January 21, 2003;
Experimental conversion of liver to pancreas.
BACKGROUND: The liver
and the pancreas
arise from adjacent regions of endoderm
in embryonic development. Pdx1
is a key transcription factor that is essential for the development of the pancreas
and is not expressed in the liver
. The aim of this study was to determine whether a gene overexpression protocol based on Pdx1
would be able to cause conversion of liver
RESULTS: We show that a modified form of Pdx1
, carrying the VP16 transcriptional activation domain, can cause conversion of liver
, both in vivo and in vitro. Transgenic Xenopus tadpoles carrying the construct TTR
-GFP were prepared. Xlhbox8
is the Xenopus homolog of Pdx1
, the TTR
(transthyretin) promoter directs expression to the liver
, and the GFP is under the control of an elastase
promoter and provides a real-time visible marker of pancreatic differentiation. In the transgenic tadpoles, part or all of the liver
is converted to pancreas
, containing both exocrine and endocrine cells, while liver
differentiation products are lost from the regions converted to pancreas
. The timing of events is such that the liver
is differentiating by the time Xlhbox8
-VP16 is expressed, so we consider this a transdifferentiation event rather than a reprogramming of embryonic development. Furthermore, this same construct will bring about transdifferentiation of human hepatocytes in culture, with formation of both exocrine and endocrine cells.
CONCLUSIONS: We consider that the conversion of liver
could be the basis of a new type of therapy for insulin
-dependent diabetes. Although expression of the transgene is transient, once the ectopic pancreas
is established, it persists thereafter.
[+] show captions
Figure 2. Endocrine and Exocrine Cells Are Both Present in Ectopic Pancreas(A–F) Expression of endocrine genes. (A–C) Insulin RNA expression; (D–F) glucagon RNA expression. (A) Wild-type insulin expression. Expression is only seen in the dorsal pancreas (black arrow) at this stage in several prominent clusters. The liver (L) is located on the opposite side of the duodenum (red arrowhead). The two dark spots on the stomach are melanocytes and not insulin-stained cells. (B) TTR-Xlhbox8-VP16 transgenic whole gut. Several ectopic clusters of insulin expression are seen in the ectopic pancreatic tissue (EP) opposite the pancreas (outlined in white). (C) Section of (B) showing the ectopic pancreas (EP, dotted line) opposite the endogenous pancreas (solid line) with two clusters of insulin-expressing cells. (D) Wild-type glucagon expression. Expression is seen in several spots within the pancreas. (E) TTR-Xlhbox8-VP16 transgenic gut. Ectopic glucagon expression is found in the position of the liver (EP, outlined in white) opposite the normal pancreas. Note that glucagon cells are normally found in stomach and intestine, as well as pancreas, but not in the liver [37–39]. (F) Section of (E) showing glucagon expression within the ectopic pancreas (dotted line).(G–I) Ectopic expression of exocrine markers in the liver of transgenic tadpoles. (G) Live image of a TTR-Xlhbox8-VP16:Elas-GFP transgenic tadpole, showing ectopic elastase-GFP fluorescence within the liver (red arrowhead), anterior to the normal pancreas (P). (H) Amylase RNA expression. Ectopic expression of amylase is seen in the position of the liver (EP) as well as in the normal pancreas (P), in an identical pattern to the Elas-GFP. (I) Section of (H) showing expression of amylase in a portion of the liver (L), as highlighted by the dotted line. Normal amylase RNA expression is seen throughout the pancreas (solid black line). L, liver; P, normal pancreas; EP, ectopic pancreas.
Figure 4. Liver Gene Expression in Transgenic Gut Preparations(A–C) Transthyretin (TTR) RNA expression. (A) In wild-type preparations, expression is only seen within the liver. (B and C) TTR-Xlhbox8-VP16 transgenics. (B) In this case, there is complete conversion of liver to pancreas, and transthyretin expression has been completely suppressed. (C) In this case, half the liver has changed into pancreas, and transthyretin expression is seen only in the other half. In all examples, nontransgenic guts were stained simultaneously with transgenic guts, and the staining reaction was stopped in both when the nontransgenic expression was clearly evident.(D) Liver differentiation begins normally in TTR-Xlhbox8-VP16 tadpoles. Xhex (blue) and GFP (magenta, driven by the elastase promoter) expression. GFP RNA is seen only in the dorsal and ventral pancreas at stage 40; no GFP expression is detected in the liver. Xhex expression is normal at this stage and is present throughout the whole liver.(E–H) Timing of liver differentiation in normal nontransgenic tadpoles: from 3 days, the liver has become a separate organ, with Xhex and AMBP expressed throughout. (E) Xhex RNA at stage 40 (3 days). (F) Xhex RNA at stage 45 (5 days). (G) AMBP RNA at stage 40 (3 days). (H) AMBP expression at stage 45 (5 days).(I and J) The TTR promoter becomes active in Xenopus tadpoles only after the liver has differentiated. This shows RNA in situ hybridization for GFP driven by the TTR promoter. (I) At stage 40 (3 days), no GFP expression is detected. (J) GFP expression is first detected at stage 44 (4 days).