Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on October 21, 2003

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Human Molecular Genetics, 2003, Vol. 12, No. 24 3307-3314
DOI: 10.1093/hmg/ddg355
© 2003 Oxford University Press

Hnf6 and Tcf2 (MODY5) are linked in a gene network operating in a precursor cell domain of the embryonic pancreas

Miguel A. Maestro¹, Sylvia F. Boj¹, Reini F. Luco¹, Christophe E. Pierreux³, Judit Cabedo¹, Joan M. Servitja¹, Michael S. German², Guy G. Rousseau³, Frédéric P. Lemaigre³ and Jorge Ferrer^1,*

¹Endocrinology, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain, ²Hormone Research Institute, University of California, San Francisco, CA, USA and ³Hormone and Metabolic Research Unit, Université Catholique de Louvain and Institute of Cellular Pathology, Brussels, Belgium

Received July 28, 2003; Accepted October 15, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
During pancreatic organogenesis endocrine cells arise from non self-renewing progenitors that express Ngn3. The precursors that give rise to Ngn3⁺ cells are presumably located within duct-like structures. However, the nature of such precursors is poorly understood. We show that, at E13–E18, the embryonic stage during which the major burst of ß-cell neogenesis takes place, pancreatic duct cells express Hnf1ß, the product of the maturity-onset diabetes of the young type 5 (MODY5) gene. Ngn3⁺ cells at this stage invariably cluster with mitotically competent Hnf1ß⁺ cells, and are often intercalated with these cells in the epithelium that lines the lumen of primitive ducts. We present several observations that collectively indicate that Hnf1ß⁺ cells are the immediate precursors of Ngn3⁺ cells. We furthermore show that Hnf1ß expression is markedly reduced in early pancreatic epithelial cells of Hnf6-deficient mice, in which formation of Ngn3⁺ cells is defective. These findings define a precursor cellular stage of the embryonic pancreas and place Hnf1ß in a genetic hierarchy that regulates the generation of pancreatic endocrine cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
The morphological development of the mouse pancreas begins at embryonic day 9 (E9) with dorsal and ventral evaginations of the foregut endoderm that later fuse to form a single organ (reviewed in 1 and 2). The epithelium of the primitive pancreas undergoes proliferation and branching morphogenesis, with the subsequent formation of ductal structures (1,3). Some epithelial cells express hormones at the earliest stages of pancreatic organogenesis, although the bulk of pancreatic endocrine cell differentiation takes place later, from E13 to E18 (3,4). Acinar cells that synthesize digestive enzymes also begin to be recognized at this point.

The understanding of the process that begins with the formation of pancreatic buds and culminates with differentiated lineages has benefited from the discovery of tissue-enriched transcription factors that define the phenotype of predifferentiated cell types (1,5–11). Pluripotent cells of the early pancreatic bud epithelium express several transcription factors, including Pdx1, Nkx2.2, Hnf6, Nkx6.1, and p48 (1,7–11). Pdx1 and p48 have been shown by lineage tracing to be present in cells that later give rise to adult exocrine and endocrine cells, and furthermore are known to be essential for early pancreas organogenesis (5,8,12–14). The basic helix–loop–helix protein neurogenin 3 (Ngn3) more specifically marks the earliest known progenitors that are committed to undergo endocrine differentiation, and is indispensable for endocrine cell formation (6,7,13).

The precise cellular stages that take place between the early Pdx1⁺/p48⁺ pluripotent stage and Ngn3⁺ endocrine commitment are poorly defined. Endocrine cells and their precursors are often seen to originate from duct-like structures during E13–E18, although information regarding the critical regulators and specific markers of the precursor cell type contained within such ducts is limited (6,13,15,16). It is nevertheless known that Hepatic Nuclear Factor 6 (Hnf6), a onecut homeodomain protein that is expressed in most early pancreatic epithelial cells, differentiated ducts and acinar cells, plays an essential role in the generation of Ngn3⁺ precursors (10,17). Consequently, mice lacking Hnf6 exhibit drastically reduced Ngn3⁺ and endocrine cells during embryogenesis (17).

Despite the morphological similarity of adult pancreatic ducts and primitive ducts that give rise to endocrine cells, a recent fate-mapping study has questioned whether these constitute a common lineage (13). An unequivocal settling of this issue is hampered by the paucity of specific markers of both primitive and adult duct cells (1,13,16). Clearly, determining the nature of the cells that possess the potential to generate differentiated pancreatic cells is crucial for the development of cell replacement and regeneration therapies of pancreatic diseases.

Several genes encoding DNA-binding proteins have been implicated in a human pancreatic ß-cell phenotype known as maturity onset of diabetes of the young (MODY) (reviewed in 18–20). Two of the known MODY genes encode for the homeodomain proteins Hnf1 and Hnf1ß (MODY3 and MODY5, respectively) (21,22). Hnf1 and ß share homologous dimerization domains that enable the formation of homo- or heterodimers, and also possess closely related homeodomains that confer identical in vitro DNA sequence recognition specificity (23,24). The distinct roles of these two proteins in the pancreas are only now beginning to be unraveled. Mouse knock-out studies have shown that Hnf1 controls a transcriptional network required to maintain differentiated functions of pancreatic ß-cells, consistent with the observation that Hnf1 is abundant in adult endocrine cells (20,25–27). The pancreatic phenotype in MODY5 is less well characterized, as Tcf2(Hnf1ß)^-/- mouse embryos fail to progress beyond the blastocyst stage (28,29). A recent report, however, indicates that MODY5 is associated with a markedly reduced pancreatic organ volume, suggesting a developmental function of Hnf1ß in the pancreas (30). One limitation to understanding the role of Hnf1ß in the pancreas lies in the limited information regarding the pancreatic cell-types expressing this protein. Tcf2(Hnf1ß) LacZ knock-in mice have shown galactosidase staining in pancreatic ducts and to a lesser extent in islets, although no coexpression analysis has been performed during pancreatic development to identify the precise cell types expressing Hnf1ß (31). Detailed knowledge of which cells express Hnf1ß and is essential to understand where these genes act during pancreatic differentiation.

In this study the expression of Hnf1ß was found to define a cellular population that forms the primitive pancreatic ducts. We show that such embryonic duct Hnf1ß⁺ cells are phenotypically distinct from earlier pancreatic bud cells, and present evidence that they are direct precursors of Ngn3⁺ endocrine progenitor cells. These results are integrated with existing information to provide a model for the lineage relationships of pancreatic cells. Furthermore, Hnf1ß in such cells is shown to be downstream of Hnf6, a gene that is required for endocrine cell formation (17). Together with the knowledge that genetic deficiency of HNF1ß can cause diabetes and pancreatic hypoplasia in humans (30), the data presented here suggests that the Hnf6–Tcf2 (Hnf1ß) hierarchy may play a key regulatory role at a defined stage of pancreatic development.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Hnf1ß expression is progressively enriched in a subset of early Pdx1⁺ pancreatic epithelial cells
At E10.5 pancreatic buds are largely formed by predifferentiated epithelial cells coexpressing Pdx1, Nkx6.1, Nkx2.2, Hnf6 and p48 (1,7,9,10,14,32). Hnf1ß is detected in such cells, although the expression levels are only slightly above the detection limit, in contrast to the high levels observed in mesonephros (Fig. 1A). As shown in Figure 1B, Hnf1 is present in E10.5 pancreatic cells, more abundantly in the dorsal bud.

View larger version (140K): [in this window] [in a new window]   Figure 1. Expression of Hnf1ß and Hnf1 in pancreatic cells. (A, B) Dual immunofluorscence analysis of E10.5 embryos for (A) Hnf1ß (red) or (B) Hnf1 (red) and Nkx2.2 (green) reveals very low expression of Hnf1ß in pancreatic buds, and higher levels of Hnf1 in the dorsal bud. dp and vp, dorsal and ventral pancreas; gu, foregut; ne, mesonephros; li, hepatic primordium. (C–E) Dual immunoanalysis of E12.5 pancreas for Hnf1ß (red) and (C) Pdx1 (green), (D) Nkx6.1 (green), or (E) Hnf1 (green) indicates Hnf1ß enrichment in a centrally located subset of Pdx1+/Nkx6.1+ cells, while Hnf1 is more abundant in peripheral cells. (F, G) Immunostaining of E13.5 pancreas for Hnf1ß (red) and (F) E-cadherin (green) or (G) Pax6 (green) shows branching Hnf1ß epithelium distinct from typical acinar structures (ac). (H–K) Immunostaining of E15.5 pancreas for (H) Hnf1ß (red) alone, or with (I) insulin (blue) and glucagon (green), (J) Pdx1 (green), or (K) Nkx6.1 (green). A dotted line indicates the Hnf1ß+ domain. H is a low magnification image. Hnf1ß+ cells at this stage are restricted to duct-like structures, expressing low levels of Pdx1 and low heterogeneous levels of Nkx6.1. (L) Immunostaining of E18.5 pancreas for Hnf1ß (red) and insulin (green). (M, N) Adult pancreas stained for Hnf1ß (red), insulin (blue) and glucagon (green). An arrow in N points to a small intraislet Hnf1ß+ cluster. (O, P) E15.5 pancreas stained for Hnf1 (green) and Hnf1ß (red) (O) or insulin (red) (P), showing opposed expression patterns of Hnf1 and ß, with enrichment of Hnf1 in hormone-producing and acinar cells. (Q) EMSA with nuclear extracts from dissected pancreas at indicated gestational ages, isolated islets, Min6 ß-cells and Hnf1ß expressing-COS7 kidney cells, using an oligonucleotide capable of binding Hnf1 and ß homo- or heterodimers (25). In this assay heterodimers can be formed in vitro even if Hnf1ß and are in separate cell compartments. Hnf1 and ß antibody supershifts are used to verify the identity of complexes. Consistent with (N, O), Hnf1ß and are enriched in early embryonic pancreas. Hnf1 is greatly increased at later stages due to the expansion of acinar and endocrine cells. Extracts from MIN6 ß-cells and islets contain predominantly Hnf1, although anti-Hnf1ß supershifts a lower abundance complex largely formed by Hnf1ß/ heterodimers. Purified islets can contain varying degrees of duct-cell contamination.

 
By E12.5 a subset of Pdx1⁺/Nkx6.1⁺ cells predominantly located in the pancreatic core exhibits more intense staining for Hnf1ß (Fig. 1C–E). At E13.5 Hnf1ß⁺ cells form branching epithelium distinct from peripheral acinar structures (Fig. 1F and G), and by E15.5, Hnf1ß is more clearly confined to cells that form discrete clusters with duct-like morphology (Fig. 1H–K). Throughout embryogenesis and in adult mice, all pancreatic structures with evident ductal morphology, including intralobular ducts, contain Hnf1ß⁺ cells (Fig. 1H–O, see also Fig. 2A and E). A lumen is not apparent in all Hnf1ß⁺ cellular clusters (Fig. 2A). This largely reflects tangential sectioning of ducts, although during early stages (<E15.5) Hnf1ß⁺ cells appear to be arranged in epithelial sheets forming more than a single layer surrounding a lumen (not shown).

View larger version (138K): [in this window] [in a new window]   Figure 2. Ngn3+ cells arise from Hnf1ß+ epithelium. Dual immunostainings of embryonic pancreas at E15.5 for Hnf1ß (red) and Ngn3 (green) at low (A) and high (B, C) magnification. In B and C the lower subpanels show only anti-Hnf1ß stainings. All Ngn3+ cells are in the immediate vicinity of Hnf1ß+ cells, often lining the lumen of ducts and costain for Hnf1ß in 28% of cells. Arrows point to Ngn3+/Hnf1ß+ cells, arrowheads to Ngn3+/Hnf1ß- cells. The proportion of Hnf1ß+/Ngn3+ cells in (C) is not typical. (D) Immunostaining of E15.5 pancreas for Hnf1ß (red), Ngn3 (blue) and Pax6 (green). Ngn3 is omitted in bottom subpanels. Most Ngn3+cells are Pax6-, but Ngn3+/Pax6+ cells are consistently Hnf1ß- (arrow). An arrowhead indicates a Ngn3+ cell fully surrounded by Pax6 or Hnf1ß cells. (E) Merged image of immunostaining of E15.5 pancreas for Hnf1ß (red) and Nkx2.2 (green). Arrows point to an intralobular duct. Endocrine precursors are associated with larger embryonic ducts, but not with intralobular ducts. (F, G) Merged images of anti-Hnf1ß (red), Ngn3 (blue) and Nkx2.2 (green) coimmunostainings in E13.5 (F) and E15.5 (G) pancreas. Ngn3+ cells are seen in different shades of blue to green depending on the level of coexpression of Nkx2.2. Hnf1ß+ cells are brownish due to costaining with Nkx2.2 at low levels. Arrowheads point to Ngn3+ cells fully surrounded by Nkx2.2hi or Hnf1ß+ cells. (H) Merged image of anti-Hnf6 (red), Ngn3 (blue) and Pax6 (green) coimmunostainings of E15.5 pancreas. Ngn3 staining is omitted from the right subpanel. Expression of Hnf6 is observed in ductal epithelial cells and acinar cells, but not in Pax6+ endocrine cells. Most Ngn3+ cells are Hnf6+ (arrows), except for the more differentiated Ngn3+–Pax6+ cells (arrowhead). (I) Costaining with nuclear marker ToPro3 (blue) showing that Ngn3- cells that do not express Hnf1ß are very rare among epithelial cells that clearly line the lumen of ducts. (J) Costainings of E13.5 pancreas with anti-Hnf1ß (red), Ngn3 (blue) and E-cadherin (green). An arrowhead points to Ngn3+ cells exclusively surrounded by Hnf1ß+ cells. (K) Representative image of a BrdU pulse label experiment of E15.5 pancreas showing 7/44 Hnf1ß+ cells (red) exhibiting BrdU incorporation (green, orange when present in Hnf1ß+ nuclei), in contrast to 0/16 Ngn3+ cells (blue).

 
Hnf1ß⁺ duct cells at E14.5–E18.5 exhibit weak Pdx1 and Nkx6.1 expression (Figs 1J and K and 2H). Pdx1 immunoreactivity in such cells is slightly lower than in acinar cells, and much lower than in earlier (e.g. E12.5) Hnf1ß⁺ cells (Fig. 1J, compare with Fig. 1C, and not shown). Nkx6.1 exhibits conspicuous cell-to-cell expression level heterogeneity within the Hnf1ß domain (Fig. 1K). Hnf6 is present in both acinar and Hnf1ß⁺ cells, although it is enriched in the latter, as judged from consecutive section stainings, while p48 cannot be detected in ductal Hnf1ß⁺ cells at this stage under conditions that readily identify expression in acinar cells (Fig. 2H, supplementary Fig. 1, and not shown).

Hnf1ß is not present in acinar cells from E14 on, and is generally undetectable in endocrine cells (Fig. 1I, L and N). Occasional very weak staining is observed in some glucagon or insulin cell nuclei (not shown). However, small clusters of one to four cells expressing Hnf1ß⁺ often forming duct-like structures are frequently seen embedded in pancreatic islets (Fig. 1N).

The expression pattern of Hnf1 in the pancreas is remarkably opposed to that exhibited by Hnf1ß. Hnf1 is present in most E12.5 epithelial cells but, in contrast to Hnf1ß, more abundantly in those located peripherally (Fig. 1E). From E13.5 on, Hnf1 is detected at very low levels in primitive ducts and Ngn3⁺ cells, only barely discernable from background staining seen in Hnf1^-/- embryos, whereas it is enriched in acinar, insulin and glucagon cells as soon as they appear (Fig. 1O and P, and not shown). In keeping with these findings, Hnf1ß and Hnf1 DNA binding activity are comparable in E14.5 pancreatic bud extracts, while only minor Hnf1ß binding activity is detected in islet-enriched preparations or MIN6 cells, where Hnf1 is present in vast excess (Fig. 1Q).

Ngn3⁺ cells arise from the Hnf1ß⁺ epithelium
Previous studies have indicated that endocrine progenitors originate from embryonic duct-like structures, although a specific marker for the precursor cells contained within such ducts is lacking (6,13,15,16). At E13.5–E18.5, Ngn3⁺ cells, the earliest known endocrine-committed progenitors (6), are invariably found either within Hnf1ß⁺ clusters, often lining the lumen of ducts, or immediately adjacent (Fig. 2A–D and F–K).

