Conditional progenitor cell ablation

The epithelial lineages of the pancreas (exocrine, endocrine and ductal) arise from cells that express the transcription factor Pdx1 (pancreatic and duodenal homeobox 1) during development¹²; after birth, Pdx1 expression becomes largely confined to insulin-producing -cells. We devised two methods to perturb the number of Pdx1-expressing (Pdx1⁺) cells in development. First, the Pdx1^tTA strain, in which the tetracycline transactivator (tTA) coding sequence is integrated in the endogenous Pdx1 locus¹³, was crossed to tetO^DTA/DTA mice, in which expression of the diphtheria toxin A chain (DTA) is controlled by upstream tetracycline operator sequences¹⁴. This cross yielded Pdx1^+/+; tetO^DTA/+ (+/DTA) and Pdx1^tTA/+; tetO^DTA/+ (tTA/DTA) embryos, in which the elimination of Pdx1⁺ progenitors could be regulated by altering the dose/timing of tetracycline addition.

Pregnant mice were given tetracycline to repress the transcriptional activity of tTA¹⁵ or left untreated to activate tTA-mediated transcription of DTA (Fig. 1a), and offspring were examined at birth (E18.5). Although body weight was maintained regardless of genotype or tetracycline treatment regimen (data not shown), tTA/DTA newborns that had not received tetracycline were virtually apancreatic (Figs 1b and 2e; see also Supplementary Fig. 1). By contrast, pancreata from tTA/DTA embryos exposed to tetracycline throughout pregnancy exhibited a normal appearance and preserved weight (Figs 1b and 2a; see also Supplementary Fig. 1).

Figure 1: Pancreatic progenitor cell ablation.

a, Experimental design. Two classes of embryos are generated by crossing Pdx1^tTA/+ and tetO^DTA/DTA mice: Pdx1^+/+; tetO^DTA/+ (Control; +/DTA) and Pdx1^tTA/+;tetO^DTA/+ (Experimental; tTA/DTA). In untreated tTA/DTA embryos (-Tet), Pdx1⁺ progenitors are killed after tTA-mediated expression of DTA. Treatment with tetracycline (+Tet) represses tTA-mediated transcription, allowing survival of Pdx1⁺ progenitors. b, Pregnant females were given plain water or water containing tetracycline throughout gestation; embryos were examined at E18.5. The midgut of a control (+/DTA) embryo is shown (right panel) to illustrate normal structures. d, duodenum; dp, dorsal pancreas; Li, liver; s, stomach; spl, spleen; vp, ventral pancreas.

Full figure and legend (123K)Figures & Tables index

Figure 2: Lack of compensatory growth during pancreas development.

a–e, Pregnant mice carrying tTA/DTA embryos were given tetracycline either throughout embryogenesis (a) or at various time points during early pancreas organogenesis (b, E9.5; c, E10.5; d, E11.5; e, no tetracycline) and embryos were examined at E18.5. Black arrows denote the period of tetracycline administration (DTA repressed); red arrows denote the period during which tetracycline was omitted, resulting in ablation. Weights were calculated as a percentage of non-ablated control (a) and are displayed along with 95% confidence intervals (CI) and the number of tTA/DTA embryos examined. Asterisk, P < 0.001.

Full figure and legend (112K)Figures & Tables index

The consequences of transient progenitor cell ablation were examined by inducing, and then repressing, DTA expression (Fig. 2). Tetracycline was withheld from pregnant females until mid-gestation and subsequently added at specific time points to halt the ablation of progenitor cells. Pancreata from tTA/DTA embryos in which ablation was halted at E9.5 appeared grossly normal at E18.5 and exhibited a small reduction in weight compared to control pancreata (Fig. 2b; see also Supplementary Fig. 1). By contrast, embryos in which progenitor cell ablation was halted starting on, or after, E11.5 had a nearly complete absence of the pancreas (Fig. 2d; see also Supplementary Fig. 1). This latter result demonstrates that if Pdx1⁺ cells are efficiently eliminated before ~E12–E12.5, formation of Pdx1⁺ pancreatic progenitor cells with the ability to rescue pancreatic development does not occur. Stated otherwise, these results suggest that all of the Pdx1⁺ progenitors needed to make the pancreas are generated during the embryonic period spanning approximately E8.5 to E12.5.

