From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

A study of the male reproductive organs of nine species of penaeid prawns (Crustacea: Penaeinae)

This paper deals with the male reproductive organs of nine species of penaeid prawns; Penaeus penicillatus Alcock, P. merguiensis De Man, P. semisulcatus De Haan, Metapenaeus affinis (H.Milne Edwards), M. monoceros (Fabricius), M. stebbingi Nobili, Parapenaeopsis hardwickii (Miers), P. sculptilis (Heller) and P. stylifera (H. Milne Edwards). The male reproductive organs exhibited structural variations, which were more pronounced at generic level. These variations are mainly due to the type of spermatophore they possess. One species of each genus, that is, P. merguiensis, M. affinis and P. sculptilis were also studied histologically to examine the internal structure of the male reproductive organs. Spermatophores of the six species belonging to the genera Penaeus and Parapenaeopsis are also described and illustrated.

Bacterial load in pond water and different organs of a Indian major carp Cirrhinus mrigala Ham.

During the study period (August, 1993 to July,l994) the mean bacterial load in surface water was found to vary from 1.39 xl05 (July'94) to 3.llxl07CFU/ml (September'93), while that of botrom water ranged from l.Olxl06 (May'94) to 5.90xl07CFU/ml (October '93). The mean total number of bacterial load in body slime, liver and kidney was found to vary from 0.58xl03 (July'94) to 2.37xl07CFU/g (March'94),from 0.22xl03(July'94)to 9.64xl06 CFU/g (March'94) from O.l5xl03 (July'94) to 9.36xl06 CFU/g (March'94), respectively. Bacterial load in slime was significantly correlated with bacterial load in liver, bacterial load in slime was significantly correlated with bacterial load in kidney and bacterial load in liver was significantly correlated with bacterial load in kidney.

Heavy metal concentrations in some organs of African catfish (Clarias gariepinus) from Eko-Ende Dam, Ikirun, Nigeria

The contents of the heavy metals Fe, Zn, Cu, Mn, Pb, Cd and As were examined in the liver, heart, gills, kidney and muscles of the fish Clarias gariepinus from Eko-Ende dam in Ikirun, the capital of Ifelodun Local Government of Osun State, Nigeria. C. gariepinus is the fish of choice and the most demanded in the southwest of Nigeria. The highest metal concentrations were in the liver and the gills while the lowest was in the muscles. The general deceasing order of metal accumulation in the organs was Fe > Zn > Cu > Mn > Cd. Lead and arsenic were not detected in any organ. The values were of lower concentrations than found in many other dams and rivers in Nigeria and some other countries. The values were also lower than the FAO/WHO recommended maximum limits in fish samples, making the fish to be safe and not of any hazards for the consumers.