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The Journal of Immunology, 2003, 171: 1297-1303.
Copyright © 2003 by The American Association of Immunologists

The Onecut Transcription Factor Hepatocyte Nuclear Factor-6 Controls B Lymphopoiesis in Fetal Liver1

Caroline Bouzin*, Frédéric Clotman*, Jean-Christophe Renauld{dagger}, Frédéric P. Lemaigre* and Guy G. Rousseau2,*

* Hormone and Metabolic Research Unit, Université Catholique de Louvain and Institute of Cellular Pathology, Brussels, Belgium; and {dagger} Experimental Medicine Unit, Ludwig Institute for Cancer Research, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Mouse genetic models have helped to identify transcription factors that are expressed by hemopoietic cells and control their differentiation into lymphoid cells. However, little is known on transcription factors that are involved in this process, but are expressed in nonhemopoietic cells of the microenvironment. We show in this study that inactivation of the gene coding for hepatocyte nuclear factor-6 (HNF-6) in mice led to B lymphopenia in the bone marrow and spleen. This phenotype disappeared shortly after birth when fetal B lymphopoiesis is no longer active, pointing to a defect in fetal liver. Indeed, the number of B cells was decreased in this organ as well. An analysis of B cell developmental markers in fetal liver cells showed that B lymphopoiesis was impaired just beyond the pre-pro B cell stage. Hemopoietic cells from hnf6-/- fetal liver could reconstitute the lymphoid system when injected into scid mice. Because parenchymal cells, but not hemopoietic cells, expressed hnf6 in normal liver, we concluded that HNF-6 controls B lymphopoiesis in fetal liver and that HNF-6 exerts this control indirectly by acting in parenchymal cells. The involvement, in the B cell defect of hnf6-/- fetuses, of genes known to exert such an indirect control was ruled out by expression analysis, including microarrays, and by in vivo rescue experiments. This work identifies HNF-6 as the first noncell-intrinsic transcription factor known to control B lymphopoiesis specifically in fetal liver.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The lymphoid system provides the basis of cellular and humoral immunity. To understand immunodeficiencies and to progress in cell therapy, one has to elucidate the molecular mechanisms of lymphocyte differentiation from hemopoietic progenitors. This process involves two types of gene expression programs that are triggered by transcription factors and control commitment, differentiation, proliferation, and cell survival. The first program, which is well documented, is endogenous to the lymphoid cells (1, 2). The second program occurs in nonhemopoietic cells, which establish with the lymphoid precursors a cross-talk that is essential for lymphopoiesis (3). Although a number of proteins that are secreted or involved in cell-cell contact to regulate B lymphopoiesis have been described (4), their regulation is poorly understood. The identification of transcription factors that control this second gene program is therefore an important step forward.

In the mouse, pluripotent progenitors become committed to the B lineage in fetal liver at embryonic day (e)3 14.5 and undergo a multistep process of differentiation to become mature B cells (5). B lymphopoiesis persists in liver for 2 wk after birth (6). Later on, B lymphopoiesis occurs in the bone marrow, wherefrom B cells migrate to the spleen and lymph nodes. Differences in gene expression programs controlling B cell development are observed between these ontogenic sites (3, 7), probably due to differences in the microenvironment (8). Thus, parenchymal cells that are specific to fetal liver are expected to play a unique role in B lymphopoiesis. At e14.5, these cells consist of hepatoblasts, which begin to differentiate into hepatocytes or into biliary epithelial cells (BEC). An influence of parenchymal cells from e14.5 murine liver on hemopoiesis has indeed been demonstrated (9, 10). These cells bear receptors for oncostatin M (OSM), a cytokine that is released by hemopoietic cells (11) and reportedly acts indirectly on B cell development (12). However, the mechanism by which hepatoblasts act in response to OSM remains unknown. In human fetal liver stromal cell-derived factor-1 (SDF-1), a cytokine known to promote B cell development, is secreted by BEC in the vicinity of B cell precursors (13, 14, 15). This suggests that BEC also play a role in B lymphopoiesis. Thus, transcription factors that are expressed in fetal hepatoblasts or in BEC might control B cell development.

