BMP4 regulates asymmetric Pkd2 distribution in mouse nodal immotile cilia and ciliary mechanosensing required for left-right determination.

BACKGROUND: Mouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear. RESULTS: The effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia. CONCLUSIONS: Experimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.

Biophysical Analysis of Mechanical Signals in Immotile Cilia of Mouse Embryonic Nodes Using Advanced Microscopic Techniques.

Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.

Initial morphological symmetry breaking in the foregut and development of the omental bursa in human embryos.

Bilaterally symmetrical primordia of visceral organs undergo asymmetrical morphogenesis leading to typical arrangement of visceral organs in the adult. Asymmetrical morphogenesis within the upper abdomen leads, among others, to the formation of the omental bursa dorsally to the rotated stomach. A widespread view of this process assumes kinking of thin mesenteries as a main mechanism. This view is based on a theory proposed already by Johannes Müller in 1830 and was repeatedly criticized, but some of the most plausible alternative views (initially proposed by Swaen in 1897 and Broman in 1904) still remain to be proven. Here, we analyzed serial histological sections of human embryos between stages 12 and 15 at high light microscopical resolution to reveal the succession of events giving rise to the development of the omental bursa and its relation to the emerging stomach asymmetry. Our analysis indicates that morphological symmetry breaking in the upper abdomen occurs within a wide mesenchymal plate called here mesenteric septum and is based on differential behavior of the coelomic epithelium which causes asymmetric paragastric recess formation and, importantly, precedes initial rotation of stomach. Our results thus provide the first histological evidence of breaking the symmetry of the early foregut anlage in the human embryo and pave the way for experimental studies of left-right symmetry breaking in the upper abdomen in experimental model organisms.

Matrix-filled microcavities in the emerging avian left-right organizer.

BACKGROUND: Hensen node of the amniote embryo plays a central role in multiple developmental processes, especially in induction and formation of axial organs. In the chick, it is asymmetrical in shape and has recently been considered to represent the left-right organizer. As mechanisms of breaking the initial left-right symmetry of the embryo are still ill-understood, analyzing the node's microarchitecture may provide insights into functional links between symmetry breaking and asymmetric morphology. RESULTS: In the course of a light- and electron-microscopic study addressing this issue we discovered novel intercellular matrix-filled cavities in the node of the chick during gastrulation and during early neurulation stages; measuring up to 45 µm, they are surrounded by densely packed cells and filled with nanoscale fibrils, which immunostaining suggests to consist of the basement membrane-related proteins fibronectin and perlecan. The cavities emerge immediately prior to node formation in the epiblast layer adjacent to the tip of the primitive streak and later, with emerging node asymmetry, they are predominantly located in the right part of the node. Almost identical morphological features of microcavities were found in the duck node. CONCLUSIONS: We address these cavities as "nodal microcavities" and propose their content to be involved in the function of the avian node by mediating morphogen signaling and storage.

Wall stress enhanced exocytosis of extracellular vesicles as a possible mechanism of left-right symmetry-breaking in vertebrate development.

In certain vertebrate species, the developing embryo breaks left-right symmetry in a transient organising structure: the "Left-Right Organiser" (LRO) known as the "node" in mice, and "Kupffer's vesicle" in fish. Directional cilia-driven flow is integral to this symmetry-breaking process, however the mechanism by which this flow is translated into an asymmetric signal remains contested; the principal theories are either flow transport of vesicles containing morphogens, or flow mechanosensing by cilia. Whilst some recent work favours the morphogen theory, other findings seem to support mechanosensing. In this study, we consider a hypothesis whereby the cilia themselves drive the release of morphogen-carrying extracellular vesicles (EVs) into the LRO; namely, that fluid stresses on the cell membrane induce/enhance exocytosis of EVs. Using a mathematical model, we calculate significant wall normal and shear stresses for a range of typical cilium parameter values comparable to levels capable of enhancing exocytosis. This mechanism may be able to reconcile the apparently conflicting experimental evidence.

An Early Chick Embryo Culture Device for Extended Continuous Observation.

Appropriate mechanical tension of the vitelline membrane as the culture substrate for the early chick embryo is frequently reported to be required for successful in vitro development. Here we describe a modified device, made of anodized aluminum, for in vitro culture which we used for studies of left-right symmetry breaking with emphasis on morphology and gene expression as readouts. The technique allows for easy, high-throughput tissue handling and provides a suitable tension in a stable and easily reproducible manner proven to be suitable for correct molecular left-right patterning and heart looping after long-term culture.