Generating human blastoids modeling blastocyst-stage embryos and implantation.

Human early development sets the stage for embryonic and adult life but remains difficult to investigate. A solution came from the ability of stem cells to organize into structures resembling preimplantation embryos-blastocysts-that we termed blastoids. This embryo model is available in unlimited numbers and could thus support scientific and medical advances. However, its predictive power depends on how faithfully it recapitulates the blastocyst. Here, we describe how we formed human blastoids that (1) efficiently achieve the morphology of the blastocyst and (2) form lineages according to the pace and sequence of blastocyst development, (3) ultimately forming cells that transcriptionally reflect the blastocyst (preimplantation stage). We employ three different commercially available 96- and 24-well microwell plates with results similar to our custom-made ones, and show that blastoids form in clinical in vitro fertilization medium and can be cryopreserved for shipping. Finally, we explain how blastoids replicate the directional process of implantation into endometrial organoids, specifically when these are hormonally stimulated. It takes 4 d for human blastoids to form and 10 d to prepare the endometrial implantation assay, and we have cultured blastoids up to 6 d (time-equivalent of day 13). On the basis of our experience, we anticipate that a person with ~1 year of human pluripotent stem cell culture experience and of organoid culture should be able to perform the protocol. Altogether, blastoids offer an opportunity to establish scientific and biomedical discovery programs for early pregnancy, and an ethical alternative to the use of embryos.

Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells.

Tension of the actomyosin cell cortex plays a key role in determining cell-cell contact growth and size. The level of cortical tension outside of the cell-cell contact, when pulling at the contact edge, scales with the total size to which a cell-cell contact can grow [J.-L. Maître et al., Science 338, 253-256 (2012)]. Here, we show in zebrafish primary germ-layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell-cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. After tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell-cell contact size is limited by tension-stabilizing E-cadherin-actin complexes at the contact.

Actin Rings of Power.

Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles.

A Serrate-Notch-Canoe complex mediates essential interactions between glia and neuroepithelial cells during Drosophila optic lobe development.

It is firmly established that interactions between neurons and glia are fundamental across species for the correct establishment of a functional brain. Here, we found that the glia of the Drosophila larval brain display an essential non-autonomous role during the development of the optic lobe. The optic lobe develops from neuroepithelial cells that proliferate by dividing symmetrically until they switch to asymmetric/differentiative divisions that generate neuroblasts. The proneural gene lethal of scute (l'sc) is transiently activated by the epidermal growth factor receptor (EGFR)-Ras signal transduction pathway at the leading edge of a proneural wave that sweeps from medial to lateral neuroepithelium, promoting this switch. This process is tightly regulated by the tissue-autonomous function within the neuroepithelium of multiple signaling pathways, including EGFR-Ras and Notch. This study shows that the Notch ligand Serrate (Ser) is expressed in the glia and it forms a complex in vivo with Notch and Canoe, which colocalize at the adherens junctions of neuroepithelial cells. This complex is crucial for interactions between glia and neuroepithelial cells during optic lobe development. Ser is tissue-autonomously required in the glia where it activates Notch to regulate its proliferation, and non-autonomously in the neuroepithelium where Ser induces Notch signaling to avoid the premature activation of the EGFR-Ras pathway and hence of L'sc. Interestingly, different Notch activity reporters showed very different expression patterns in the glia and in the neuroepithelium, suggesting the existence of tissue-specific factors that promote the expression of particular Notch target genes or/and a reporter response dependent on different thresholds of Notch signaling.

The actin-binding protein Canoe/AF-6 forms a complex with Robo and is required for Slit-Robo signaling during axon pathfinding at the CNS midline.

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.

Canoe functions at the CNS midline glia in a complex with Shotgun and Wrapper-Nrx-IV during neuron-glia interactions.

Vertebrates and insects alike use glial cells as intermediate targets to guide growing axons. Similar to vertebrate oligodendrocytes, Drosophila midline glia ensheath and separate axonal commissures. Neuron-glia interactions are crucial during these events, although the proteins involved remain largely unknown. Here, we show that Canoe (Cno), the Drosophila ortholog of AF-6, and the DE-cadherin Shotgun (Shg) are highly restricted to the interface between midline glia and commissural axons. cno mutant analysis, genetic interactions and co-immunoprecipitation assays unveil Cno function as a novel regulator of neuron-glia interactions, forming a complex with Shg, Wrapper and Neurexin IV, the homolog of vertebrate Caspr/paranodin. Our results also support additional functions of Cno, independent of adherens junctions, as a regulator of adhesion and signaling events in non-epithelial tissues.

Characterization and regulation of basal calcium influx in human peripheral blood lymphocytes.

The basal 45Ca2+ influx into resting human blood lymphocytes was measured. This process showed biphasic kinetics with first rapid phase followed by the second long-lasting and markedly slower phase. Further, it showed signs of saturability and reaches maximal values at 37 degrees C and extracellular pH 7.2. The basal 45Ca2+ influx was stimulated by addition of submicromolar concentrations of 4 beta-phorbol-12-myristate-13-acetate, and this effect was abolished by protein kinase C (PKC) inhibitor Ro-31-8220. In the regulation of basal 45Ca2+ influx is probably only partially involved adenylate cyclase pathway as show results with intracellular c-AMP elevating agents (dB-c-AMP, 3-isobutyl-1-metylxantine and forskolin). Uncoupler 3,3',4',5-tetrachloro-salicylanilide (TCS) in micromolar concentrations stimulated basal 45Ca2+ influx and its effect was more significant in media with high extracellular concentration of K+.