Dr. Sarder N. Uddin
1. Neural Circuit Development and Processing of Sensory Information.
Behaviors result from information processing by neural circuits. An in‐depth understanding of any given behavior will thus require knowledge of both the identities of the component neurons of the underlying neural circuit and the synaptic connectivity relationships among them. Mapping neural circuits has proven challenging due to their complexity and the lack of efficient circuit mapping methodologies. Current neural circuit mapping strategies include the use of electron microscopy and electrophysiology, but these methods limit the rate of progress in mapping neural circuits because both are labor intensive and electrophysiology can only be performed on relatively large, anatomically accessible neurons.
A novel technique, GFP Reconstitution Across Synaptic Partners (GRASP) (Feinberg et al, 2008), identifies synaptic contacts between neurons and does not have the limitations of electron microscopy or electrophysiology. GRASP is thus a complementary approach with tremendous potential for rapidly advancing the pace of neural circuit mapping. Unfortunately, the original GRASP method has the caveat that it detects both synaptic and non-‐synaptic contacts between neurons and other cell types and thus it has a low reliability for identifying functional synaptic connections. mGRASP, a second-‐generation version of GRASP that preferentially identifies functional synaptic contacts, was recently developed in mice (Kim J et al, 2012). Since mGRASP does not have the caveat of the original GRASP of detecting neuronal contacts that are not synaptic, it is thus a much more reliable tool for neural circuit mapping.
One of the major challenges is to find out a strategy for adapting mGRASP to Drosophila. Since mGRASP identifies sites of synaptic connectivity via direct GFP fluorescence or as mGRASP‐specific immunostaining (Yamagata et al, 2008), and thousands of functional synaptic contacts are potentially identifiable in a single experiment, it is much more efficient and higher throughput than existing neural circuit mapping approaches. Adapting mGRASP to Drosophilawould thus fill the gap for a badly needed efficient neural circuit mapping methodology in this important and widely used model system. Drosophilaadults and larva exhibit a large and varied repertoire of behaviors elicited by the same sensory stimuli detected by humans (light, sound, taste, smell, touch) that have been extensively characterized over the past century.
So, identifying the neuron that control the underline neural circuit as well as identifying the synaptic connectivity relationships among them is a prerequisite to understand how sensory information is processed. Available traditional approaches for neural circuit mapping include electrophysiology and electron microscopy. Both are labor intensive. I’d like to develop a GFP reconstitution methods that target GFP 11 construct to axon/pre-synaptic terminals and GFP 1-10 construct to dendrites/post-synaptic terminals. This technique will exhibit reconstruction of GFP fluorescence preferentially at synaptic contract sites and will be much higher throughput than traditional methods. Data will be presented demonstrating the functionality of the techniques and its use in identifying second order neurons of Drosophila larval accordion behavior elicited by mechanosensory stimulation. The methods should be generally applicable for determining neural connectivity arrangements in any area of Drosophilaneurobiology including development, physiology and disease model.
2. Molecular Mechanism for the Development of Digestive Tract.
The developmental genetics of Drosophila has been intensely studied from early 80s onward, and the principles and knowledge accumulated during the period have brought about revolution in the functional study of genes of multicellular animals. The Drosophilaembryonic digestive tract (gut) is fruitful territory for investigation of events common to many types of organogenesis and a robust system for the study of patterning (establishment) and morphogenesis of epithelial organs.
The gut of Drosophilais one of the most evolutionarily ancient and conserved organs among embryos of the multicellular animals across the protostomes and deuterostomes, and hindgut is most important part of its. Molecular and genetic mechanisms of cell differentiation of hindgut have recently been a target of intense study in the field of Drosophila developmental biology. One of the most important progresses is the finding of brachyenteron(byn), transcription factors which is thought to act as a master gene for hindgut development of Drosophila. orthopedia (otp)is non-HOM-C homeobox gene (homeobox containing gene), which acts as a transcription factor. In vertebrate embryos, otp is responsible for development of the specific territory of the CNS. In Drosophila, otpis expressed in the hindgut, but the otpexpression is not detected in the hindgut remnant in bynmutant embryo. A few essential steps in the pathway of digestive tract (gut) development are revealed including the differentiation of the small intestine in Drosophila(Uddin et al, 2011), noticeably, drmexpression pattern, drmmutation phenotypes, cell-non-autonomous activity of drmetc. drmactivity eventually leads to the activation of Bowl, and the latter is essential for determining tissue identity of the small intestine. Though, there is no direct evidence that demonstrates specific roles of Bowl in this process. All these circumstantial knowledges suggest that there may be some unknown factor that defines identity of the small intestine. However, mechanisms of the specification of small intestine as well as details mode of action of drmare still rather ambiguous and puzzling. Regarding small intestine development, the most important issue remains to be solved is: extracellular signaling factor generated under the control of drm, and, gene that determines tissue identity of the small intestine. Interestingly, the similar mechanism of drmmay implies in the development of anterior gut as drmexpression was observed in junctional region of foregut and anterior midgut which also deserve to investigate. Also,the details mechanism of transcription as well as the downstream (target) of both drmand otpis unknown A major aim of this proposed study program is to elucidate function of byn, drmand otpgene during the hindgut development of Drosophila.
The proposed research interest is relevant to the molecular and genetic mechanisms of the pattern formation and cell differentiation of multicellular animals. Principles and techniques that will be learnt through the proposed research working are essentially be applicable to various fields of life sciences including more practical and applied ones.