Other markers that become activated concomitantly or subsequent to Ngn3 in the endocrine lineage (reviewed in 1) are consistently found in cells surrounding Hnf1ß⁺ and Ngn3⁺ cell clusters (Fig. 2D–H). Of these, Pax6, Hb9 and Isl1 are not at all detectable within the Hnf1ß⁺ domain (Fig. 2D and data not shown). Pax6 is detected in a small number of Ngn3⁺ cells, consistent with its activation at a late stage of the Ngn3⁺ cell differentiation process (Fig. 2D). Nkx2.2 is detected at very low and heterogeneous levels within the Hnf1ß⁺ domain, while strong expression (Nkx2.2^hi) is elicited in most Ngn3⁺ cells, as well as in Ngn3^- endocrine cells surrounding the Hnf1ß⁺ domain (Fig. 2E–G). Similarly, intensely staining Nkx6.1⁺ and Pdx1⁺ cells (Nkx6.1^hi, Pdx1^hi), representing non-alpha endocrine lineage cells, are seen in the vicinity of Hnf1ß⁺ clusters (Fig. 1J and K). The average distance between Ngn3⁺ cells and the closest Hnf1ß cell at E15.5 is 3.74±0.28 µm, while for insulin and glucagon cells it is 16.89±0.63 and 20.74±1.81 µm, respectively. Taken together these results imply that Ngn3⁺ cells originate from within Hnf1ß⁺ cellular clusters and then migrate outwards as they differentiate.

Several observations collectively indicate that Hnf1ß⁺ cells constitute direct precursors of Ngn3⁺ endocrine progenitors. Most Ngn3⁺ cells express no Hnf1ß or only trace amounts. However, 28% do coexpress Ngn3 and Hnf1ß (Fig. 2B and C). Interestingly, Ngn3⁺ cells expressing Pax6, a late differentiation marker, do not express Hnf1ß. Analogously, Hnf6, a duct-enriched factor that is not present in endocrine cells, is present in >50% of Ngn3⁺ cells, and is undetectable in Ngn3⁺ cells that have initiated expression of Pax6 (Fig. 2H). These findings are precisely as expected for a transition state between Hnf6⁺/Hnf1ß⁺/Ngn3^- cells and Hnf6^-/Hnf1ß^-/Ngn3⁺ endocrine committed progenitors.

Ngn3⁺ cells are non self-renewing and short lived (13), and clearly appear to migrate outwards from the ductal epithelium as they differentiate. This requires the existence of a pool of precursor cells that is at least as abundant as Ngn3⁺ cells in pancreatic ductal epithelium. Costaining with the nuclear marker ToPro3 or the epithelial marker E-cadherin plus Ngn3 and Hnf1ß reveals that Ngn3⁺ cells constitute the vast majority of Hnf1ß-negative cells that line discernable lumina of ducts (Fig. 2I). This finding argues against the existence of an alternate non-Hnf1ß⁺ source of endocrine precursors in the epithelium that lines primitive ducts. Further support for this notion comes from the elevated frequency of Ngn3⁺ cells that are either exclusively surrounded by Hnf1ß⁺ cells or, much more commonly, by cells expressing Hnf1ß, Pax6 or Nkx2.2^hi, the latter two being markers of cells already engaged in the endocrine lineage (Fig. 2D, F, G and J) (1,7,32).

Consistent with a role as a precursor with self-renewal activity, Hnf1ß cells exhibit a very high proliferation rate, unlike Ngn3 and hormone positive cells (22.4% Hnf1ß⁺ versus 1% Ngn3⁺ cell BrdU labeling indexes; Fig. 2K).

Hnf1ß expression is defective in the primitive ducts of Hnf6^-/- embryos
Hnf1ß expression was next assessed in the pancreas of Hnf6^-/- embryos. This was prompted by knowledge of the role of Hnf6 in embryonic development: (a) it is enriched in the Hnf1ß⁺ domain (Supplementary Fig. A); (b) it is required for embryonic formation of Ngn3⁺ cells (33); and (c) it is upstream of Hnf1ß in a cascade that controls bile duct differentiation (34). As previously described (17), E12.5–E14.5 Hnf6^-/- embryos exhibit disturbed ductal morphogenesis and markedly reduced numbers of Ngn3⁺ cells (Fig. 3A and B, and data not shown). Hnf1ß staining in pancreatic ducts at this stage is markedly reduced (Fig. 3A and B). Hnf1ß mRNA in dissected pancreas from Hnf6^-/- E14.5 embryos was reduced 2.4±0.2-fold relative to controls. By E17.5 Hnf1ß expression in Hnf6^-/- pancreas has partially recovered, although staining intensity remains below that of control embryos (Fig. 3C). Interestingly, the reduction of Ngn3⁺ and endocrine cells in Hnf6^-/- embryos is clearly also less pronounced at this stage as compared with earlier time-points (Fig. 3C, and data not shown).

