Nickel-Catalyzed Antibody Bioconjugation.

New biocompatible methods for post-translational protein modification are challenging to develop but crucial to create improved chemical probes and optimize next-generation biologic therapies such as antibody-drug conjugates (ADCs). Herein, we describe the bottom-up construction of an aqueous nickel-catalyzed cross-coupling for the chemospecific arylation of cysteine residues on peptides and proteins and its use for the preparation of ADCs. A variety of arene linkages are exemplified, enabling the incorporation of small molecules, probes, and cytotoxic payloads. The utility of this new bioconjugation platform in a drug discovery setting is highlighted by the construction of novel ADCs with target-mediated in vitro cytotoxic activity.

Epithelial-Mesenchymal Micro-niches Govern Stem Cell Lineage Choices.

Adult tissue stem cells (SCs) reside in niches, which, through intercellular contacts and signaling, influence SC behavior. Once activated, SCs typically give rise to short-lived transit-amplifying cells (TACs), which then progress to differentiate into their lineages. Here, using single-cell RNA-seq, we unearth unexpected heterogeneity among SCs and TACs of hair follicles. We trace the roots of this heterogeneity to micro-niches along epithelial-mesenchymal interfaces, where progenitors display molecular signatures reflective of spatially distinct local signals and intercellular interactions. Using lineage tracing, temporal single-cell analyses, and chromatin landscaping, we show that SC plasticity becomes restricted in a sequentially and spatially choreographed program, culminating in seven spatially arranged unilineage progenitors within TACs of mature follicles. By compartmentalizing SCs into micro-niches, tissues gain precise control over morphogenesis and regeneration: some progenitors specify lineages immediately, whereas others retain potency, preserving self-renewing features established early while progressively restricting lineages as they experience dynamic changes in microenvironment.

Rac1 plays an essential role in axon growth and guidance and in neuronal survival in the central and peripheral nervous systems.

BACKGROUND: Rac1 is a critical regulator of cytoskeletal dynamics in multiple cell types. In the nervous system, it has been implicated in the control of cell proliferation, neuronal migration, and axon development. RESULTS: To systematically investigate the role of Rac1 in axon growth and guidance in the developing nervous system, we have examined the phenotypes associated with deleting Rac1 in the embryonic mouse forebrain, in cranial and spinal motor neurons, in cranial sensory and dorsal root ganglion neurons, and in the retina. We observe a widespread requirement for Rac1 in axon growth and guidance and a cell-autonomous defect in axon growth in Rac1 (-/-) motor neurons in culture. Neuronal death, presumably a secondary consequence of the axon growth and/or guidance defects, was observed in multiple locations. Following deletion of Rac1 in the forebrain, thalamocortical axons were misrouted inferiorly, with the majority projecting to the contralateral thalamus and a minority projecting ipsilaterally to the ventral cortex, a pattern of misrouting that is indistinguishable from the pattern previously observed in Frizzled3 (-/-) and Celsr3 (-/-) forebrains. In the limbs, motor-neuron-specific deletion of Rac1 produced a distinctive stalling of axons within the dorsal nerve of the hindlimb but a much milder loss of axons in the ventral hindlimb and forelimb nerves, a pattern that is virtually identical to the one previously observed in Frizzled3 (-/-) limbs. CONCLUSIONS: The similarities in axon growth and guidance phenotypes caused by Rac1, Frizzled3, and Celsr3 loss-of-function mutations suggest a mechanistic connection between tissue polarity/planar cell polarity signaling and Rac1-dependent cytoskeletal regulation.

Partial interchangeability of Fz3 and Fz6 in tissue polarity signaling for epithelial orientation and axon growth and guidance.

In mammals, a set of anatomically diverse polarity processes - including axon growth and guidance, hair follicle orientation, and stereociliary bundle orientation in inner ear sensory hair cells - appear to be mechanistically related, as judged by their dependence on vertebrate homologues of core tissue polarity/planar cell polarity (PCP) genes in Drosophila. To explore more deeply the mechanistic similarities between different polarity processes, we have determined the extent to which frizzled 3 (Fz3) can rescue the hair follicle and Merkel cell polarity defects in frizzled 6-null (Fz6(-/-)) mice, and, reciprocally, the extent to which Fz6 can rescue the axon growth and guidance defects in Fz3(-/-) mice. These experiments reveal full rescue of the Fz6(-/-) phenotype by Fz3 and partial rescue of the Fz3(-/-) phenotype by Fz6, implying that these two proteins are likely to act in a conserved manner in these two contexts. Stimulated by these observations, we searched for additional anatomical structures that exhibit macroscopic polarity and that might plausibly use Fz3 and/or Fz6 signaling. This search has revealed a hitherto unappreciated pattern of papillae on the dorsal surface of the tongue that depends, at least in part, on redundant signaling by Fz3 and Fz6. Taken together, these experiments provide compelling evidence for a close mechanistic relationship between multiple anatomically diverse polarity processes.