Direct evidence that DTA expression reduces the pancreatic progenitor pool was obtained by staining sectioned pancreatic buds with a Pdx1 antibody. Pdx1⁺ precursors are first detected at approximately E8.5–9.0 (ref. 16), so the absence of tetracycline up to and including E9.5 is expected to have a minor effect. Indeed, embryos subjected to ablation through E9.5 exhibited a mild reduction (8%) in the size of the Pdx1⁺ progenitor pool, whereas a more marked reduction (61%) was observed after ablation through E10.5 (Supplementary Fig. 2a, c). Ablation through later time points resulted in the loss of pancreatic buds and the near absence of pancreatic progenitors (Supplementary Fig. 2a, c). These results show that death of pancreatic progenitor cells, and resulting loss of pancreatic tissue mass, can be regulated by tetracycline in these mice.

Lack of significant growth compensation

To assess the capacity for compensatory growth, we generated embryos in which many, but not all, progenitor cells were ablated. On the basis of the results above, progenitor ablation up to and including E10.5 was predicted to provide an appropriate experimental condition for testing the ability of pancreas size to normalize. Indeed, ablation of progenitor cells in tTA/DTA embryos through E10.5, followed by a 2-day recovery, resulted in pancreatic buds that were significantly smaller than controls (Supplementary Fig. 2b).

Under these conditions, a reproducible 60–80% reduction in newborn pancreas size was observed (Fig. 2c; see also Supplementary Fig. 1). This result is surprising because it shows that, after a reduction in the Pdx1⁺ progenitor pool at the early bud stage, the remaining pancreatic progenitors are able to grow, divide and differentiate, but cannot increase the rate or number of cell divisions to make an organ of normal size. The DTA-mediated death of a fraction of Pdx1⁺ progenitor cells, coupled with an inability of the remaining cells to compensate for this reduced cell number, causes the pancreas to be small at birth. The distal stomach and proximal intestine, which normally express Pdx1 during development and which therefore might be expected to show some loss, were not noticeably affected in tTA/DTA embryos under any ablation conditions; this may be a result of silencing of the tetO^DTA locus in these tissues (data not shown).

One potential explanation for a small pancreas after transient progenitor ablation is the possibility that a subset of progenitor cells was selectively eliminated. Because exocrine cells constitute over 80% of tissue mass, loss of exocrine progenitor cells might account for the reduced size¹². To address this possibility, we determined the relative area occupied by exocrine, ductal and endocrine cells from tTA/DTA pancreata in which progenitor ablation had been halted at E10.5. These pancreata contained all lineages, although the area occupied by insulin-positive -cells was decreased approximately twofold compared to +/DTA littermates (Supplementary Fig. 3). Because -cells make up a small percentage (<5%) of overall pancreatic mass, faulty specification of endocrine progenitor cells does not account for the reduction in pancreas size.

Another possibility is that compensatory growth occurs, but does so very slowly. Specifically, it might take longer than the 8 days between DTA repression at E10.5 and analysis at E18.5 for size normalization to occur. To address this, we followed cohorts of mice (in which progenitor cell ablation had been halted at E10.5) for approximately 1, 3, 4, or 11 weeks after birth. Similar to their newborn counterparts, the pancreata of these mice remained proportionately small, exhibiting a growth rate that matched that of littermate controls (Fig. 3). Mice in these cohorts displayed marked glucose intolerance, possibly reflecting the relative and absolute decrease in -cells noted above. Thus, a reduction in the number of progenitor cells during an early and brief window of pancreatic development has a lasting impact on pancreas size in adult animals, persisting for 3 months after the original developmental deficit.

Figure 3: Small pancreata do not exhibit catch-up growth.

To generate adult mice with small pancreata, pregnant females carrying Pdx1^tTA/+; tetO^DTA/+ (tTA/DTA; pink) and Pdx1^+/+; tetO^DTA/+ (+/DTA; blue) embryos were given tetracycline at E10.5 to halt ablation. Pancreas weights were measured at birth and approximately 1, 3, 4, or 11 weeks of age. Mean (s.e.m.) pancreas weights of experimental and control littermates were plotted on a log₁₀ scale against age, and best-fit curves were generated. Experimental pancreata grow at approximately the same rate as control pancreata but remain proportionately small. The number of animals analysed at each experimental time point is shown below.

Full figure and legend (80K)Figures & Tables index

Complementing the pancreatic epithelium

These observations support the notion that pancreatic progenitor cell growth follows an intrinsic or autonomous programme, one that does not aim to achieve a certain final size for the whole organ. However, it is conceivable that other factors contributed to the failure of a limited number of progenitor cells to compensate. For example, dead cells generated by DTA expression might have inhibited expansion of remaining Pdx1⁺ progenitors, or a subset of progenitor cells with a greater capacity for compensatory growth may have been selectively killed by the DTA ablation scheme. To address these possibilities, we developed a system wherein progenitor number could be approached from the opposite direction, where we added back progenitors to dictate the starting number of cells in the pancreatic anlage.

Because homozygous inactivation of Pdx1 prevents pancreas development^{17, 18}, we hypothesized that a chimaeric embryo derived by combining Pdx1-deficient blastocysts with wild-type embryonic stem (ES) cells would exhibit selective growth and maturation of ES cell progeny during pancreatic development. Furthermore, if the Pdx1 mutation acts cell-autonomously—as suggested by in vitro recombination experiments¹⁹—the entire pancreatic epithelium generated by such a complementation scheme would be derived from the ES cells. A similar approach has been used successfully in mice to create an ES-cell-derived lymphoid system²⁰ and lens²¹.

We used Pdx1^lacZ mice, which harbour a -galactosidase cDNA in place of Pdx1 coding sequences¹⁸. Wild-type ES cells were injected into blastocysts generated by an intercross of Pdx1^lacZ/+ heterozygous mice, and embryos were collected at later stages (Fig. 4a). As predicted, pancreata from Pdx1^+/+ and Pdx1^lacZ/+ embryos appeared normal. Notably, several chimaeric embryos derived from Pdx1^lacZ/lacZ blastocysts also appeared to have a normal pancreas. The structures that express Pdx1—stomach, pancreas and duodenum—stain blue in Pdx1^lacZ/+ embryos (Fig. 4c), allowing us to distinguish between blastocyst-derived and ES-derived epithelial cells. In all cases, the pancreas arising from a rescued Pdx1^lacZ/lacZ blastocyst lacked -galactosidase activity (Fig. 4e), indicating that it was formed from donor ES cells. Pdx1^lacZ/lacZ embryos without ES cell contribution had a blue 'dorsal ductule', representing the arrested product of a dorsal pancreatic bud as previously noted¹⁸, but no pancreas (Fig. 4d). When ES cells tagged with yellow fluorescent protein (YFP)²² were used to make chimaeras, the entire pancreatic epithelium of complemented Pdx1-deficient chimaeras was YFP⁺ (Fig. 4e, inset; see also Supplementary Fig. 4). These results demonstrate unambiguously the cell-autonomous nature of Pdx1 function and the ability of wild-type ES cells to give rise to a normal pancreas when introduced into a Pdx1 mutant blastocyst.

Figure 4: Pdx1-deficient blastocyst complementation.

a, Strategy. YFP-tagged ES cells are injected into blastocysts generated from an intercross of Pdx1^lacZ/+ mice. Homozygous Pdx1^lacZ/lacZ embryos exhibiting pancreas complementation should express YFP but not -galactosidase. b–e, Glucagon (brown) and -galactosidase (blue) staining of E14.5 midguts; pancreata are outlined in red. b, Chimaeric Pdx1^+/+ embryos exhibit pancreatic glucagon staining (arrowhead) but no -galactosidase activity. c, d, Glucagon staining and -galactosidase activity are both present in heterozygous Pdx1^lacZ/+ pancreata (c) and in the dorsal ductules¹⁸ of Pdx1^lacZ/lacZ embryos (d). e, Complemented Pdx1^lacZ/lacZ pancreata lack -galactosidase activity but are YFP⁺ (inset). d, duodenum; dd, dorsal ductule; e, epithelium; m, mesenchyme; s, stomach.