View larger version (32K): [in this window] [in a new window]   Figure 3. Reduced Hnf1ß content in early pancreatic duct epithelial cells of Hnf6-/- embryos. Coimmunostaing of Hnf1ß (red) with Ngn3 (green) in E13.5 (A), E14.5 (B) and E17.5 (C) control and Hnf6-/- embryonic pancreas. Hnf1ß expression is reduced in E13.5 and E14.5 Hnf6-/- embryos, at which time virtually no Ngn3+ cells are observed. At E17.5 Hnf1ß expression is only slightly decreased in Hnf6-/- embryos. Some endocrine lineage cells that do not migrate from the ductal epithelium are seen at this stage in Hnf6-/- embryos.

 

	DISCUSSION

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Hnf1ß is a specific marker for primitive and adult pancreatic duct epithelium
Several markers, including R-cadherin, cytokeratin 20, and V₃B₂ integrin receptors, are enriched in embryonic ductal cells, although their value as specific cell markers is limited because they are also present in endocrine and/or acinar cells (13,16,35,36). In the current study we have shown that Hnf1ß immunostaining allows for unequivocal identification of a population of embryonic epithelial cells that form pancreatic ducts, thus providing an informative molecular marker as opposed to relying solely on the identification of an ill-defined morphological structure.

One immediate benefit of having such a marker lies in the ability to trace the embryonic development of pancreatic ducts. It has been suggested that embryonic duct-like structures that give rise to Ngn3⁺ cells might entirely coalesce to form endocrine-cell islets, rather than also providing the precursors of definitive duct cells (13). Our results show that from E14.5 on Hnf1ß marks all identifiable embryonic ductal structures. Thus, Hnf1ß⁺ epithelium as early as E14.5 lines small intralobular ducts connected with larger ducts, forming a ductal tree analogous to that seen in the adult pancreas (Fig. 2E). Larger ducts initially cluster with endocrine lineage cells. Following such large ducts throughout consecutive embryonic ages from E14.5 on reveals that they contain progressively fewer Ngn3⁺ cells and are instead increasingly found in the vicinity of islets of hormone-expressing cells (Fig. 1L and data not shown), but otherwise maintain the same ductal tree architecture. Such findings support embryonic ducts containing endocrine progenitors and adult ducts that represent two developmental stages of the same anatomical network structure.

The Hnf1ß⁺ cell as a novel stage in pancreatic development
There is currently a gap in our knowledge concerning the cellular stages that lead from Pdx1⁺/p48⁺ stem cells in the early pancreatic bud to the Ngn3⁺ progenitor stage. On the basis of the results presented here, we propose that the Hnf1ß⁺ pancreatic duct epithelium arises as a cellular stage distinct from primitive Pdx1⁺/p48⁺ pluripotent cells. Embryonic duct Hnf1ß⁺ cells are proposed to be self-renewing precursors of both Ngn3⁺ endocrine-specific progenitors and definitive duct cells (Fig. 4). This model does not imply that the Hnf1ß⁺ domain is homogenous. It will be interesting to determine, for example, if Hnf1ß⁺ endocrine progenitors are segregated from other Hnf1ß⁺ cells earlier than Ngn3 protein detection.

View larger version (43K): [in this window] [in a new window]   Figure 4. Pancreatic cell lineage model based on the results from the current and previous studies. Top panel: early Pdx1+/p48+ pluripotent precursors give rise to embryonic duct precursors marked by Hnf1ß. During the major embryonic stage of endocrine cell formation this Hnf1ß+ cell domain gives rise to Ngn3+ endocrine-committed progenitors (only the ß-cell lineage is depicted here), as well as to differentiated ductal cells. A Pdx1+ acinar lineage cell is represented beyond E12.5 based on results from Gu et al. (13). Bottom panel: summary of expression results from a broader selection of transcription factors in the major developmental stages outlined here. Hnf1ß+ embryonic duct cells exhibit a transcription factor phenotype that is distinct from early Pdx1+ cells. Transitional cells and other transcription factors are not depicted for simplicity. A plus sign indicates moderate or high expression, plus/minus, low but consistent expression; minus, no detectable expression. Nkx2.2hi, Nkx6.1hi and Pdx1hi alludes to high level expression observed in endocrine compared to early bud cells, as described in the text.

