Origin and morphogenesis of the murine spleen

• Main Author: Burn, Sally
• Format: Dissertation
• Language: English
• Published: University of Edinburgh 2007

This thesis establishes methods to investigate early spleen development in the mouse embryo. An expression analysis of early Nkx2.5 expression is reported, along with the finding that Nkx2.5 may be the earliest marker of splenic precursors in the mouse. Expression of Nkx2.5 is also shown for the first time to overlap considerably with that of Nkx3.2 – a major gut development gene upstream of Nkx2.5. An in silico analysis of the evolutionary conserved regions upstream of Nkx2.5 is presented along with the establishment and analysis of stable transgenic reporter lines expressing LacZ under the control of an Nkx2.5 gut regulatory sequence (NGRS). NGRS confers spleen, posterior stomach, and pyloric sphincter expression, with very little of the cardiac expression associated with endogenous Nkx2.5. This enhancer is thus ideal for gut studies, providing a tool for directing gut-specific expression and genetic manipulations. NGRS was also found not to require Nkx3.2 for its activity. Finally, NGRS is demonstrated to have the potential to mark abnormal spleen development, in a previously unreported splenic mutant: the Rwhs mutant. Potential uses for NGRS are explored. An approach was taken to mark and follow spleen development, using NGRS-LacZ in an organ culture system. Data generated from these experiments shed some light on how the E11.5 spleen develops, providing evidence for migration of splenic precursors along the stomach, and suggesting that an inhibitory “anchor” effect is normally exerted by the posterior spleno-pancreatic mesenchyme, disruption of which permits precocious spleen development. Finally, an analysis of the role of Wnt signalling in development of the spleno-pancreatic region is presented. Wnt signalling is active in the developing spleen at E11.5, E12.5 and E14.5. A number of Wnt and Frz genes are expressed in the E14.5 spleen.