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. Laser ablations of actomyosin networks reveal that constricting cells have higher cortical tension, generate tension anisotropies and morphological changes in adjacent regions of neighboring cells, and require their neighbors to coordinate their own changes in shape. Thus, tensile forces determine the first spatial segregation of cells during mammalian development. We propose that, unlike more cohesive tissues, the early embryo dissipates tensile forces required by constricting cells via their neighbors, thereby allowing confined cell repositioning without jeopardizing global architecture.Fil: Samarage, Chaminda R.. Monash University; AustraliaFil: White, Melanie D.. Monash University; AustraliaFil: Alvarez, Yanina Daniela. Monash University; Australia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fierro González, Juan Carlos. Monash University; AustraliaFil: Henon, Yann. Monash University; AustraliaFil: Jesudason, Edwin C.. National Health Service Scotland; Reino UnidoFil: Bissiere, Stephanie. Monash University; Australia. Institute of Molecular and Cell Biology; SingapurFil: Fouras, Andreas. Monash University; AustraliaFil: Plachta, Nicolas. Monash University; Australia. Institute of Molecular and Cell Biology; Singapu

How cells change shape and position in the early mammalian embryo

During preimplantation development, cells of the mammalian embryo must resolve their shape and position to ensure the future viability of the fetus. These initial changes are established as the embryo expands from one to thirty-two cells, and a group of originally spherical cells is transformed into a more polarized structure with distinct cell geometries and lineages. Recent advances in the application of non-invasive imaging technologies have enabled the discovery of mechanisms regulating patterning of the early mammalian embryo. Here, we review recent findings revealing cell protrusions that trigger early changes in cell shape and embryo compaction, and how anisotropies in mechanical forces drive the first spatial segregation of cells in the embryo to form the pluripotent inner mass

The rostral anterior cingulate cortex modulates depression but not anxiety-related behaviour in the rat.

A growing body of functional imaging studies suggests that human depression and anxiety symptoms are associated with functional abnormalities in the circuitry formed by the rostral anterior cingulate cortex (rACC) and its direct limbic and paralimbic connections. In rodents however, the role of the rACC (rCG1/rCG2) remains unknown in depression-related behaviours and elusive in acute anxiety. In order to address this, we specifically lesioned the rat rCG1/rCG2, and assessed the behavioural outcome using a modified forced swim test (FST) and the elevated plus maze (EPM), tests for depression and anxiety related behaviours respectively. Lesions of the rostral anterior cingulate cortex significantly increased the time spent immobile in the FST without affecting climbing or swimming performances, suggesting a pro-depressant effect. On the contrary, none of the parameters measured in the EPM was affected by the lesion. These data point to an involvement of the rCG1/rCG2 in depression-related coping behaviours

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. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation. Expanding actin rings zipper the mouse embryo to seal it and allow formation of the blastocyst cavity.Fil: Zenker, Jennifer. Institute Of Molecular And Cell Biology; SingapurFil: White, Melanie D.. Institute Of Molecular And Cell Biology; SingapurFil: Gasnier, Maxime. Institute Of Molecular And Cell Biology; SingapurFil: Alvarez, Yanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Lim, Hui Yi Grace. Institute Of Molecular And Cell Biology; SingapurFil: Bissiere, Stephanie. Institute Of Molecular And Cell Biology; SingapurFil: Biro, Maté. Institute Of Molecular And Cell Biology; SingapurFil: Plachta, Nicolas. Institute Of Molecular And Cell Biology; Singapu

Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos

10.1186/s12915-016-0331-9BMC Biology14

Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos

Probing dynamic processes occurring within the cell nucleus at the quantitative level has long been a challenge in mammalian biology. Advances in bio-imaging techniques over the past decade have enabled us to directly visualize nuclear processes in situ with unprecedented spatial and temporal resolution and single-molecule sensitivity. Here, using transcription as our primary focus, we survey recent imaging studies that specifically emphasize the quantitative understanding of nuclear dynamics in both time and space. These analyses not only inform on previously hidden physical parameters and mechanistic details, but also reveal a hierarchical organizational landscape for coordinating a wide range of transcriptional processes shared by mammalian systems of varying complexity, from single cells to whole embryos

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. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.Fil: Zhao, Ziqing Winston. Institute of Molecular and Cell Biology; SingapurFil: White, Melanie D.. Institute of Molecular and Cell Biology; SingapurFil: Alvarez, Yanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Institute of Molecular and Cell Biology; SingapurFil: Zenker, Jennifer. Institute of Molecular and Cell Biology; SingapurFil: Bissiere, Stephanie. Institute of Molecular and Cell Biology; SingapurFil: Plachta, Nicolas. Institute of Molecular and Cell Biology; Singapu

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

10.1038/nprot.2017.051NATURE PROTOCOLS1271458-147

The rostral anterior cingulate cortex modulates the efficiency of amygdala-dependent fear learning.

BACKGROUND: The rostral anterior cingulate cortex (rACC) and the amygdala consistently emerge from neuroimaging studies as brain regions crucially involved in normal and abnormal fear processing. To date, however, the role of the rACC specifically during the acquisition of auditory fear conditioning still remains unknown. The aim of this study is to investigate a possible top-down control of a specific rACC sub-region over amygdala activation during pavlovian fear acquisition. METHODS: We performed excitotoxic lesions, temporal inactivation, and activation of a specific sub-region of the rACC that we identified by tracing studies as supporting most of the connectivity with the basolateral amygdala (r(Amy)-ACC). The effects of these manipulations over amygdala function were investigated with a classical tone-shock associative fear conditioning paradigm in the rat. RESULTS: Excitotoxic lesions and transient inactivation of the r(Amy)-ACC pre-training selectively produced deficits in the acquisition of the tone-shock associative learning (but not context). This effect was specific for the acquisition phase. However, the deficit was found to be transient and could be overcome by overtraining. Conversely, pre-training transient activation of the r(Amy)-ACC facilitated associative learning and increased fear expression. CONCLUSIONS: Our results suggest that a subregion of the rACC is key to gating the efficiency of amygdala-dependent auditory fear conditioning learning. Because r(Amy)-ACC inputs were confirmed to be glutamatergic, we propose that recruitment of this brain area might modulate overall basolateral amygdala excitatory tone during conditioned stimulus-unconditioned stimulus concomitant processing. In the light of clinical research, our results provide new insight on the effect of inappropriate rACC recruitment during emotional events