanoparticles are better for intracellular or tissue delivery. Here, we evaluate deformable hydrogel microparticles as carriers for transporting nanoparticles to a diseased vascular wall. Depending on microparticle modulus, nanoparticle-loaded poly(ethylene glycol)-based hydrogel microparticles delivered significantly more 50-nm nanoparticles to the vessel wall than freely injected nanoparticles alone, resulting in >3000% delivery increase. This work demonstrates the benefit of optimizing microparticles' efficient margination to enhance nanocarriers' transport to the vascular wall.

Sox17 and ß-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and ß-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and ß-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses ß-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/ß-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and ß-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

Exploring deformable particles in vascular-targeted drug delivery: Softer is only sometimes better.

The ability of vascular-targeted drug carriers (VTCs) to localize and bind to a targeted, diseased endothelium determines their overall clinical utility. Here, we investigate how particle modulus and size determine adhesion of VTCs to the vascular wall under physiological blood flow conditions. In general, deformable microparticles (MPs) outperformed nanoparticles (NPs) in all experimental conditions tested. Our results indicate that MP modulus enhances particle adhesion in a shear-dependent manner. In low shear human blood flow profiles in vitro, low modulus particles showed favorable adhesion, while at high shear, rigid particles showed superior adhesion. This was confirmed in vivo by studying particle adhesion under venous shear profiles in a mouse model of mesenteric inflammation, where MP adhesion was 127% greater (p < 0.0001) for low modulus particles compared to more rigid ones. Mechanistically, we establish that particle collisions with leukocytes drive these trends, rather than differences in particle deformation, localization, or detachment. Overall, this work demonstrates the importance of VTC modulus as a design parameter for enhanced VTC interaction with vascular walls, and thus, contributes important knowledge for development of successful clinical theranostics with applications for many diseases.

Neutrophil-Particle Interactions in Blood Circulation Drive Particle Clearance and Alter Neutrophil Responses in Acute Inflammation.

Although nano- and microparticle therapeutics have been studied for a range of drug delivery applications, the presence of these particles in blood flow may have considerable and understudied consequences to circulating leukocytes, especially neutrophils, which are the largest human leukocyte population. The objective of this work was to establish if particulate drug carriers in circulation interfere with normal neutrophil adhesion and migration. Circulating blood neutrophils in vivo were found to be capable of rapidly binding and sequestering injected carboxylate-modified particles of both 2 and 0.5 µm diameter within the bloodstream. These neutrophil-particle associations within the vasculature were found to suppress neutrophil interactions with an inflamed mesentery vascular wall and hindered neutrophil adhesion. Furthermore, in a model of acute lung injury, intravenously administered drug-free particles reduced normal neutrophil accumulation in the airways of C57BL/6 mice between 52% and 60% versus particle-free mice and between 93% and 98% in BALB/c mice. This suppressed neutrophil migration resulted from particle-induced neutrophil diversion to the liver. These data indicate a considerable acute interaction between injected particles and circulating neutrophils that can drive variations in neutrophil function during inflammation and implicate neutrophil involvement in the clearance process of intravenously injected particle therapeutics. Such an understanding will be critical toward both enhancing designs of drug delivery carriers and developing effective therapeutic interventions in diseases where neutrophils have been implicated.

Evaluation of Receptor-Ligand Mechanisms of Dual-Targeted Particles to an Inflamed Endothelium.

Vascular-targeted carriers (VTCs) are designed as leukocyte mimics, decorated with ligands that target leukocyte adhesion molecules (LAMs) and facilitate adhesion to diseased endothelium. VTCs require different design considerations than other targeted particle therapies; adhesion of VTCs in regions with dynamic blood flow requires multiple ligand-receptor (LR) pairs that provide particle adhesion and disease specificity. Despite the ultimate goal of leukocyte mimicry, the specificity of multiple LAM-targeted VTCs remains poorly understood, especially in physiological environments. Here, we investigate particle binding to an inflamed mesentery via intravital microscopy using a series of particles with well-controlled ligand properties. We find that the total number of sites of a single ligand can drive particle adhesion to the endothelium, however, combining ligands that target multiple LR pairs provides a more effective approach. Combining sites of sialyl Lewis A (sLeA) and anti-intercellular adhesion molecule-1 (aICAM), two adhesive molecules, resulted in ~3-7-fold increase of adherent particles at the endothelium over single-ligand particles. At a constant total ligand density, a particle with a ratio of 75% sLeA: 25% aICAM resulted in more than 3-fold increase over all over other ligand ratios tested in our in vivo model. Combined with in vitro and in silico data, we find the best dual-ligand design of a particle is heavily dependent on the surface expression of the endothelial cells, producing better adhesion with more particle ligand for the lesser-expressed receptor. These results establish the importance of considering LR-kinetics in intelligent VTC ligand design for future therapeutics.

Emergence and Utility of Nonspherical Particles in Biomedicine.

The importance of the size of targeted, spherical drug carriers has been previously explored and reviewed. Particle shape has emerged as an equally important parameter in determining the in vivo journey and efficiency of drug carrier systems. Researchers have invented techniques to better control the geometry of particles of many different materials, which have allowed for exploration of the role of particle geometry in the phases of drug delivery. The important biological processes include clearance by the immune system, trafficking to the target tissue, margination to the endothelial surface, interaction with the target cell, and controlled release of a payload. The review of current literature herein supports that particle shape can be altered to improve a system's targeting efficiency. Non-spherical particles can harness the potential of targeted drug carriers by enhancing targeted site accumulation while simultaneously decreasing side effects and mitigating some limitations faced by spherical carriers.

Drug carrier interaction with blood: a critical aspect for high-efficient vascular-targeted drug delivery systems.

Vascular wall endothelial cells control several physiological processes and are implicated in many diseases, making them an attractive candidate for drug targeting. Vascular-targeted drug carriers (VTCs) offer potential for reduced side effects and improved therapeutic efficacy, however, only limited therapeutic success has been achieved to date. This is perhaps due to complex interactions of VTCs with blood components, which dictate VTC transport and adhesion to endothelial cells. This review focuses on VTC interaction with blood as well as novel 'bio-inspired' designs to mimic and exploit features of blood in VTC development. Advanced approaches for enhancing VTCs are discussed along with applications in regenerative medicine, an area of massive potential growth and expansion of VTC utility in the near future.

Cas9-based genome editing in Xenopus tropicalis.

Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.