Full figure and legend (379K)Figures & Tables index

Size variation in complemented pancreata

Chimaeras generated by blastocyst injection exhibit a wide range of ES-cell contributions. Although the factors that determine the extent of chimaerism are poorly understood, it seems that only one or two ES cells contribute to each chimaeric embryo²³. Because of this inherent variation, the tissue complementation system allowed us to ask whether a limited number of competent cells can undergo compensatory growth. If a small number of progenitor cells are able to compensate, we would expect to observe a normal-sized pancreas in all cases, regardless of the degree of chimaerism. Conversely, a lack of compensatory growth would result in variation in pancreas size (Fig. 5a).

Figure 5: Size variation after pancreas complementation.

a, Injection of wild-type ES cells into Pdx1^-/- blastocysts yields chimaeras with variable ES contribution. The total number of cells within the pancreatic anlage is the same in chimaeric and wild-type (ctrl) mice; however, the relative percentage of competent Pdx1⁺ cells (green) and non-competent Pdx1-deficient cells (black) varies. The ability of a reduced number of competent cells to give rise to a normal pancreas is tested by examining newborn pancreata from multiple chimaeras. b–d, Dissected guts from three complemented Pdx1^-/- embryos (E18.5), showing variation in pancreas size (outlined). e–g, Top row: three complemented pancreata exhibiting size variation (different from those above) were stained with amylase (red), yielding microscopic (e), small (f) and normal (g) pancreata. Bottom row: the degree of chimaerism (green; arrows) correlates with pancreas size.

Full figure and legend (337K)Figures & Tables index

Although the vast majority of chimaeras generated from Pdx1^-/- blastocysts contained some pancreatic tissue (23 out of 27; 85%), significant variation in pancreas size was observed (Fig. 5b–d). In each case, the pancreas, when formed, had exocrine, endocrine and duct cells, but the overall size of the organ varied considerably, ranging from normal to microscopic (Supplementary Table 1). Furthermore, the extent of chimaerism in complemented embryos, measured by YFP fluorescence in extra-pancreatic tissues, was correlated with the size of the pancreas (Fig. 5e–g; see also Supplementary Fig. 5). These results are consistent with the hypothesis that a limited number of competent pancreatic progenitor cells come to populate the developing endodermal gut tube and that the fates of these ES-derived cells are fixed at a certain time. Subsequently, these cells and their progeny are unable to undergo compensatory growth to produce a pancreas of normal size.

Growth compensation in the liver

We wished to determine whether other tissues develop with similar constraints on growth. The liver was chosen for comparison because of its close developmental relationship to the pancreas. Both organs are derived from adjacent regions of endoderm and there is evidence for a bi-potential progenitor cell with the capacity to give rise to either liver or pancreas²⁴. Furthermore, the adult liver undergoes robust regeneration after injury or resection²⁵, in contrast to the pancreas, which has a more limited capacity for regeneration.

To ablate hepatic progenitor cells, we used a transgenic strain in which the promoter for LAP (liver-enriched transcriptional activator protein), a marker of early hepatic progenitor cells²⁶, drives tTA-mediated gene expression in the liver at a level at least 200-fold higher than that of any other tissue^{15, 27}. Using this strain, the elimination of LAP⁺ hepatic progenitors could be regulated by mating LAP^tTA mice to tetO^DTA mice and altering the dose/timing of tetracycline addition. Ablation through E13.5 resulted in a reduction in the hepatic progenitor pool of at least 65% (Supplementary Figs 6 and 7), enabling us to perform an experiment comparable to the transient ablation of pancreatic progenitors described above. After an initial pulse of ablation²⁸, DTA expression was repressed with tetracycline, and liver size was determined 4 days later. Under these conditions, and distinct from the corresponding result in the pancreas, the livers of LAP-tTA/DTA embryos appeared normal (compare orange boxed panels in Figs 6 and 2c). This 'catch-up' growth occurred rapidly and with great precision (Fig. 6b; see also Supplementary Fig. 7). Thus, in contrast to the inability of the pancreas to normalize size long after a loss of early progenitor cells, the liver regulates growth to achieve a normal size after only 4 days (compare Figs 6b and 3).