Hepatocyte nuclear factor-6 (HNF-6) (16) is the prototype of the Onecut class of homeodomain transcription factors, whose members contain a single cut domain and a divergent homeodomain (17). HNF-6 is expressed in mouse liver and pancreas as soon as these organs develop from the foregut endoderm, in which HNF-6 is also expressed (18, 19). The role of HNF-6 in the liver and in the pancreas is illustrated by the phenotype of hnf6-/- mice. These mice exhibit defects in the expression of genes by hepatocytes (20) and in the differentiation of the BEC (19) and of the endocrine pancreas (21). Unexpectedly, inactivation of the hnf6 gene also affects the spleen, which, in newborn mice, is smaller than normal. This raised the possibility that HNF-6 also controls lymphopoiesis. The aim of this work was to investigate this hypothesis.

We report in this work that the splenic anomaly of hnf6-/- mice is due to a decrease of the B cell population and that this results from a defective B lymphopoiesis in fetal liver. Our data show that B lymphopoiesis involves a HNF-6-dependent control exerted by liver parenchymal cells on B cell progenitors. These results provide the first example of a liver-specific, noncell-autonomous, hemopoietic defect and show that hnf6 knockout mice are a new model to study the cross-talk between hemopoietic cells and cells of their microenvironment in liver.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals

The mice, raised in our animal facility, were hnf6+/+ or hnf6+/- (controls) or hnf6-/- (knockout) mice (129Sv/Swiss background), in which the hnf6 gene had been inactivated by homologous recombination (21). The cyto-Met transgenic mice (22) were kindly provided by M. Weiss (Pasteur Institute, Paris, France) and M. Tripodi (Universita La Sapienza, Rome, Italy). Mice were genotyped by PCR on tail DNA. The transgenic and the scid mice were housed in sterile cages in pathogen-free animal quarters and were given autoclaved food and water. For reconstitution of the lymphoid system in scid mice, livers from control or hnf6-/- fetuses were harvested aseptically. Cells obtained as described below were resuspended in 100 µl of PBS and injected i.p. to newborn mice. The spleen of scid mice injected with cells from e12.5, e14.5, or e15.5 fetuses was examined after 4.5, 7, or 6 wk, respectively. The bone marrow of scid mice injected with cells from e12.5 fetuses was examined after 4.5 wk. All the mice were treated according to the principles of laboratory animal care of the University animal welfare committee.

Flow cytometric analysis

Cell suspensions of spleen and of liver were prepared by dissociation of the organs with tweezers and then with a Pasteur pipette in culture IMDM or DMEM medium (Life Technologies, San Diego, CA) containing 3% FBS. Bone marrow cells were obtained by flushing two femora and two tibiae with PBS. Erythrocytes were lysed with a 0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2–7.4 solution (spleen, bone marrow, and e16.5 fetal liver) or were eliminated by centrifugation in Ficoll (e17.5 and e18.5 fetal liver). Cells were filtered through a nylon membrane (80-µm pores) and counted. For flow cytometric analysis, cell suspensions were labeled with a biotin-conjugated anti-B220 mAb (clone RA3-6B2) and then with PE-conjugated streptavidin (BD PharMingen, San Diego, CA), or with a FITC-conjugated anti-CD43 mAb (clone S7); or else with an APC-conjugated anti-B220 mAb (clone RA3-6B2), with a PE-conjugated anti-BP-1 mAb, with a biotin-conjugated anti-heat-stable Ag (HSA) mAb (clone 30-F1), and then with Red670-conjugated streptavidin (Invitrogen, San Diego, CA). All Abs were from BD PharMingen. Negative controls for double or triple labeling were cells incubated with the respective single or double label. Labeled cells were fixed in 1.25% paraformaldehyde, and fluorescence intensity was measured using a FACScan apparatus (BD Biosciences, San Diego, CA) and was analyzed by the CellQuest software (BD Biosciences).

RNA isolation and RT-PCR

Total RNA was isolated from cells with the High Pure RNA Isolation kit and from liver with the Tri-Pure Isolation Reagent, both from Roche (Mannheim, Germany). Total RNA was reverse transcribed for 1 h at 37°C with random primers (Life Technologies), and PCR was performed in the linear range of amplification with the following primers: 5'-CAGCACCTCACGCCCACCTC-3' and 5'-CTTCCCATGTTCTTGCTCTTTCC-3' for HNF-6 (446 and 521 bp (17)), 5'-ATGACTCCCTCCATCTCATGG-3' and 5'-CTGTGGCAATGCTCACTGGGG-3' for {alpha}1-AT (230 bp), 5'-TCGTATTCCAACAGGAGG-3' and 5'-AGGCTTTTGCTTCACCAG-3' for {alpha}FP (173 bp), 5'-TCCTCGGACCATCAGGACAG-3' and 5'-CCTGTTGATGGAGCTGACGC-3' for Pax5 (219 and 384 bp), 5'-CTGACCCATAGAGTTCATCC-3' and 5'-TCATGTGGACAGAAACATTG-3' for OSMR (177 bp), 5'-TTACGGACCCAACCATGAGC-3' and 5'-TCTGACGTCCCTAGATTAGC-3' for c-Met (209 bp), 5'-CTTCACCAGCTCCACCACAG-3' and 5'-CTTCCTTTCCTTCTCCTTTGC-3' for insulin-like growth factor-1 (IGF-1) (335 bp), 5'-GAGCTTGTTGACACGCTTCAGTTTG-3' and 5'-GTTTGGCCTCTCTGAACTCTTTGAG-3' for IGF-2 (355 bp), 5'-ACGCCAAGGTCGTCGCCGTGCTGG-3' and 5'-GTTAGGGTAATACAATTCCTTAGA-3' for SDF-1 (539 bp), 5'-CTTGTTCTGCTGCCTGTCAC-3' and 5'-CTTGCGAGCAGCACGATTTAG-3' for IL-7 (209 bp), 5'-GGGCTGAAGAATACTCCACC-3' and 5'-GAGCCCCAGCTGATGACTCC-3' for factor XII (231 bp), 5'-AGATGCATCCTTTCCTGACC-3' and 5'-CACAGGTGTTCTTCACCAGC-3' for inhibitor of the HGF activator (HAI) (191 bp). The amount of RNA was controlled by amplification of a 190-bp-long TATA box-binding protein (TBP) cDNA fragment with the primers 5'-ACCCTTCACCAATGACTCCTATG-3' and 5'-ATGATGACTGCAGCAAATCGC-3'.

