Mechanics of head fold formation: investigating tissue-level forces during early development.

During its earliest stages, the avian embryo is approximately planar. Through a complex series of folds, this flat geometry is transformed into the intricate three-dimensional structure of the developing organism. Formation of the head fold (HF) is the first step in this cascading sequence of out-of-plane tissue folds. The HF establishes the anterior extent of the embryo and initiates heart, foregut and brain development. Here, we use a combination of computational modeling and experiments to determine the physical forces that drive HF formation. Using chick embryos cultured ex ovo, we measured: (1) changes in tissue morphology in living embryos using optical coherence tomography (OCT); (2) morphogenetic strains (deformations) through the tracking of tissue labels; and (3) regional tissue stresses using changes in the geometry of circular wounds punched through the blastoderm. To determine the physical mechanisms that generate the HF, we created a three-dimensional computational model of the early embryo, consisting of pseudoelastic plates representing the blastoderm and vitelline membrane. Based on previous experimental findings, we simulated the following morphogenetic mechanisms: (1) convergent extension in the neural plate (NP); (2) cell wedging along the anterior NP border; and (3) autonomous in-plane deformations outside the NP. Our numerical predictions agree relatively well with the observed morphology, as well as with our measured stress and strain distributions. The model also predicts the abnormal tissue geometries produced when development is mechanically perturbed. Taken together, the results suggest that the proposed morphogenetic mechanisms provide the main tissue-level forces that drive HF formation.

Microbial Processes and Microbial Communities in the Water Column of the Polar Meromictic Lake Bol'shie Khruslomeny at the White Sea Coast.

Microbiological, molecular ecological, biogeochemical, and isotope geochemical research was carried out at the polar Lake Bol'shie Khruslomeny at the coast of the Kandalaksha Bay, White Sea in March and September 2017. The uppermost mixolimnion was oxic, with low salinity (3-5%). The lower chemocline layer was brown-green colored, with very high content of particulate organic matter (up to 11.8 mg C L-1). The lowermost monimolimnion had marine salinity (22-24%) and very high concentrations of sulfide (up to 18 mmol L-1) and CH4 (up to 1.8 mmol L-1). In the chemocline, total microbial abundance and the rate of anoxygenic photosynthesis were 8.8 × 106 cells mL-1 and 34.4 µmol C L-1 day-1, respectively. Both in March and September, sulfate reduction rate increased with depth, peaking (up to 0.6-1.1 µmol S L-1 day-1) in the lower chemocline. Methane oxidation rates in the chemocline were up to 85 and 180 nmol CH4 L-1 day-1 in March and September, respectively; stimulation of this process by light was observed in September. The percentages of cyanobacteria and methanotrophs in the layer where light-induced methane oxidation occurred were similar, ∼2.5% of the microbial community. Light did not stimulate methane oxidation in deeper layers. The carbon isotope composition of particulate organic matter (δ13C-Corg), dissolved carbonates (δ13C-DIC), and methane (δ13C- CH4) indicated high microbial activity in the chemocline. Analysis of the 16S rRNA gene sequences revealed predominance of Cyanobium cyanobacteria (order Synechococcales) in the mixolimnion. Green sulfur bacteria Chlorobium phaeovibrioides capable of anoxygenic photosynthesis constituted ∼20% of the chemocline community both in March and in September. Methyloprofundus gammaptoteobacteria (family Methylomonaceae) were present in the upper chemocline, where active methane oxidation occurred. During winter, cyanobacteria were less abundant in the chemocline, while methanotrophs occurred in higher horizons, including the under-ice layer. Chemolithotrophic gammaproteobacteria of the genus Thiomicrorhabdus, oxidizing reduced sulfur compounds at low oxygen concentrations, were revealed in the chemocline in March. Both in March and September archaea constituted up to 50% of all microorganisms in the hypolimnion. The percentage of putative methanogens in the archaeal community was low, and they occurred mainly in near-bottom horizons.

Electrophysiology of the rhythmic defecation program in nematode Heterorhabditis megidis.

The nervous system controls most rhythmic behaviors, with a remarkable exception. In Caenorhabditis elegans periodic defecation rhythm does not appear to involve the nervous system. Such oscillations are studied in detail with genetic and molecular biology tools. The small size of C. elegans cells impairs the use of standard electrophysiological methods. We studied a similar rhythmic pacemaker in the noticeably larger gut cells of Heterorhabditis megidis nematode. H. megidis defecation cycle is driven by a central pattern generator (CPG) associated with unusual all-or-none hyper-polarization "action potential". The CPG cycle period depends on the membrane potential and CPG cycling also persisted in experiments where the membrane potential of gut cells was continuously clamped at steady voltage levels. The usual excitable tissue description does not include the endoderm or imply the generation of hyper-polarization spikes. The nematode gut cells activity calls for a reevaluation of the excitable cells definition.

