CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient biallelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.
CRISPR-Cas9 and related technologies can efficiently alter genomic DNA at targeted positions and have far-reaching implications for functional screening and therapeutic gene editing. Understanding and unlocking this potential requires accurate evaluation of editing efficiency. Here, we show that methodological decisions for analyzing sequencing data can significantly affect mutagenesis efficiency estimates and we provide a comprehensive R-based toolkit, CrispRVariants and an accompanying web tool CrispRVariantsLite, that resolve and localize individual mutant alleles with respect to the endonuclease cut site. CrispRVariantsenabled analyses of newly generated and existing genome editing datasets underscore how careful consideration of the full variant spectrum gives insight toward effective guide and amplicon design as well as the mutagenic process.
Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin ( drl ) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl -based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona , and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.
Much has been written in recent years about the changes in corporate strategies and industry structures associated with electronic coordination of market activities. This paper considers the advent of electronic market coordination in the home mortgage industry, focusing on Computerized Lean Origination (CL O) systems. Case studies of five CLOs (First Boston's Shelternet, PRC's LoanExpress, American Financial Network's Rennie Mae, Prudential's CLOS, and Citicorp's Mortgage Power Plus) reveal a range of system functionalities. Predictions from the Electronic Markets Hypothesis (EMH) are tested against the empirical results of the five case studies. As suggested by the EMH, financial intermediaries have been threatened by the in-"troduction of CLOS, and in some cases opposition has been mounted against the systems. On the other hand, despite the availability of the technology and mortgages' seemingly favorable characteristics as an electronically mediated market product, the industry has not been fundamentally changed by the introduction of these systems, despite more than a decade of experience with them. Of the two case studies that could be characterized as electronic markets, neither continues to exist in that form today. And the system with the largest dollar volume of mortgages of the five is best characterized as an electronic hierarchy. These results suggest that either the full results predicted by the EMH require a longer gestation period or that the underlying hypothesis will require augmentation in order to fully explain the results in the home mortgage market.
Mutant Estrogen Receptor (ERT2) ligand-binding domain fusions with Cre recombinase are a key tool for spatio-temporally controlled genetic recombination with the Cre/lox system. CreERT2 is efficiently activated in a concentration-dependent manner by the Tamoxifen metabolite trans-4-OH-Tamoxifen (trans-4-OHT). Reproducible and efficient Cre/lox experimentation is hindered by the gradual loss of CreERT2 induction potency upon prolonged storage of dissolved trans-4-OHT, which potentially results from gradual trans-to-cis isomerization or degradation. Here, we combined zebrafish CreERT2 recombination experiments and cell culture assays to document the gradual activity loss of trans-4-OHT and describe the alternative Tamoxifen metabolite Endoxifen as more stable alternative compound. Endoxifen retains potent activation upon prolonged storage (3 months), yet consistently induces half the ERT2 domain fusion activity compared to fresh trans-4-OHT. Using 1H-NMR analysis, we reveal that trans-4-OHT isomerization is undetectable upon prolonged storage in either DMSO or Ethanol, ruling out isomer transformation as cause for the gradual loss of trans-4-OHT activity. We further establish that both trans-4-OHT and Endoxifen are insensitive to light exposure under regular laboratory handling conditions. We attribute the gradual loss of trans-4-OHT potency to precipitation over time, and show that heating of aged trans-4-OHT aliquots reinstates their CreERT2 induction potential. Our data establish Endoxifen as potent and reproducible complementary compound to 4-OHT to control ERT2 domain fusion proteins in vivo, and provide a framework for efficient chemically controlled recombination experiments.
Intestinal smooth muscle cells (iSMCs) are a crucial component of the adult gastrointestinal tract and support intestinal differentiation, peristalsis and epithelial homeostasis during development. Despite these crucial roles, the origin of iSMCs and the mechanisms responsible for their differentiation and function remain largely unknown in vertebrates. Here, we demonstrate that iSMCs arise from the lateral plate mesoderm (LPM) in a stepwise process. Combining pharmacological and genetic approaches, we show that TGFβ/Alk5 signaling drives the LPM ventral migration and commitment to an iSMC fate. The Alk5-dependent induction of zeb1a and foxo1a is required for this morphogenetic process: zeb1a is responsible for driving LPM migration around the gut, whereas foxo1a regulates LPM predisposition to iSMC differentiation. We further show that TGFβ, zeb1a and foxo1a are tightly linked together by miR-145. In iSMC-committed cells, TGFβ induces the expression of miR-145, which in turn is able to downregulate zeb1a and foxo1a. The absence of miR-145 results in only a slight reduction in the number of iSMCs, which still express mesenchymal genes but fail to contract. Together, our data uncover a cascade of molecular events that govern distinct morphogenetic steps during the emergence and differentiation of vertebrate iSMCs.
In jawed vertebrates (gnathostomes), Hox genes play an important role in patterning head and jaw formation, but mechanisms coupling Hox genes to neural crest (NC) are unknown. Here we use cross-species regulatory comparisons between gnathostomes and lamprey, a jawless extant vertebrate, to investigate conserved ancestral mechanisms regulating Hox2 genes in NC. Gnathostome Hoxa2 and Hoxb2 NC enhancers mediate equivalent NC expression in lamprey and gnathostomes, revealing ancient conservation of Hox upstream regulatory components in NC. In characterizing a lamprey hoxα2 NC/hindbrain enhancer, we identify essential Meis, Pbx, and Hox binding sites that are functionally conserved within Hoxa2/Hoxb2 NC enhancers. This suggests that the lamprey hoxα2 enhancer retains ancestral activity and that Hoxa2/Hoxb2 NC enhancers are ancient paralogues, which diverged in hindbrain and NC activities. This identifies an ancestral mechanism for Hox2 NC regulation involving a Hox-TALE regulatory circuit, potentiated by inputs from Meis and Pbx proteins and Hox auto-/cross-regulatory interactions.
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