Microarray analysis

Total RNA was extracted from the liver of hnf6+/+ or hnf6-/- fetuses at e12.5 using the Tri-Pure Isolation Reagent (Roche, Basel, Switzerland), according to manufacturer’s instructions. RNA extracted from three livers of the same phenotype were pooled. For first strand cDNA synthesis, 5 µg of RNA was mixed with 2 µg of a HPLC-purified anchored oligo(dT) + T7 promoter (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24(ACG)-3') (Eurogentec, Seraing, Belgium), 40 U of RNaseOUT (Invitrogen), and 0.9 M of D+ trehalose (Sigma-Aldrich, Bornem, Belgium) in a total volume of 11 µl, and heated at 75°C for 5 min. To this mixture, 4 µl of 5x first strand buffer (Invitrogen), 2 µl of 0.1 M DTT, 1 µl of 10 mM dNTP mix, 1 µl of 1.7 M D+ trehalose (Sigma-Aldrich), and 200 U of SuperScript II (Invitrogen) were added. The samples were incubated at 37°C for 5 min, 45°C for 10 min, 10 cycles at 60°C for 2 min, and at 55°C for 2 min. To the first strand reaction mixture, 103.8 µl of water, 33.4 µl of 5x second strand synthesis buffer (Invitrogen), 3.4 µl of 10 mM dNTP mix, 1 µl of 10 U/µl Escherichia coli DNA ligase (Invitrogen), 4 µl of 10 U/µl E. coli DNA polymerase I (Invitrogen), and 1 µl of 2 U/µl E. coli RNase H (Invitrogen) were added, and incubated at 16°C for 2 h. Antisense RNA synthesis was done with the AmpliScribe T7 high yield transcription kit (Epicentre Technologies, Madison, WI) in a total volume of 20 µl, according to the manufacturer’s instructions. The RNA was purified with RNeasy purification kit (Qiagen, Hilden, Germany). From this RNA, 5 µg was labeled by reverse transcription using random nonamer primers (Genset, Evry, France), 0.1 mM d(G/T/A)TPs, 0.05 mM dCTP (Amersham BioSciences, Little Chalfont, Buckinghamshire, U.K.), 0.05 mM Cy3-dCTP or Cy5-dCTP (Amersham BioSciences), 10 mM DTT, and 200 U of SuperScript II (Invitrogen) in first strand buffer (20 µl total volume). The RNA and primers were denatured at 75°C for 5 min and cooled on ice before adding the remaining reaction components. After 2 h of incubation at 42°C, mRNA was hydrolyzed in 250 mM NaOH for 15 min at 37°C. The sample was neutralized with 10 µl of 2 M MOPS, and the synthesized double-stranded cDNA was purified with Qiaquick (Qiagen). The mouse gene set consisted of five separate microarrays containing in total 21,492 cDNA fragments (www.microarrays.be). The clone set was composed from the 6K collection of Incyte (Mouse Gem I; Incyte, Palo Alto, CA) and from the 15K collection of the National Institute of Aging (http://lgsun.grc.nia.nih.gov). Unique cDNAs (4, 300) were spotted in duplicate on type VIIstar silane-coated slides (Amersham BioSciences). The cDNA inserts were PCR amplified using M13 primers, purified with Multi-Screen-PCR plate (Millipore, Brussels, Belgium), and arrayed on the slides using a Molecular Dynamics Generation III printer (Amersham BioSciences). Slides were blocked in 3.5% SSC, 0.2% SDS, and 1% BSA for 10 min at 60°C. The probes were resuspended in 30 µl of hybridization solution (50% formamide, 5x SSC, 0.1% SDS, 100 µg/ml salmon sperm DNA) and prehybridized with 1 µl of poly(dT) (1 mg/ml) at 42°C for 30 min to block hybridization on the poly(A)/T tails of the cDNA on the arrays. Mouse Cot-1 DNA (Invitrogen) was added (1 mg/ml) to the mixture and placed on the array under a glass coverslip. Slides were incubated for 18 h at 42°C in a humid hybridization cabinet (Amersham BioSciences). Posthybridization washings were performed for 10 min at 56°C in 1x SSC, 0.1% SDS, twice for 10 min at 56°C in 0.1x SSC, 0.1% SDS, and for 2 min at 37°C in 0.1x SSC. Arrays were scanned at 532 and 635 nm using a Generation III scanner (Amersham BioSciences). Image analysis was performed with ArrayVision (Imaging Research, Ontario, Canada). Spot intensities were measured as artifact-removed total intensities (ARVol) minus median intensity of the local background of each spot.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Depletion of B cells in the spleen and bone marrow of hnf6-/- mice

Two weeks after birth, at a time when spleen development is complete (6), the spleen of hnf6-/- mice was smaller than that of control littermates. Spleen histology showed typical B cell-containing follicles in control mice, but only a few small follicles in hnf6-/- mice, pointing to a B cell defect. To quantify this defect, splenocytes were analyzed by FACS after labeling with an anti-B220 Ab. The B220 surface Ag is a marker of all cells of the B lineage, from primitive B cell progenitors to mature B lymphocytes (5). As shown in Table I and Fig. 1A, the proportion of B cells in the spleen was decreased by 60% in hnf6-/- mice. The size and granularity profiles of the splenocytes were similar in hnf6-/- and control mice, indicating that this decrease did not result from cell death. The decrease in the proportion of B cells was not due to an increased number of the other cell types, because the total number of splenocytes was also decreased by 60% in the knockout mice (Table I). In contrast, 5 wk after birth, the spleen of hnf6-/- mice contained as many B220+ cells as that of control mice (Table I and Fig. 1A). Similar observations were made in the bone marrow. Two weeks after birth, only 10% of the bone marrow cells belonged to the B cell lineage in hnf6-/- mice, as compared with 30% for control mice (Table I and Fig. 1A). In 5-wk-old mice, the proportion of bone marrow B220+ cells was no longer affected by the hnf6 knockout (Table I and Fig. 1A).


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  		Table I. Effect of inactivating the hnf6 gene on B lymphocytesa

 


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  		FIGURE 1. Inactivation of the hnf6 gene decreases the proportion of cells of the B lineage in the spleen and bone marrow of 2-wk-old, but not 5-wk-old, mice and in e17.5 and e18.5, but not e16.5, fetal liver. FACS tracings show representative experiments (see Table I) for hemopoietic cells selected with the anti-B220 Ab. The percentages of B220+ cells are given. The gray area corresponds to the fluorescence of cells not incubated with the Abs.

 
The depletion of B cells in 2-wk-old hnf6 knockout mice was confirmed with the cell surface marker IgM. The percentage of IgM+ splenocytes was 12.1 ± 2.0 SEM (n = 6) in hnf6 knockout mice as compared with 26.5 ± 2.6 SEM (n = 6) in control mice. In the bone marrow, the corresponding values were 3.5% ± 1.0 SEM (n = 5) for knockout mice and 8.2% ± 0.8 SEM (n = 4) for control mice. All these data showed that the lack of HNF-6 leads to B lymphopenia. They also showed that this defect is obvious at a time when the B cell population in bone marrow and spleen originates from the liver, but that the defect is no longer present when the bone marrow is the only site of hemopoiesis. These observations suggested that it is in fetal liver that HNF-6 is involved in B lymphopoiesis.

Defective B lymphopoiesis in the liver of hnf6-/- fetuses

To investigate the role of HNF-6 in B lymphopoiesis, we therefore studied B cells in the liver of hnf6-/- fetuses. We found that the proportion of B220+ cells in liver hemopoietic cells of hnf6-/- fetuses was less than that of control fetuses at e18.5 and e17.5 (Table I). Representative experiments are shown in Fig. 1B. However, the proportion of B220+ cells was not decreased in the liver of knockout fetuses at e16.5 (Table I and Fig. 1B). This pointed to a defect between e16.5 and e17.5 in the B cell differentiation program. To confirm this, we examined fetal liver B220+ cells for developmental markers. B cell maturation proceeds through the pre-pro B, pro B, pre B, and immature B stages (5). Transition from the pro B stage to the subsequent stages is characterized by the loss of the CD43 marker. We first used this marker to determine the maturation stage that B cells reach in fetal liver. We found that the percentage of CD43- cells in the B220+ population was only 0.5% ± 0.04 SEM (n = 4) in e17.5 fetuses and 2.2% ± 0.3 SEM (n = 4) in e18.5 fetuses, indicating that B cell maturation in the liver has not gone significantly beyond the pro B stage at the end of gestation. We therefore resorted to markers of earlier stages, namely HSA and BP-1. HSA-BP-1- (pre-pro B) cells differentiate into HSA+BP-1- (early pro B) cells, which become HSA+BP-1+ (late pro B) cells (23). The results of this analysis are shown in Table II and Fig. 2. In control fetuses at e16.5, these three subsets of B220+ cells were present, with very few cells in the third, more mature, fraction. At e17.5, there was a striking increase in the third fraction and a decrease in the first fraction (pre-pro B cells). In hnf6-/- fetuses at e16.5, the three cell populations were detected in the same proportions as in control fetuses, consistent with the lack of B lymphopenia at that stage. In contrast, the distribution of B220+ cells between the three fractions was abnormal in hnf6-/- liver at e17.5. There was an increased proportion in the first two fractions and a decreased proportion in the third fraction, as compared with control fetuses. These results showed that the absence of HNF-6 affects B lymphopoiesis just beyond the pre-pro B cell stage. We concluded that the absence of HNF-6 leads to B lymphopenia in fetal liver because of a differentiation defect, with consequences that are detectable after birth in the bone marrow and in the spleen as long as these organs depend on liver hemopoiesis.


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  		Table II. Effect of inactivating the hnf6 gene on B lymphocyte differentiation in fetal livera

 


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  		FIGURE 2. Inactivation of the hnf6 gene affects the differentiation of B lymphocytes just beyond the pre-pro B cell stage in fetal liver. B220+ cells were analyzed for the expression of HSA and BP-1 at the embryonic days indicated. The percentages of HSA-BP-1-, HSA+BP-1-, and HSA+BP-1+ cells among B220+ cells are given for a representative FACS experiment (see Table II).

 
The defective B lymphopoiesis in hnf6-/- fetal liver is noncell autonomous

To investigate further how HNF-6 controls B lymphopoiesis in liver, we determined the cellular distribution of HNF-6 mRNA in fetal liver. RNA was extracted for RT-PCR from the pool of isolated liver cells and from hemopoietic cells sorted out from this pool by FACS on the basis of cell size and granularity. The efficiency of these procedures was assessed by measuring cell-specific gene products. The pool of isolated liver cells expressed the {alpha}-fetoprotein and {alpha}1-antitrypsin genes, as expected for hepatoblasts and hepatocytes (24, 25), and the pax5 gene, which is a B cell marker (26) (Fig. 3A). Hemopoietic cells, sorted out from the pool of isolated liver cells, expressed only the pax5 gene, indicating that this fraction was not contaminated by parenchymal cells. As to HNF-6 mRNA, we detected it in the nonfractionated liver cell population, but not in the hemopoietic cell fraction (Fig. 3A). This showed that the impairment of B lymphopoiesis in the hnf6-/- mice is not due to a primary defect in the hemopoietic compartment.



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  		FIGURE 3. HNF-6 is expressed in fetal liver parenchymal cells, but not hemopoietic cells, and liver cells from hnf6-/- fetuses can reconstitute B lymphopoiesis in scid mice. A, Total RNA was extracted at e15.5 from the total pool of liver cells (L) isolated from control fetuses, or from the hemopoietic cells (H) sorted out from that pool by FACS on the basis of size and granularity. The mRNA of HNF-6, of the parenchymal cell markers {alpha}1-antitrypsin ({alpha}1-AT) and {alpha}-fetoprotein ({alpha}FP), of the B cell marker Pax-5, and of TBP as a reference were detected by RT-PCR, followed by visualization on an agarose gel stained with ethidium bromide. B, Representative experiments of FACS analysis of the spleen of scid mice after reconstitution with liver cells taken from control or hnf6-/- fetuses at the embryonic days indicated. The percentages refer to B220+ cells in the splenocytes. Values for control normal mice were 6.7 and 9.4% at e12.5, 31.5% ± 5.2 SEM (n = 9) at e14.5, and 40.0% ± 4.6 SEM (n = 5) at e15.5.

 
These observations supported the hypothesis that B cell development in fetal liver is regulated indirectly by an HNF-6-dependent control exerted by nonhemopoietic cells. If this is so, hemopoietic cells from hnf6-/- mice should be as competent as hemopoietic cells from control mice to differentiate into B cells, provided they are in a hnf6+/+ environment. To test this hypothesis in vivo, we performed adoptive transfer experiments in scid mice. These mice lack lymphocytes, but can reconstitute lymphopoiesis from foreign hemopoietic cells, which they tolerate because of their immunodeficiency (27). Cells from the liver of hnf6-/- or control fetuses were injected to newborn scid mice, and the spleen of the injected mice was examined. Hemopoietic cells from hnf6-/- mice were as competent as those from control fetuses to reconstitute the B cell population in the spleen (Fig. 3B) and in the bone marrow (data not shown). These experiments confirmed that the B lymphopenia of hnf6-/- mice is not cell autonomous, but is due to the absence of HNF-6 in hepatic cells other than hemopoietic cells.

Lack of involvement of signaling pathways known to control B lymphopoiesis

To address the mechanism by which HNF-6 controls B cell lymphopoiesis in fetal liver, we first analyzed gene products that are expressed by nonhemopoietic cells in fetal liver and are known to control B lymphopoiesis. The five candidate genes expressed by parenchymal cells were those coding for the OSM receptor (11, 12), the hepatocyte growth factor (HGF) receptor c-Met (28, 29), and the growth factors IGF-I and IGF-II (30, 31) and SDF-1 (13, 15). Their expression was determined by RT-PCR on liver RNA taken from hnf6-/- or control fetuses at five stages before detection of the B lymphopenia. However, we did not find changes in the expression in any of these genes in hnf6-/- fetuses as compared with controls (Fig. 4A).



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  		FIGURE 4. Gene expression in the liver of hnf6-/- or control fetuses and study of the role of the HGF pathway in the hemopoietic phenotype of hnf6-/- mice (see text for details). A and B, Total RNA was extracted from the liver of control (c) or hnf6-/- (ko) fetuses at the embryonic days indicated (A) or at e12.5 (B). The mRNA for the factors indicated, with TBP mRNA as a reference, were detected by RT-PCR, followed by visualization on an agarose gel stained with ethidium bromide. B, FXII, factor XII. C, FACS analysis of liver hemopoietic cells obtained at e17.5 from fetuses of the indicated genotype. The percentage of B220+ cells is given for the representative experiment shown and as means ± SEM for the number of mice indicated in parentheses.

 
The differentiation of hepatoblasts into hepatocytes exerts a feedback control on hemopoiesis (10). We therefore explored the idea that inactivation of the hnf6 gene perturbs the time course of hepatocyte maturation, thereby hampering this feedback. Because the expression of {alpha}1-antitrypsin progressively increases during hepatocyte differentiation, we determined its pattern in hnf6-/- liver as a marker of hepatocyte maturation. As expected, the expression of {alpha}1-antitrypsin in control liver increased with time during liver development, as did expression of the OSMR (Fig. 4A). However, this increase was not altered in hnf6-/- liver (Fig. 4A), ruling out the hypothesis that hepatocyte maturation is perturbed in the hnf6-/- fetuses.

Another possibility was that HNF-6-expressing cells in fetal liver act on hemopoietic cells indirectly via stromal cells. It was conceivable that an aberrant development of the fetal hepatic architecture in hnf6-/- fetuses perturbs the lymphopoietic potential of supporting fetal stroma. This was unlikely, as the histology of the liver of hnf6-/- fetuses appeared to be normal, except for the premature differentiation of hepatoblasts into BEC around the branches of the portal vein (19) (data not shown). To further document a possible involvement of the stromal cells, we determined the expression of IL-7. This cytokine is produced by stromal cells in the bone marrow, where it is required for B cell differentiation in the adult, as demonstrated by studies on IL-7-/- (32, 33) and on IL-7R-/- (34) mice. To our knowledge, the profile of expression of IL-7 had not been described in mouse fetal liver. Therefore, we first determined this profile in control fetuses. IL-7 mRNA was detectable as early as e12.5, just before commitment of pluripotent progenitor cells to the B lineage (Fig. 4A). However, we did not detect differences in IL-7 expression between hnf6-/- and control fetuses at any of the stages tested (Fig. 4A). We concluded that inactivation of the hnf6 gene did not lead to detectable changes in the extrinsic pathways possibly involved in B lymphopoiesis in fetal liver.

Microarray analysis

We therefore undertook a large-scale search for changes in gene expression in the liver of hnf6-/- fetuses by microarray analysis. Hybridization of fetal liver cDNA to 21,492 clones showed that expression of ~100 mRNAs was up-regulated and of ~150 mRNAs was down-regulated in hnf6-/- liver as compared with control liver. Among these products, an examination of known genes pointed to two candidates for controlling lymphopoiesis not yet tested, both of them being involved in the HGF pathway. There was a 75% decrease in mRNA for Hageman factor (coagulation factor XII), a serine protease that catalyzes the cleavage of pro-HGF into HGF (35) and an increased expression (85%) of the HAI. These changes were confirmed by RT-PCR (Fig. 4B). As HAI is probably produced by BEC, this increase in HAI expression was consistent with the increased number of BEC observed in hnf6-/- fetal liver (19). It may seem surprising that the inactivation of the hnf6 gene affects the expression of proteins that control the same signaling pathway, but are structurally unrelated. However, this is consistent with the recent observation that the organization of gene networks to which HNF-6 participates entails the forward and feedback control of activators and inhibitors of a given developmental program (36). We therefore investigated the possibility that a combined reduction of HGF activation and of c-met activity in fetal liver might explain the defect in B lymphopoiesis of hnf6-/- mice.