 
The model shown in Figure 4 is based on the integration of immunostaining analysis of embryonic pancreas with existing genetic data (5–8,12–15), and can now be independently assessed using further genetic fate-mapping experiments. It is nevertheless worth emphasizing that it can be fully reconciled with the results of an existing transgenic lineage tracing study that employed a Pdx1 promoter-driven inducible Cre-recombinase coupled to a Cre-dependent reporter to mark pancreatic progenitor cells (13). In that study the progenitors of adult ducts, like those of acinar and ß-cells, were marked upon induction of Cre during E10–E12.5. However, after E12.5 recombination efficiency dropped markedly in the precursors of adult duct cells, but not in endocrine progenitors. This was taken to indicate that the two lineages cannot be derived from a common pool of precursors after E12.5 (13). The integration of those Pdx1–Cre lineage tracing results with the expression findings presented in the current study instead support an alternate interpretation that is entirely consistent with a common endocrine/duct precursor lineage. Thus, the downregulation of Pdx1 in the Hnf1ß⁺ domain observed after E12.5 is expected to result in reduced Pdx1-promoter-driven recombination in duct and endocrine precursors in the above mentioned lineage tracing study. However, ß-cells derived from the Hnf1ß⁺ epithelium during this developmental stage selectively undergo strong Pdx1 activation, thus enabling a high rate of Cre-mediated recombination exclusively in the ß-cell lineage.

The lack of acinar-specific committed progenitor markers precludes us from addressing in greater detail if acinar cells are also derived from the Hnf1ß⁺ epithelium, or if they are derived from dedicated progenitors that do not go through the Hnf1ß⁺ ductal epithelium stage. The finding that early (E13.5) acini appear to bud off Hnf1ß⁺ duct-like epithelium raises the intriguing possibility that the former may be true (e.g. Fig. 1F and G).

The MODY5 phenotype suggests a regulatory role of Hnf1ß in pancreatic precursors
Because Hnf1ß is a transcriptional regulator its function may be relevant to the morphogenesis and differentiation of primitive pancreatic ductal cells. Cell-specific inactivation of Hnf1ß in the liver has demonstrated that it is essential for the development of a related tubular epithelial structure, the biliary duct (37). While experiments that produce early pancreatic inactivation of Hnf1ß are clearly required, the available human genetic data is consistent with the notion that Hnf1ß is also critical for pancreatic cell formation. Thus, patients with MODY5 diabetes mellitus exhibit mild exocrine cell dysfunction and a markedly reduced pancreatic volume (30). The specific expression of Hnf1ß in a precursor domain suggests that MODY5 could result from decreased generation of both endocrine and exocrine pancreatic cells during embryogenesis. Another mechanism compatible with the expression pattern of Hnf1ß is defective postnatal neogenesis, as many studies have suggested that endocrine cells can be derived from adult ducts (16,35,38–40). The intraislet Hnf1ß⁺ cell clusters shown here (Fig. 1N) can be regarded as a particularly interesting candidate pool of postnatal endocrine precursors.

This proposed role for Hnf1ß sharply contrasts with the notion that Hnf1, the MODY3 gene product, becomes enriched in acinar and endocrine cells as soon as these cells initiate differentiation, and is required to maintain a transcriptional network in differentiated ß-cells (20,25–27). Thus, despite the structural homology of Hnf1 and Hnf1ß and that both lead to a ß-cell haploinsufficient phenotype in humans, genetic and expression analysis suggest that many of their critical functions most likely occur during distinct stages of pancreatic development (18,19,30).

Tcf2 (Hnf1ß) is downstream of Hnf6 in early pancreatic epithelial cells
Hnf6 is the only known transcription factor, aside from Ngn3, whose null mutant phenotype exhibits defective generation of Ngn3 and endocrine cells. Hnf6^-/- embryos also exhibit pancreatic hypoplasia and disturbed duct morphogenesis (17,33). On the other hand, Hnf6 is required for the development of the biliary duct epithelium, and this effect has been linked to a direct regulation of the Tcf2 (Hnf1ß) gene by Hnf6 in biliary ducts (34).

