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A long-standing challenge in drug development is the identification of the mechanisms of action of small molecules with therapeutic potential. A number of methods have been developed to address this challenge, each with inherent strengths and limitations. We here provide a brief review of these methods with a focus on chemical-genetic methods that are based on systematically profiling the effects of genetic perturbations on drug sensitivity. In particular, application of these methods to mammalian systems has been facilitated by the recent advent of CRISPR-based approaches, which enable one to readily repress, induce, or delete a given gene and determine the resulting effects on drug sensitivity.

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.

It has been suggested that monocytes/macrophages represent the pivotal cell type during early adaptive growth of pre‐existent arterial anastomoses toward functional collateral arteries (arteriogenesis) upon arterial occlusion. This hypothesis was supported by previous studies providing evidence that elevation of the peripheral monocyte count, increased monocyte survival (e.g., granulocyte macrophage‐colony stimulating factor), as well as enhanced attraction or adhesion (e.g., monocyte chemoattractant protein 1; intercellular adhesion molecule 1) of the latter cells correlates directly with the arteriogenic response to restore tissue perfusion. However, the experimental proof of the essential role of monocytes/macrophages remains to be given. We therefore hypothesized that arteriogenesis is reduced in a genuine, nonpharmocologically induced monocyte/macrophage‐deficient model of femoral artery occlusion in osteopetrotic (op/op) mice. Total leukocyte count did not differ between op/op mice and control (B6C3Fe a/a‐Csf1+/+) mice. op/op mice show a significant monocytopenia (0.67%±0.38% vs. 1.53%±0.32%), granulocytosis (33.66%±6.67% vs. 22.83±7.47%), and a concomitant, relative lymphopenia (65.67%±6.58% vs. 75.65%±7.31%). Microsphere‐based perfusion measurement 7 days after femoral artery occlusion demonstrated a significantly reduced perfusion restoration upon femoral artery occlusion in op/op mice as compared with controls (28.19%±0.91% vs. 47.88%±2.49%). The application of a novel method of high resolution (microfocus X‐ray system) angiography revealed a reduction of proliferation and diameter of collateral arteries. Quantitative immunohistology showed significantly lower numbers of macrophages in the surrounding tissue of proliferating arteries. This study provides additional evidence for the preeminent role of monocytes/macrophages during arteriogenesis in a genuine model of monocyte deficiency, i.e., without pharmacological intervention.

Cardiovascular and cerebrovascular disease represent the two most common causes of mortality and morbidity in western countries, and the treatment for these is generally by the mechanical restoration of blood flow in the affected tissues. Stimulation of collateral artery growth (arteriogenesis) provides a potential alternative option for the treatment of patients suffering from occlusive artery disease. Therefore, researchers have established several angiogenesis and arteriogenesis animal models to investigate basic mechanisms and pharmacological modulation of collateral artery growth. The authors highlight the most important aspects of vascular growth, discuss different methods and techniques for examining the process, and review the advantages and disadvantages associated with the animal models available for studying this phenomenon.

Detection and purification of novel bioactive peptides from biological sources is a scientific task that led to a substantial number of important discoveries. One major laborious approach used is the repetitive stepwise separation of the test sample into several fractions followed by the determination of their bioactivity, until purity allows for sequence identification. We tested whether functional peptidomics, a combination of biological read-outs with differential peptide display (DPD) is a suitable strategy to isolate bioactive peptides at lower workload and with improved success. Additionally, we evaluated the use of DPD to monitor the processing status of proinsulin by inhibition of the insulin processing pathway. The rat insulinoma cell line INS-1 stimulated either with 2 mmol/l or 10 mmol/l glucose was used as model to generate differential peptide displays. In parallel, the bioactivity of the supernatants from the INS-1 cells was measured by glucose uptake and lipolysis assays using the adipocyte cell line 3T3-L1. We were able to quickly and elegantly trace the known activity of insulin to increase glucose uptake and inhibit lipolysis. Following re-chromatography of selected fractions, relevant peptides were identified by DPD and bioassays: the rat insulin-1 precursor and two different insulin peptides. We demonstrated in a semi-quantitative fashion that inhibition of proinsulin processing leads to accumulation of the insulin precursor, and reduced secretion of insulin-1. Thus, we conclude that DPD is an attractive support technology in peptide purification strategies aiming to identify bioactive compounds, and is superior to ELISA in discriminating between the processing status of insulin and its precursor.

Essential genes are the hubs of cellular networks, but lack of high-throughput methods for titrating gene expression has limited our understanding of the fitness landscapes against which their expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISPRi activity and demonstrated its broad applicability. Using libraries of mismatched sgRNAs predicted to span the full range of knockdown levels, we characterized the expression-fitness relationships of most essential genes in Escherichia coliand Bacillus subtilis. We find that these relationships vary widely from linear to bimodal but are similar within pathways. Notably, despite ∼2 billion years of evolutionary separation between E. coliand B. subtilis, most essential homologs have similar expression-fitness relationships with rare but informative differences. Thus, the expression levels of essential genes may reflect homeostatic or evolutionary constraints shared between the two organisms.

Discriminating between phenotypically indistinguishable heterozygous and wild-type mice presents a difficult situation for researchers attempting to find suitable breeding pairs. The authors describe a fast, reliable, nonradioactive method that combines PCR with capillary electrophoresis to determine genotypical differences among heterozygous (+/op), homozygous (op/op), and wild-type (+/+) mice.