Figure 6: Compensatory growth during liver development.

a, Pregnant mice carrying LAP-tTA/DTA and +/DTA embryos were given plain drinking water or water containing tetracycline at designated time points and embryos were examined at E17.5. Black arrows denote the period of tetracycline administration (DTA repressed); red arrows denote the period during which tetracycline was omitted (ablation). Repression of DTA expression from E13.5–E17.5 results in a complete recovery of liver size (boxed panel). Scale bar, 1 mm. b, Tetracycline was provided either throughout gestation (blue) or beginning at E13.5 (pink). LAP-tTA/DTA liver weight was plotted as a percentage of +/DTA littermate control weight (mean  s.e.m.) using best-fit analysis. The number of embryos examined at each time point (experimental/control) is shown, along with the number of litters examined (parentheses). ND, not determined.

Full figure and legend (155K)Figures & Tables index

Discussion

Given the highly regulative nature of vertebrate development, we expected that reducing the number of early tissue-specific progenitor cells would not perturb final organ size. This expectation was based on numerous precedents; for example, removing most of the cells in the embryonic limb field in amphibians or chickens has no effect on final limb size because compensatory proliferation makes up for the loss²⁹. Similar compensatory proliferation occurs in Drosophila imaginal discs after cell loss, resulting in normal tissue size³⁰. And in the blood and central nervous system, growth factors and apoptotic factors regulate final organ size by controlling cell number^{31, 32}. Contrary to these predictions, our studies show that compensatory growth during pancreas development is either quite limited or does not occur at all. Thus, embryonic progenitor cells represent a critical and limiting determinant of pancreas size.

Although the prediction of growth compensation was not borne out in our pancreas studies, it proved to be true for liver size. The adult liver exhibits robust recovery of mass after injury or partial removal. We find that the embryonic liver shares this capacity for compensatory growth and does so with an impressive speed and accuracy. Four days after losing roughly two-thirds of its mass, the embryonic liver exhibits a size that is almost identical to that of controls. By contrast, the pancreas fails to compensate after a similar loss of mass 3 months after the initial insult. Thus, two closely related endoderm derivatives—the pancreas and liver—differ markedly in their response to developmental alterations in progenitor cell number.

We did not perform tissue complementation in the liver, although comparable studies have recently been reported³³. Chimaeric embryos containing a mixture of wild-type and Hex-deficient liver progenitor cells exhibit progressive loss of the Hex-null cells during liver morphogenesis. In these chimaeras, the remaining wild-type cells increase their proliferation and compensate for this loss, giving rise to a normally sized liver bud³³.

Factors that regulate cellular proliferation are undoubtedly involved in pancreatic growth control. FGF10 is an important mediator of pancreas growth³⁴, acting through Notch signalling to balance proliferation and differentiation^{35, 36}. Wnt signalling also has a principal role in pancreas growth during development^{37, 38}, and recent observations suggest that stabilization of -catenin results in an increase in pancreas size through effects on proliferation³⁹. The results reported here suggest that despite a developmental requirement for growth factors, these factors cannot mediate a significant compensatory response in the pancreas.

Our analysis draws attention to the embryonic progenitor cell in setting final organ size. Committed progenitor cells, in contrast to stem cells, are defined by their limited capacity for division and self-renewal. Thus, it is possible that pancreatic progenitor cell proliferation is autonomously restricted and that each progenitor cell is capable of giving rise to only a fixed amount of tissue. In the central nervous system, for example, a 'counting mechanism' limits the number of times a glia precursor cell divides⁴⁰. Pancreas size might be dictated through a similar mechanism, such that the size of the progenitor pool specified during endoderm patterning might dictate final organ size. Conversely, size information might be encoded within the pancreatic mesenchyme, which in turn might control the growth of the epithelium in a manner that is not subject to compensatory growth.