Frizzled3 is required for the development of multiple axon tracts in the mouse central nervous system.

Targeted mutation of the Frizzled3 (Fz3) gene in mice has been shown to disrupt the growth and guidance of a subset of peripheral and central axons. Here we used conditional deletion of Fz3 to explore the forebrain territories in which Fz3 action is required for the development of the anterior commissure and the corticothalamic, corticospinal, and thalamocortical tracts. Experiments with region-specific deletion of Fz3 using a variety of Cre lines show that proper routing of corticothalamic and thalamocortical axons in the internal capsule requires Fz3 expression in the ventral telencephalon. The pattern of defects among forebrain axon tracts that are induced by conditional deletion of Fz3 conforms closely to the pattern previously observed with analogous conditional deletion of Celsr3, implying a close mechanistic link between Fz3 and Celsr3 in axon guidance. We further found that several central nervous system axon tracts require Fz3 function as early as embryonic day 11.5, and that Fz3 is required for pathfinding by dopaminergic and serotonergic axons in the brain and by a subset of optic tract axons. In addition, conditional deletion of Fz3 in all tissues caudal to the neck eliminates the spinothalamic tract and the transmission of somatosensory information from the spinal cord to the brain, as determined by neuroanatomic tracing and behavioral testing.

Frizzled3 controls axonal development in distinct populations of cranial and spinal motor neurons.

Disruption of the Frizzled3 (Fz3) gene leads to defects in axonal growth in the VII(th) and XII(th) cranial motor nerves, the phrenic nerve, and the dorsal motor nerve in fore- and hindlimbs. In Fz3(-/-) limbs, dorsal axons stall at a precise location in the nerve plexus, and, in contrast to the phenotypes of several other axon path-finding mutants, Fz3(-/-) dorsal axons do not reroute to other trajectories. Affected motor neurons undergo cell death 2 days prior to the normal wave of developmental cell death that coincides with innervation of muscle targets, providing in vivo evidence for the idea that developing neurons with long-range axons are programmed to die unless their axons arrive at intermediate targets on schedule. These experiments implicate planar cell polarity (PCP) signaling in motor axon growth and they highlight the question of how PCP proteins, which form cell-cell complexes in epithelia, function in the dynamic context of axonal growth. DOI: http://dx.doi.org/10.7554/eLife.01482.001.

New mouse lines for the analysis of neuronal morphology using CreER(T)/loxP-directed sparse labeling.

BACKGROUND: Pharmacologic control of Cre-mediated recombination using tamoxifen-dependent activation of a Cre-estrogen receptor ligand binding domain fusion protein [CreER(T)] is widely used to modify and/or visualize cells in the mouse. METHODS AND FINDINGS: We describe here two new mouse lines, constructed by gene targeting to the Rosa26 locus to facilitate Cre-mediated cell modification. These lines should prove particularly useful in the context of sparse labeling experiments. The R26rtTACreER line provides ubiquitous expression of CreER under transcriptional control by the tetracycline reverse transactivator (rtTA); dual control by doxycycline and tamoxifen provides an extended dynamic range of Cre-mediated recombination activity. The R26IAP line provides high efficiency Cre-mediated activation of human placental alkaline phosphatase (hPLAP), complementing the widely used, but low efficiency, Z/AP line. By crossing with mouse lines that direct cell-type specific CreER expression, the R26IAP line has been used to produce atlases of labeled cholinergic and catecholaminergic neurons in the mouse brain. The R26IAP line has also been used to visualize the full morphologies of retinal dopaminergic amacrine cells, among the largest neurons in the mammalian retina. CONCLUSIONS: The two new mouse lines described here expand the repertoire of genetically engineered mice available for controlled in vivo recombination and cell labeling using the Cre-lox system.