ABSTRACT
Autonomous animal locomotion, such as swimming, is modulated by neuronal networks acting on cilia or muscles. Understanding how these networks are formed and coordinated is a complex scientific problem, which requires various technical approaches. Among others, behavioral studies of developing animals treated with exogenous substances have proven to be a successful approach for studying the functions of neuronal networks. One such substance crucial for the proper development of the nervous system is the vitamin A-derived morphogen retinoic acid (RA). In the larva of the marine annelid Platynereis dumerilii , for example, RA is involved in the specification and differentiation of individual neurons and responsible for orchestrating the swimming behavior of the developing larva. Here, we report a workflow to analyze the effects of RA on the locomotion of the P. dumerilii larva. We provide a protocol for both the treatment with RA and the recording of larval swimming behavior. Additionally, we present a pipeline for the analysis of the obtained data in terms of swimming speed and movement trajectory. This chapter thus summarizes the methodology for analyzing the effects of a specific drug treatment on larval swimming behavior. We expect this approach to be readily adaptable to a wide variety of pharmacological compounds and aquatic species.
Subject(s)
Annelida/physiology , Neurons/cytology , Tretinoin/pharmacology , Animals , Aquatic Organisms/physiology , Behavior, Animal/drug effects , Body Patterning , Cell Differentiation/drug effects , Larva/physiology , Neurons/drug effects , Swimming/physiology , WorkflowABSTRACT
To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.
Subject(s)
Cell Nucleus/metabolism , Histones/metabolism , Interphase/physiology , Acetylation , Algorithms , Centromere/ultrastructure , Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 14/ultrastructure , Chromosomes, Human, Pair 22/genetics , Chromosomes, Human, Pair 22/ultrastructure , Chromosomes, Human, X/genetics , Chromosomes, Human, X/ultrastructure , Fibroblasts/metabolism , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , In Situ Hybridization, Fluorescence , MethylationABSTRACT
Composing microscopic images of very high resolution from several parts posed some problems. One of them was the necessity to adjust the focusing level when moving from one part to another. Re-focusing lead to problems with joining the image parts, which did not correspond exactly, and the area of image fusion was noticeable. A computer program was developed to overcome these problems. Our program worked with all the image parts together to find their optimal order for image fusion. Individual image parts were joined using a steep gradient running along a randomly generated curve. This method gave good results even in images with background or holes in the tissue. The method of composing large images from individual parts was used for digitizing the skin lymphoma collection of the Institute of Dermatology, University Hospital, Zürich. This collection of digital images is a part of the 6th version of Hypertext atlas of Dermatopathology at www.muni.cz/atlases.
