Tuning collagen nonlinear mechanics with interpenetrating networks drives adaptive cellular phenotypes in three dimensions.

In living tissues, collagen networks rarely exist alone because they are embedded within other biological matrices. When combined, collagen networks rigidify via synergistic mechanical interactions and stiffen only with higher mechanical loads. However, how cells respond to the nonlinear elasticity of collagen in hybrid networks remains largely unknown.

Genomic mosaicism reveals developmental organization of trunk neural crest-derived ganglia.

The neural crest generates numerous cell types, but conflicting results leave developmental origins unresolved. Here using somatic mosaic variants as cellular barcodes, we infer embryonic clonal dynamics of trunk neural crest, focusing on the sensory and sympathetic ganglia. From three independent adult neurotypical human donors, we identified 1,278 mosaic variants using deep whole-genome sequencing, then profiled allelic fractions in 187 anatomically dissected ganglia.

A Lifeact-EGFP quail for studying actin dynamics in vivo.

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis.

Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation.

Keratins are asymmetrically inherited fate determinants in the mammalian embryo.

To implant in the uterus, the mammalian embryo first specifies two cell lineages: the pluripotent inner cell mass that forms the fetus, and the outer trophectoderm layer that forms the placenta. In many organisms, asymmetrically inherited fate determinants drive lineage specification, but this is not thought to be the case during early mammalian development. Here we show that intermediate filaments assembled by keratins function as asymmetrically inherited fate determinants in the mammalian embryo.

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing.

Cell Fate Decisions During Preimplantation Mammalian Development.

The early mouse embryo offers a phenomenal system to dissect how changes in the mechanisms controlling cell fate are integrated with morphogenetic events at the single-cell level. New technologies based on live imaging have enabled the discovery of dynamic changes in the regulation of single genes, transcription factors, and epigenetic mechanisms directing early cell fate decision in the early embryo. Here, we review recent progress in linking molecular dynamic events occurring at the level of the single cell in vivo, to some of the key morphogenetic changes regulating early mouse development.

Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy.

Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems.

Sustained Exocytosis after Action Potential-Like Stimulation at Low Frequencies in Mouse Chromaffin Cells Depends on a Dynamin-Dependent Fast Endocytotic Process.

Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles.

Long-Lived Binding of Sox2 to DNA Predicts Cell Fate in the Four-Cell Mouse Embryo.

Transcription factor (TF) binding to DNA is fundamental for gene regulation. However, it remains unknown how the dynamics of TF-DNA interactions change during cell-fate determination in vivo. Here, we use photo-activatable FCS to quantify TF-DNA binding in single cells of developing mouse embryos.

Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo.

Every cell in our body originates from the pluripotent inner mass of the embryo, yet it is unknown how biomechanical forces allocate inner cells in vivo. Here we discover subcellular heterogeneities in tensile forces, generated by actomyosin cortical networks, which drive apical constriction to position the first inner cells of living mouse embryos. Myosin II accumulates specifically around constricting cells, and its disruption dysregulates constriction and cell fate.

Influence of gold nanoparticles on the kinetics of α-synuclein aggregation.

α-synuclein (AS) is a small (140 amino acids), abundant presynaptic protein, which lacks a unique secondary structure in aqueous solution. Amyloid aggregates of AS in dopaminergic neurons of the midbrain are the hallmark of Parkinson's disease (PD). The process of aggregation involves a series of complex structural transitions from innocuous monomeric AS to oligomeric, presumably neurotoxic, forms and finally to fibril formation.

The immediately releasable pool of mouse chromaffin cell vesicles is coupled to P/Q-type calcium channels via the synaptic protein interaction site.

It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca(2+) channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca(2+) current. Accordingly, in the present work we found that the Ca(2+) current flowing through P/Q-type Ca(2+) channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels.

The immediately releasable vesicle pool: highly coupled secretion in chromaffin and other neuroendocrine cells.

In neuroendocrine cells, such as adrenal chromaffin cells, the exocytosis of hormone-filled vesicles is triggered by a localized Ca(2+) increase that develops after the activation of voltage-dependent Ca(2+) channels. To reach the fusion competent state, vesicles have to go through a series of maturation steps that involve the detachment from cytoskeletal proteins, docking and priming. However, the fusion readiness of vesicles will also depend on their proximity to the calcium source.

P/Q Ca2+ channels are functionally coupled to exocytosis of the immediately releasable pool in mouse chromaffin cells.

Chromaffin cell exocytosis is triggered by Ca(2+) entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca(2+) channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements.