Spleen versus pancreas: strict control of organ interrelationship revealed by analyses of Bapx1-/- mice

Abstract

During early stages of pancreatic development, the mesenchyme that contributes to the spleen overlies the dorsal pancreatic endoderm. Here, we show that interactions between splenic mesenchyme and pancreas proceed via a highly orchestrated morphogenetic program. Disruption of morphogenesis, as occurs in the Bapx1(Nkx3.2)(-/-) embryo, results in transformation of these tissues into well-organized, ectopic gut-like structures. Bapx1 plays a crucial organizing role effecting position and separation of the spleen and pancreas to prevent this metaplastic transformation. Similar transformations occur in organ cultures employing wild-type pancreatic endoderm and spleen mesenchyme, revealing the developmental plasticity of the pancreas and that precise spatial and temporal control of tissue interactions are required for development of both organs.

Figure 1.

The splenic mesenchyme does not separate from the pancreas in Bapx1-null embryos. (A) Schematic representation of early splenopancreatic development. The mesenchyme that accumulates around the dorsal pancreas promotes growth and differentiation of the pancreatic epithelium and also contributes to development of the spleen. The splanchnic mesodermal plate directs laterality to the splenopancreatic region, and the spleen forms as a gradual condensation/separation of the distal mesenchyme. (B) Bapx1 in situ hybridization (ISH) on a transverse section at the pancreatic anterior–posterior level from an e9.5 wild-type embryo. (C) Whole-amount ISH (wild type) showing Bapx1 expression restricted to the splenic primordium and pyloric sphincter associated mesenchyme at e12.5. (D – G) OPT-generated iso-surface reconstructions depicting splenopancreatic development in wild-type (D–F) and Bapx1^−/− embryos (G) at e11.25 (D), e11.5 (E), and e13.5 (F,G). Broken red line corresponds to blow up views. In C through G, the splenic mesenchyme has been digitally colored in bright red. duo indicates duodenum; pam, pancreas-associated mesenchyme; st, stomach; dpe, dorsal pancreatic epithelium; dpm, dorsal pancreatic mesenchyme; splm, splenic mesenchyme; sm, stomach mesenchyme; se, stomach epithelium; li, liver; bd, bile duct; and vpe, ventral pancreatic epithelium.

Figure 2.

Cystic structures are formed from the pancreatic endoderm in Bapx1-null mice. In all figures the cyst lumen is marked by an asterisk. (A – C) The splenopancreatic region, including stomach, from e14.5 Bapx1^+/+ (A) and Bapx1^−/− embryos forming (B) and not forming (C) cystic structures. The approximate position of rudimentary splenic mesenchyme is marked by broken line (C). (D) Whole mount of pancreatic cysts labeled for HNF3β (red) and smooth muscle α-actin (ASMA, green). (E) OPT section of specimen seen in D showing continuation between a developing cyst and the pancreatic endoderm (*¹). The cyst marked *² has pinched off from the endoderm. Concomitant with cyst formation, smooth muscle is induced in the splenic mesenchyme (arrowhead). (F) Iso-surface reconstruction based on HNF3β signal of OPT sections as seen in E. Blue plane corresponds to E. (G) Cryosection through two cysts labeled for E-cadherin (red) and Ptf1A (green). Ptf1a is expressed in the pancreatic endoderm but not in the cysts. (H) Photomicrograph of the splenopancreatic region and stomach from e18.5 Bapx1^+/+ and Bapx1^−/− embryos. (I – K) Cryosections through the cyst of the center mutant seen in H labeled for DAPI (blue, I) and ASMA (green, I) and C-kit (red, J). K corresponds to white box in I and J and shows overlay of the ASMA and C-kit signal. C-kit-positive cells are intercalated in the muscle layer surrounding the cyst (arrowheads, K). dp indicates dorsal pancreas; vp, ventral pancreas; spl, spleen; st, stomach; and d, duodenum. Bar, 50 μm (G), 108 μm (I, J), 23 μm (K).

Figure 3.

Shh is induced in the cystic structures formed from the dorsal pancreas in Bapx1^−/− mice. ISH on sections of the splenopancreatic region from e14.5 Bapx1^+/+ (A – C) and Bapx1^−/− (E – G) embryos using antisense probes for Ihh (A,E), Shh (B,F), and Patched ([Ptc] C,G). Sections are obtained as illustrated in D and H. In E, the cyst epithelium is indicated by a broken line and the cyst lumen by *. In contrast to Bapx1^+/+ mice, Shh is expressed in the cystic epithelium (arrowhead in F) and Ptc in the surrounding splenic mesenchyme (arrowhead in G). splm indicates splenic mesenchyme; st, stomach; and dp, dorsal pancreas. Bar, 100 μm.

Figure 4.

In vitro recombination between embryonic pancreas and spleen results in transformation of pancreatic epithelium (see text for details). (A – E) Photomicrographs showing explants of wPE (A,C,E) and bPE (B) recombined with wPM (A, B), bSM (C), and wSM (E) isolated at e13.5 and cultured for 4 d. D is a Bapx1^−/−−derived cyst isolated at e14.5 and cultured for 4 d. (F ) Shh RT-PCR on the above tissues. (G) Activin βA RT-PCR on wild-type and Bapx1^−/− splenic mesenchyme. (H – V) Immunohistochemistry on sections from explant recombinations as seen in A (H–L), B (M–Q), and E (R–V). wPE indicates wild-type pancreatic epithelium; bPE, Bapx1^−/− pancreatic epithelium; wPM, wild-type pancreatic mesenchyme; bSM, Bapx1^−/− spleen mesenchyme; wSM, wild-type spleen mesenchyme; H/E hematoxylin/eosin; E-cad, E-cadherin; Ins, insulin; Glu, glucagon; and Amy, amylase. Bar, 140 μm (A), 250 μm (B,D), 240 μm (C), 210 μm (E), 90 μm (H–V), 30 μm (insets, H–T).

Figure 5.

Model for cyst formation in the Bapx1 mutant. (A–C) During normal development, the splenic mesenchyme condense and dislocate from the dorsal pancreas, thereby preventing agitating interactions between the two organs. (A′–D′) In Bapx1 mutants, the splenic mesenchyme stays in proximity of the dorsal pancreas. As a consequence, cystic structures are induced in the dorsal pancreatic epithelium (gray). Onset of Shh (blue) in the cyst epithelial component in turn results in gut specific differentiation in surrounding mesenchyme as marked by smooth muscle (green). PE indicates pancreatic epithelium; PM, pancreatic mesenchyme; and SM, splenic mesenchyme.