Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).     

Conditional restoration and inactivation of Rbm47 reveal its tissue‐context requirement for viability and growth

Rbm47 encodes a RNA binding protein that is necessary for Cytidine to Uridine RNA editing. Rbm47^gt/gt mutant mice that harbor inactivated Rbm47 display poor viability. Here it was determined that the loss of Rbm47^gt/gt offspring is due to embryonic lethality at mid‐gestation. It was further showed that growth of the surviving Rbm47^gt/gt mutants is impaired. Rbm47 is expressed in both the visceral endoderm and the definitive endoderm. Using the utility of the switchable FlEx gene‐trap cassette and the activity of Cre and FLP recombinases to generate mice that conditionally inactivate and restore Rbm47 function in tissue‐specific manner, it was demonstrated that Rbm47 function is required in the embryo proper, and not the visceral endoderm, for viability and growth. genesis 54:115–122, 2016. © 2016 Wiley Periodicals, Inc.     

Computational analysis of a flapping uvula on aerodynamics and pharyngeal wall collapsibility in sleep apnea

Studying the airflows and the resultant aerodynamic pressure/force in the pharyngeal airway is critical for understanding the pathophysiology of snoring and sleep apnea. In this work, an experiment-driven computational study was conducted to examine the aerodynamics in human pharyngeal airway. An anatomically accurate pharynx model associated with different uvula kinematics was reconstructed from human magnetic resonance image (MRI) and high-speed photography. An immersed-boundary-method (IBM)-based direct numerical simulation (DNS) flow solver was adopted to simulate the corresponding unsteady flows in all their complexity. Analyses were performed on vortex dynamics and pressure fluctuations in the pharyngeal airway and force oscillations on the pharyngeal wall under the influence of varying airway obstructions, uvula flapping mode, and uvula flapping frequencies. It was found the vortex formation, aerodynamic pressure, and pharyngeal wall force were significantly affected by the width of the pharyngeal airway. By contrast, the influences from the uvula flapping mode were insignificant when other parameters were similar. Fast Fourier transformation (FFT) and continuous wavelet transform (CWT) analysis of the pressure time history revealed the existence of higher order harmonics of base frequency with significant pressure amplitudes and energy intensities. It was also found the airway pressure and pharyngeal wall force oscillate more dramatically at higher uvula flapping frequencies, which tends to promote the collapse of pharyngeal wall and initiates sleep apnea.     

Ultrastructure and histochemistry of the foregut in<Emphasis Type="Italic"> Wirenia argentea</Emphasis> and<Emphasis Type="Italic"> Genitoconia rosea</Emphasis> (Mollusca,Solenogastres)

Wirenia argentea and Genitoconia rosea feed on Cnidaria like most representatives of the molluscan taxon Solenogastres (Aplacophora, Neomeniomorpha sensu Scheltema). The structure and histochemistry of the foregut are described based on histologic, semithin, and ultrathin section series. The ultrastructure was analyzed by means of transmission electron microscopy. There are two sets of unicellular glands: a narrow row of preoral gland cells opening to the preoral area, and pharyngeal gland cells in high numbers. Preoral gland cells produce serous secretions in W. argentea, but mucosubstances in G. rosea, whereas pharyngeal gland cells are similar in structure and histochemistry in both species. Based on the size and electron density of gland vesicles, five distinct types of pharyngeal gland cells can be defined. In contrast to earlier assumptions, all types of pharyngeal gland cells produce serous secretions, most probably representing digestive ferments, but no mucosubstances.     

Relationship Between Male Size and Newborn Size in the Seaweed Pipefish, Syngnathus Schlegeli

Relationships between male size, number, and weight of newborn were determined in the seaweed pipefish, Syngnathus schlegeli, with paternal care of embryos in the brood pouch. While the number of newborn increased linearly with the increase in male size, the pouch volume increased exponentially with the male size. This resulted in embryo density in the pouch negatively correlating with the male size. Newborn dry weight was negatively correlated with the embryo density in the pouch. Thus, larger males were considered to brood embryos at lower densities and give birth to heavier newborn. Neither paternal size nor embryo density seemed to have an effect upon developmental stage and total length of the newborn. Substantial weight loss of embryos during incubation indicated that paternal nutritional contribution to embryos is insignificant; therefore, the tendency of larger males to produce heavier newborn may not relate to allocation of paternal nourishment. Because egg weight increased with the female size, larger males are considered to receive the eggs of larger females and give birth to heavier newborn that developed from heavier eggs. Lower embryo density in larger males is attributable to the larger size of the heavier eggs. Assuming the pouch to be a cylindrical tube and the egg to be a sphere, geometrical calculations revealed that the relationship between the volumetric fraction of the pouch used by eggs (i.e. functional volume) and the male size was negatively quadric and had a peak. Increase in egg diameter increased the male size at which the functional volume of the pouch is maximized. Thus it appears that the size relationship of mates in pipefish is related to effective use of the pouch space.     

Analysis of spatial and temporal gene expression patterns in blastula and gastrula stage chick embryos

Studies on the genetic basis of rostral-caudal specification, neural induction, and head development require knowledge of the relevant gene expression patterns. Gaps in our understanding of gene expression have led us to examine the detailed spatiotemporal expression patterns of 19 genes implicated in early development, to learn more about their potential role in specifying and patterning early developmental processes leading to head formation. Here, we report the expression patterns of these markers in blastula- and gastrula-stage chick embryos, using whole-mount in situ hybridisation. Nodal, Fgf8, Bmp7, Chordin, Lim1, Hnf3beta, Otx2, Goosecoid, Cerberus, Hex, Dickkopf1, and Crescent are all already expressed by the time the egg is laid. When the primitive streak has reached its full length, a later group of genes, including Ganf, Six3, Bmp2, Bmp4, Noggin, Follistatin, and Qin (BF1), begins to be expressed. We reassess current models of early rostral patterning based on the analysis of these dynamic spatiotemporal expression patterns.     

Cytokeratins 8 and 19 in the mouse placental development

To investigate the expression and biological roles of cytokeratin 19 (K19) in development and in adult tissues, we inactivated the mouse K19 gene (Krt1-19) by inserting a bacterial beta-galactosidase gene (lacZ) by homologous recombination in embryonic stem cells, and established germ line mutant mice. Both heterozygous and homozygous mutant mice were viable, fertile, and appeared normal. By 7.5-8.0 days post coitum (dpc), heterozygous mutant embryos expressed lacZ in the notochordal plate and hindgut diverticulum, reflecting the fact that the notochord and the gut endoderm are derived from the axial mesoderm-originated cells. In the adult mutant, lacZ was expressed mainly in epithelial tissues. To investigate the possible functional cooperation and synergy between K19 and K8, we then constructed compound homozygous mutants, whose embryos died approximately 10 dpc. The lethality resulted from defects in the placenta where both K19 and K8 are normally expressed. As early as 9. 5 dpc, the compound mutant placenta had an excessive number of giant trophoblasts, but lacked proper labyrinthine trophoblast or spongiotrophoblast development, which apparently caused flooding of the maternal blood into the embryonic placenta. These results indicate that K19 and K8 cooperate in ensuring the normal development of placental tissues.