The mechanisms that regulate organ size will probably be understood in the context of two related and general questions: (1) to what extent do different organs develop from self-renewing stem cells versus committed progenitor cells; and (2) to what extent is size determined by cell-autonomous and non-cell-autonomous mechanisms. Although it has been suggested that tissues are generated during fetal life by a hierarchy of stem cell to progenitor cell to progeny cell⁴¹, it is unknown for most organs whether it is stem cells or committed progenitors that constitute the substrate for organogenesis. Our previous studies of the pancreas suggested that stem cells do not contribute significantly to the maintenance of adult islets⁴². The current study highlights the possibility that this adult state is presaged by an absence of pancreatic stem cells during embryonic development. Moreover, these results raise the question of whether there are two kinds of tissues: some, like the liver, that exhibit regulated growth in development and robust regeneration in adulthood because of a reliance on extrinsic signals; and others, like the pancreas, that exhibit reduced regenerative capacity in adulthood because of intrinsic growth constraints that are imprinted early in development.

Methods

A detailed description of materials and methods is provided in Supplementary Information.

1. Gilbert, S. F. Developmental Biology (Sinauer, Sunderland, Massachusetts, 2000)
2. Bryant, P. J. & Simpson, P. Intrinsic and extrinsic control of growth in developing organs. Q. Rev. Biol. 59, 387–415 (1984) | Article | PubMed | ISI | ChemPort |
3. Conlon, I. & Raff, M. Size control in animal development. Cell 96, 235–244 (1999) | Article | PubMed | ISI | ChemPort |
4. Britton, J. S., Lockwood, W. K., Li, L., Cohen, S. M. & Edgar, B. A. Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2, 239–249 (2002) | Article | PubMed | ISI | ChemPort |
5. Rulifson, E. J., Kim, S. K. & Nusse, R. Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science 296, 1118–1120 (2002) | Article | PubMed | ISI | ChemPort |
6. Ikeya, T., Galic, M., Belawat, P., Nairz, K. & Hafen, E. Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12, 1293–1300 (2002) | Article | PubMed | ISI | ChemPort |
7. Colombani, J. et al. Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science 310, 667–670 (2005) | Article | PubMed | ChemPort |
8. Twitty, V. C. & Schwind, J. L. The growth of eyes and limbs transplanted heteroplastically between two species of Ambystoma. J. Exp. Zool. 59, 61–86 (1931) | Article |
9. Wolpert, L. Cellular basis of skeletal growth during development. Br. Med. Bull. 37, 215–219 (1981) | PubMed | ChemPort |
10. Muller-Sieburg, C. E., Cho, R. H., Sieburg, H. B., Kupriyanov, S. & Riblet, R. Genetic control of hematopoietic stem cell frequency in mice is mostly cell autonomous. Blood 95, 2446–2448 (2000) | PubMed | ChemPort |
11. Robin, C. et al. An unexpected role for IL-3 in the embryonic development of hematopoietic stem cells. Dev. Cell 11, 171–180 (2006) | Article | PubMed | ChemPort |
12. Gu, G., Dubauskaite, J. & Melton, D. A. Direct evidence for the pancreatic lineage: NGN3⁺ cells are islet progenitors and are distinct from duct progenitors. Development 129, 2447–2457 (2002) | PubMed | ISI | ChemPort |
13. Holland, A. M., Hale, M. A., Kagami, H., Hammer, R. E. & MacDonald, R. J. Experimental control of pancreatic development and maintenance. Proc. Natl Acad. Sci. USA 99, 12236–12241 (2002) | Article | PubMed | ChemPort |
14. Lee, P. et al. Conditional lineage ablation to model human diseases. Proc. Natl Acad. Sci. USA 95, 11371–11376 (1998) | Article | PubMed | ChemPort |
15. Kistner, A. et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl Acad. Sci. USA 93, 10933–10938 (1996) | Article | PubMed | ChemPort |
16. Guz, Y. et al. Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. Development 121, 11–18 (1995) | PubMed | ISI | ChemPort |
17. Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371, 606–609 (1994) | Article | PubMed | ISI | ChemPort |