Lacrimal Gland Repair Using Progenitor Cells.

In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous-deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild-type mouse LGs by transplanting them into the LGs of TSP -1-/- mice, which represent a novel mouse model for ADDE. TSP-1-/- mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c-kit-positive epithelial cell adhesion molecule (EpCAM+ ) populations sorted from mouse LGs, the c-kit+ dim/EpCAM+ /Sca1 - /CD34 - /CD45 - cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three-dimensional cultures. Moreover, when transplanted into injured or "diseased" LGs, they engraft into acinar and ductal compartments. EPCP-injected TSP-1-/- LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88-98.

Tracking morphogenetic tissue deformations in the early chick embryo.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to study the physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

The role of mechanical forces in the torsional component of cardiac looping.

During early development, the initially straight heart tube (HT) bends and twists (loops) into a curved tube to lay out the basic plan of the mature heart. The physical mechanisms that drive and regulate looping are not yet completely understood. This paper reviews our recent studies of the mechanics of cardiac torsion during the first phase of looping (c-looping). Experiments and computational modeling show that torsion is primarily caused by forces exerted on the HT by the primitive atria and the splanchnopleure, a membrane that presses against the ventral surface of the heart. Experimental and numerical results are described and integrated to propose a hypothesis for cardiac torsion, and key aspects of our hypothesis are tested using experiments that perturb normal looping. For each perturbation, the models predict the correct qualitative response. These studies provide new insight into the mechanisms that drive and regulate cardiac looping.

On modeling morphogenesis of the looping heart following mechanical perturbations.

Looping is a crucial early phase during heart development, as the initially straight heart tube (HT) deforms into a curved tube to lay out the basic plan of the mature heart. This paper focuses on the first phase of looping, called c-looping, when the HT bends ventrally and twists dextrally (rightward) to create a c-shaped tube. Previous research has shown that bending is an intrinsic process, while dextral torsion is likely caused by external forces acting on the heart. However, the specific mechanisms that drive and regulate looping are not yet completely understood. Here, we present new experimental data and finite element models to help define these mechanisms for the torsional component of c-looping. First, with regions of growth and contraction specified according to experiments on chick embryos, a three-dimensional model exhibits morphogenetic deformation consistent with observations for normal looping. Next, the model is tested further using experiments in which looping is perturbed by removing structures that exert forces on the heart--a membrane (splanchnopleure (SPL)) that presses against the ventral surface of the heart and the left and right primitive atria. In all cases, the model predicts the correct qualitative behavior. Finally, a two-dimensional model of the HT cross section is used to study a feedback mechanism for stress-based regulation of looping. The model is tested using experiments in which the SPL is removed before, during, and after c-looping. In each simulation, the model predicts the correct response. Hence, these models provide new insight into the mechanical mechanisms that drive and regulate cardiac looping.

Cardiac looping in experimental conditions: effects of extraembryonic forces.

The chick embryo is a popular experimental model used to study the mechanisms of cardiac looping. To facilitate oxygen transport, researchers typically culture the embryo on the surface of the medium. Such preparations, however, expose the embryo and the heart to surface tension that is not present in ovo. This study investigates the influence that surface and extraembryonic membrane tensions have on looping morphology. To eliminate surface tension, we developed a technique in which the embryo is cultured under a thin layer of fluid. To eliminate membrane tension, the membrane was removed. Our results show that both tensions can affect looping, with surface tension potentially having a much greater effect. Moreover, we show that surface tension can alter results in one classic looping experiment.

The role of mechanical forces in dextral rotation during cardiac looping in the chick embryo.

Cardiac looping is a vital morphogenetic process that transforms the initially straight heart tube into a curved tube normally directed toward the right side of the embryo. While recent work has brought major advances in our understanding of the genetic and molecular pathways involved in looping, the biophysical mechanisms that drive this process have remained poorly understood. This paper examines the role of biomechanical forces in cardiac rotation during the initial stages of looping, when the heart bends and rotates into a c-shaped tube (c-looping). Embryonic chick hearts were subjected to mechanical and chemical perturbations, and tissue stress and strain were studied using dissection and fluorescent labeling, respectively. The results suggest that (1) the heart contains little or no intrinsic ability to rotate, as external forces exerted by the splanchnopleure (SPL) and the omphalomesenteric veins (OVs) drive rotation; (2) unbalanced forces in the omphalomesenteric veins play a role in left-right looping directionality; and (3) in addition to ventral bending and rightward rotation, the heart tube also bends slightly toward the right. The results of this study may help investigators searching for the link between gene expression and the mechanical processes that drive looping.