To this end, we designed an experiment to rescue the B cell phenotype resulting from inactivation of the hnf6 gene. We used transgenic mice (cyto-Met) that express in liver a constitutively active form of c-Met, so that their HGF pathway is turned on, irrespective of events upstream of the receptor. In these mice, a cDNA coding for a truncated form of human c-Met is expressed under the control of {alpha}1-antitrypsin regulatory sequences (22). As shown in Fig. 4A, the {alpha}1-antitrypsin promoter is active in liver cells from both control and hnf6-/- fetuses as early as e12.5. We crossed hnf6+/- mice with cyto-Met mice to obtain cyto-Met/hnf6-/- mice. Their phenotype was indistinguishable from that of hnf6-/- mice. The percentage of liver hemopoietic cells that express the anti-B220 Ab was determined at e17.5, namely at a time when the B cell lineage in fetal liver is fully established. The results are shown in Fig. 4C. As expected, hnf6-/- mice had less B cells than control mice. Constitutive expression of c-Met per se did not influence the proportion of B cells, as this value (45.6% ± 7 SEM, n = 4) was the same in cyto-Met/hnf6+/+ mice as in control mice. However, the cyto-Met/hnf6-/- mice had no more B cells than hnf6-/- mice (Fig. 4C). This failure to rescue the B cell defect characterized in this study in hnf6-/- mice did not support the suggestion that it results from a defect in the HGF pathway.

We conclude from the present work that the transcription factor HNF-6 controls B lymphopoiesis in liver. Our data indicate that HNF-6 exerts this control via an indirect mechanism by which liver parenchymal cells would express on their membrane or release in their environment factor(s) that acts on hemopoietic precursors. Indeed, we show that the lymphopenia in hnf6-/- mice is observed only during the course of liver hemopoiesis, that hnf6 is not expressed in liver hemopoietic cells, and that hemopoietic cells from hnf6-/- mice differentiate normally in a hnf6+/+ background. To our knowledge, HNF-6 is the first noncell-intrinsic transcription factor identified as controlling B lymphopoiesis specifically in fetal liver. The original mouse genetic model for an extrinsic defect in liver B lymphopoiesis described in this work should provide new leads for elucidating the cross-talk beween parenchymal cells and hemopoietic cells at the onset of lymphopoiesis.


   Acknowledgments
 
We thank A. Tonon, A. Vink, G. Warnier, and Y. Peignois for technical help, and P. Van Hummelen for advice on microarray experiments. cyto-Met mice were a kind gift from M. Weiss and M. Tripodi.


   Footnotes
 
1 This work was supported by grants from the Belgian State Program on Interuniversity Poles of Attraction, from the D. G. Higher Education and Scientific Research of the French Community of Belgium, from the Human Frontier Science Program (Grant N° GR0303/2000-M), and from the Fund for Scientific Medical Research (Belgium). C.B. held a fellowship from the Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture. Back

2 Address correspondence and reprint requests to Dr. Guy G. Rousseau, Université Catholique de Louvain-Institute of Cellular Pathology 7529, 75 Avenue Hippocrate, B-1200 Brussels, Belgium. E-mail address: rousseau{at}horm.ucl.ac.be Back

3 Abbreviations used in this paper: e, embryonic day; BEC, biliary epithelial cell; HAI, inhibitor of HGF activator; HGF, hepatocyte growth factor; HNF-6, hepatocyte nuclear factor-6; HSA, heat-stable Ag; IGF, insulin-like growth factor; OSM, oncostatin M; SDF, stromal cell-derived factor-1; TBP, TATA box-binding protein. Back

Received for publication November 21, 2002. Accepted for publication May 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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