18. Offield, M. F. et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122, 983–995 (1996) | PubMed | ISI | ChemPort |
19. Ahlgren, U., Jonsson, J. & Edlund, H. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development 122, 1409–1416 (1996) | PubMed | ISI | ChemPort |
20. Chen, J., Lansford, R., Stewart, V., Young, F. & Alt, F. W. RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc. Natl Acad. Sci. USA 90, 4528–4532 (1993) | Article | PubMed | ChemPort |
21. Liegeois, N. J., Horner, J. W. & DePinho, R. A. Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo. Proc. Natl Acad. Sci. USA 93, 1303–1307 (1996) | Article | PubMed | ChemPort |
22. Hadjantonakis, A. K., Macmaster, S. & Nagy, A. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2, 11 (2002) | Article | PubMed |
23. Wang, Z. & Jaenisch, R. At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation. Dev. Biol. 275, 192–201 (2004) | Article | PubMed | ISI | ChemPort |
24. Deutsch, G., Jung, J., Zheng, M., Lora, J. & Zaret, K. S. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development 128, 871–881 (2001) | PubMed | ISI | ChemPort |
25. Taub, R. Liver regeneration: from myth to mechanism. Nature Rev. Mol. Cell Biol. 5, 836–847 (2004) | Article |
26. Westmacott, A., Burke, Z. D., Oliver, G., Slack, J. M. & Tosh, D. C/EBP and C/EBP are markers of early liver development. Int. J. Dev. Biol. 50, 653–657 (2006) | Article | PubMed | ChemPort |
27. Lavon, I. et al. High susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-B activation. Nature Med. 6, 573–577 (2000) | Article |
28. Furth, P. A. et al. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl Acad. Sci. USA 91, 9302–9306 (1994) | Article | PubMed | ChemPort |
29. Kalthoff, K. Analysis of Biological Development (McGraw-Hill, New York, 1996)
30. Gallant, P. Myc, cell competition, and compensatory proliferation. Cancer Res. 65, 6485–6487 (2005) | Article | PubMed | ChemPort |
31. Hidalgo, A. & ffrench-Constant, C. The control of cell number during central nervous system development in flies and mice. Mech. Dev. 120, 1311–1325 (2003) | Article | PubMed | ChemPort |
32. Morrison, S. J., Uchida, N. & Weissman, I. L. The biology of hematopoietic stem cells. Annu. Rev. Cell Dev. Biol. 11, 35–71 (1995) | Article | PubMed | ISI | ChemPort |
33. Bort, R., Signore, M., Tremblay, K., Martinez Barbera, J. P. & Zaret, K. S. Hex homeobox gene controls the transition of the endoderm to a pseudostratified, cell emergent epithelium for liver bud development. Dev. Biol. 290, 44–56 (2006) | Article | PubMed | ChemPort |
34. Bhushan, A. et al. Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128, 5109–5117 (2001) | PubMed | ISI | ChemPort |
35. Norgaard, G. A., Jensen, J. N. & Jensen, J. FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development. Dev. Biol. 264, 323–338 (2003) | Article | PubMed | ChemPort |
36. Hart, A., Papadopoulou, S. & Edlund, H. Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells. Dev. Dyn. 228, 185–193 (2003) | Article | PubMed | ChemPort |
37. Papadopoulou, S. & Edlund, H. Attenuated Wnt signaling perturbs pancreatic growth but not pancreatic function. Diabetes 54, 2844–2851 (2005) | PubMed | ChemPort |
38. Murtaugh, L. C., Law, A. C., Dor, Y. & Melton, D. A. -catenin is essential for pancreatic acinar but not islet development. Development 132, 4663–4674 (2005) | Article | PubMed | ChemPort |
39. Heiser, P. W., Lau, J., Taketo, M. M., Herrera, P. L. & Hebrok, M. Stabilization of -catenin impacts pancreas growth. Development 133, 2023–2032 (2006) | Article | PubMed | ChemPort |
40. Temple, S. & Raff, M. C. Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions. Cell 44, 773–779 (1986) | Article | PubMed | ISI | ChemPort |
41. Weissman, I. L., Anderson, D. J. & Gage, F. Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. Annu. Rev. Cell Dev. Biol. 17, 387–403 (2001) | Article | PubMed | ISI | ChemPort |
42. Dor, Y., Brown, J., Martinez, O. I. & Melton, D. A. Adult pancreatic -cells are formed by self-duplication rather than stem-cell differentiation. Nature 429, 41–46 (2004) | Article | PubMed | ISI | ChemPort |