Impaired spleen formation perturbs morphogenesis of the gastric lobe of the pancreas

Abstract

Despite the extensive use of the mouse as a model for studies of pancreas development and disease, the development of the gastric pancreatic lobe has been largely overlooked. In this study we use optical projection tomography to provide a detailed three-dimensional and quantitative description of pancreatic growth dynamics in the mouse. Hereby, we describe the epithelial and mesenchymal events leading to the formation of the gastric lobe of the pancreas. We show that this structure forms by perpendicular growth from the dorsal pancreatic epithelium into a distinct lateral domain of the dorsal pancreatic mesenchyme. Our data support a role for spleen organogenesis in the establishment of this mesenchymal domain and in mice displaying perturbed spleen development, including Dh +/-, Bapx1-/- and Sox11-/-, gastric lobe development is disturbed. We further show that the expression profile of markers for multipotent progenitors is delayed in the gastric lobe as compared to the splenic and duodenal pancreatic lobes. Altogether, this study provides new information regarding the developmental dynamics underlying the formation of the gastric lobe of the pancreas and recognizes lobular heterogeneities regarding the time course of pancreatic cellular differentiation. Collectively, these data are likely to constitute important elements in future interpretations of the developing and/or diseased pancreas.

Figure 1. The gastric lobe constitutes a significant portion of the mouse pancreas and is formed by perpendicular growth from the stalk of the dorsal pancreas into a lateral mesenchymal domain positioned in close proximity to the pyloric sphincter and pyloric antrum.

(A) Photomicrograph of a gut segment including the stomach, duodenum, spleen and pancreas from a C57Bl/6 mouse at 8 weeks. (B) Graph depicting the 10log values of the epithelial volume of the DP (yellow diamond), VP (green square), GL (yellow triangle) and the entire pancreas (black cross) between e10.5 and 8 weeks. (C) Relative lobular volumes between e10.5 and 8 weeks. From e15.5 onwards, the gastric lobe constitutes more than 10% of the pancreatic epithelium. DP: red; VP: green; GL: yellow. Data in (B, C) is derived from analyses of C57Bl/6 mice. Values in (C) are given ± SEM. (D through Q) OPT generated iso-surface reconstructions of gut segments including the stomach, duodenum, spleen and pancreas between e10.5 to e17.5 based on the signal from E-cadherin antibodies (epithelium, D to J) and the signal from tissue autofluorescence (mesenchyme, K to Q). The condensation and migration of primordial spleen cells in an anterior direction results in the formation of a lateral mesenchymal domain which provides the template for growth from the stalk of the dorsal pancreatic epithelium. (D to J) The dorsal, ventral and gastric epithelium have been pseudocolored red, green and yellow respectively. (L to N) The dorsal mesenchyme and the spleen primordium have been pseudocolored blue and pink respectively. The specimens in (D–Q) are not depicted to scale. Scale bar in A is 3 mm.

Figure 2. Disturbed splenogenesis perturbs formation of the gastric lobe mesenchymal domain.

(A through O) OPT generated iso-surface reconstructions of the e14.5 pancreatic region from normal C57/Bl6 (A, F, K), Dh+/− (B, G, L), Bapx1−/− (E, H, M), Sox11−/− (D, I, N) and Pdx1−/− (E, J, O) mice. Reconstructions include the stomach, duodenum, spleen and pancreas and is based on the signal from; E-cadherin antibodies (epithelium – light grey, A to E and K to O) and the signal from tissue auto fluorescence (mesenchyme – dark grey, F to J and K to O). The incapacity to form a proper spleen primordium in Dh+/−, Bapx1−/− and Sox11−/− mice results in various degree of failure to separate the dorsal and gastric lobe mesenchyme. Thereby development of the entire dorsal epithelial region is perturbed (compare B to D with A). The gastric lobe is indicated by an arrow in (A). As indicated by mesenchymal morphogenesis in the Pdx1 mutant, formation of the GL mesenchymal domain is an autonomous event independent of pancreatic epithelial development (arrowheads, F and J). In (A to G) the dorsal, ventral and gastric lobes have been pseudocolored red, green and yellow respectively. The specimens are not depicted to scale.

Figure 3. Development of the dorsal pancreatic region is dependent on sequential interaction with neighboring tissues.

(A) The initial steps of pancreas development include the induction of the pancreatic anlage by signals originating from the notochord and dorsal aorta , and are followed by the migration of splanchnic mesenchyme to cover the dorsal epithelium . The ventral pancreas forms caudally to the presumptive liver anlage, distal from the cardiac mesoderm , , . The left SMP is formed under influence of the L/R genetic cascade. (B to D) During subsequent development the DP and VP buds grow branch and differentiate as they invade the surrounding mesenchyme. (B) On the dorsal side, the SMP mediates leftward growth of the spleno-pancreatic region. (C) Spleen precursor cells migrate and condense (white arrows) over the left side of the greater curvature of the stomach and this process mediates the subdivision of the pancreatic mesenchyme into a dorsal and a gastric domain. (D) The GL mesenchymal domain provides a template for epithelial growth from the stalk of the DP epithelium. (E) In absence of proper spleen formation, the GL does not form and the morphology of the entire dorsal epithelium is perturbed.