In this study we have shown that early pancreatic duct epithelial cells in Hnf6^-/- embryos exhibit defective Hnf1ß expression, thus linking two key pancreatic transcriptional regulators in a common genetic hierarchy. The finding that Hnf1ß is specifically expressed in presumptive precursor cells, together with the pancreatic defect of heterozygous TCF2 (HNF1ß) mutations, suggests that Hnf1ß-deficiency may be instrumental in the pancreatic phenotype observed in Hnf6^-/- embryos.

	MATERIALS AND METHODS

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Animals, tissue isolation, DNA binding and RNA analysis
Microdissected pancreas or whole embryos were obtained from time-mated pregnant mice and fixed in 4% paraformaldehyde for immunostaining or immediately processed for nuclear extracts or RNA analysis (25,34,41). BrdU labeling was performed by i.p. injection of 50 mg/kg 2 h prior to animal dissection. Hnf6^-/- mice have been described previously (17). Pancreatic islets were purified using a Histopaque gradient and further selected under a stereomicroscope (41). Electrophoretic mobility shift assays (EMSA) were carried out as described previously (25). Real-time PCR was performed with SYBR Green PCR Core Reagents (Perkin Elmer) on a MyiQ cycler(Bio-Rad). Hnf1ß mRNA was quantitated in triplicate in four control and four Hnf6^-/- samples after normalization to ß-actin mRNA. Primers are available upon request.

Immunofluorescence
This was performed as described (25,41) except that tyramide signal amplification (TSA^TM Fluorescence Systems, PerkinElmer) was employed for anti-Hnf6 and anti-Hnf1ß antisera following the manufacturer's instructions. For double or triple immunofluorescence staining with the non-amplified primary antibodies was performed after the enzymatic amplification reaction. Images were collected using a Leica TCS 4D confocal microscope.

Antisera
Rabbit antisera directed against Hnf1 peptides GLIEEPTGDELPTK and EASSEPGLHEPPSPA were immunoaffinity purified using CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) chromatography. Antibodies used were as follows (titer and source in brackets): guinea pig anti-insulin (1 : 5000; Chris Van Schravendijk), mouse anti-E-Cadherin (1 : 200, transduction labs), rabbit anti Hb9 (1 : 1000, Samuel Pfaff) , rabbit anti-idx1 (Pdx1; 1 : 1000; Joel Habener), rabbit anti-nkx6.1 (1 : 1000; Ole Madsen), rabbit anti-p48 (1 : 100, Francisco X. Real), goat anti-Hnf1ß (1 : 100, Santacruz), rabbit anti-HNF6 (1 : 300, Santacruz), rabbit anti-glucagon (1 : 200 Dako), mouse anti-nkx2.2, -Pax6, -isl1, and -BrdU (1 : 20, Developmental Hybridoma Bank). Rabbit and guinea-pig anti-Ngn3 were described previously (15). Secondary antibodies preabsobred for multiple labeling purposes were obtained from Jackson Immunolabs and Molecular Probes.

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
We are indebted to C. VanSchravendijk (Vrije Universiteit Brussel), O. Madsen (Hagedorn Research Institute), J. Habener (Harvard Medical School), F.X. Real (Institut Municipal d'Investigacions Biomediques), and S. Pfaff (Salk Institute) for antisera and J. Miyazaki (Osaka University) for Min6 cells. Antibodies were also obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa. Imaging support from the Cell Biology department and the confocal microscopy unit (SCT) of the University of Barcelona School of Medicine is acknowledged. This work was supported by the Juvenile Diabetes Research Foundation (JDRF 1-2002-21), Ministerio de Ciencia y Tecnología (SAF01-2457), a group of excellence aid from Instituto de Salud Carlos III (GO3/212), and the Belgian State Program on Interuniversity Poles of Attraction, the D. G. Higher Education and Scientific Research of the French Community of Belgium, and the Fund for Scientific Medical Research (Belgium). C.E.P. is Senior Research Assistant of the FNRS.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Endocrinology, Hospital Clínic i Universitari, Villarroel 170, Barcelona-08036, Spain. Tel: +34 2275400(3028); Fax: +34 934516638; Email: jferrer{at}medicina.ub.es

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