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Human pluripotent stem cells (hPSCs) are an important tool in the field of regenerative medicine due to their ability to differentiate towards all tissues of the adult organism. An important task in the study of hPSCs is to understand the factors that influence the maintenance of pluripotent and clonal characteristics of colonies represented by their morphological phenotype. Such factors include the ability of colonies to migrate during growth. In this work, we measured and analyzed the migration trajectories of hPSC colonies obtained from bright-field images of three cell lines, including induced hPSC lines AD3 and HPCASRi002-A (CaSR) and human embryonic stem cell line H9. To represent the pluripotent status, the colonies were visually phenotyped into two classes having a "good" or "bad" morphological phenotype. As for the migration characteristics, we calculated the colony speed and distance traveled (mobility measures), meandering index (motion persistence measures), outreach ratio (trajectory tortuosity characteristic), as well as the velocity autocorrelation function. The analysis revealed that the discrimination of phenotypes by the migration characteristics depended on both the cell line and growth environment. In particular, when the mTESR1/Matrigel culture environment was used, "good" AD3 colonies demonstrated a higher average migration speed than the "bad" ones. The reverse relationship between average speeds of "good" and "bad" colonies was found for the H9 line. The CaSR cell line did not show significant differences in the migration speed between the "good" and "bad" phenotypes. We investigated the type of motion exhibited by the colonies by applying two diffusion models to the mean squared displacement dynamics, one model corresponding to normal and the other to anomalous diffusion. The type of diffusion and diffusion parameter values resulting from the model fitting to data demonstrated a similar cell line, environment, and phenotype dependency. Colonies mainly showed a superdiffusive behavior for the mTESR1/Matrigel culture conditions, characterized by longer migration steps compared to the normal random walk. The specific properties of migration features and the patterns of their variation demonstrated in our work can be useful for the development and/or improvement of automated solutions for quality control of hPSCs. [ABSTRACT FROM AUTHOR]

Genetic studies have revealed that segment determination in Drosorphila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. We present a detailed analysis of regulatory interactions involved in gap gene regulation based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. Our models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. Our models predict activation of Kr by Cad and clarify several other regulatory interactions. Our analysis suggests a central role for repressive feedback loops between complementary gap genes. We observe that repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, our models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system.

Author(s): Johannes Jaeger [1]; Svetlana Surkova [2]; Maxim Blagov [2]; Hilde Janssens [1]; David Kosman [3]; Konstantin N. Kozlov [2]; Manu [1]; Ekaterina Myasnikova [2]; Carlos E. Vanario-Alonso [1, 4]; [...]

Quantitative measurements derived using sophisticated microscopy techniques are essential for understanding the basic principles that control the behavior of biological systems. We have developed a five-step data pipeline to extract quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. This protocol describes the preparation of Drosophila embryos for imaging by confocal microscopy. Embryos are collected at the appropriate developmental stage and fixed. They are then stained with both primary antibodies and secondary antibodies conjugated with fluorophores to reveal the segmentation gene expression patterns.