Development 113, 589-599 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

589

Hepatic Nuclear Factor 1 (HNF1) shows a wider distribution than products

of its known target genes in developing mouse

MARTABLUMENFELD1, MARTINE MAURY2, TANGUY CHOUARD1, MOSHE YANTV1 and

HUBERT CONDAMINE2

lUnit6 des Virus Oncogenes, UA 1149 du CNRS, De~partement des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex15, France2Uniti de Ginitique Cellulaire, UA 1148 du CNRS, Dipartement de Biologie Moliculaire, Institut Pasteur, 25 rue du Dr Roux, 75724 Pariscedex 15, France

Summary

Hepatic nuclear factor 1 (HNF1) is a highly divergedhomeoprptein that is crucial for transcription of manyliver-specific genes including albumin. In particular, aminimal promoter, consisting of an HNFl-binding-siteand a TATA box, is highly active only in hepatoma celllines. The expression of the HNF1 and albumin genes hasbeen examined in mouse embryos by in situ hybridiz-ation. At 10.5 days of gestation, the HNF1 mRNA wasdetected in both the hepatic primordia and visceralendoderm of the yolk sac whereas the albumin transcriptwas present only in the nascent liver. At later stages ofdevelopment, HNF1 was detected in liver, in the

epithelial cells of most of the digestive tract and in thecortex of the kidney, whereas albumin was again foundonly in the liver. The presence of HNF1 protein in adultkidney was demonstrated by immunodetection in gel-retardation assays and western blot analysis. Theseexperiments show that, even though the HNF1 homeo-protein is essential for expression of many liver-specificgenes, it cannot, by itself, force high expression levels ofthese genes, in non-hepatic tissues.

Key words: Tissue-specific transcription, homeodomainprotein, in situ hybridization, albumin.

Introduction

HNF1 (Hepatocyte Nuclear Factor 1; also named APF,LFB1 or HP1) has been described as a sequence-specific DNA-binding protein from rat liver thatinteracts with promoter elements present in many liver-restricted genes, such as albumin, alpha- and beta-fibrinogen, alpha-1-antitrypsin, alpha-fetoprotein, pyr-uvate kinase, transthyretin and aldolase B, amongothers (Cereghini et al. 1988; Schorpp et al. 1988;Courtois et al. 1987, 1988; Baumhueter et al. 1988; DeSimone et al. 1987; Hardon et al. 1988; Jose-Estanyoland Danan, 1988; Vaulont et al. 1989; Costa etal. 1988;Tsutsumi et al. 1989; Frain et al. 1990; Toniatti et al.1990; Rodriguez de Cordoba et al. 1991; reviewed inRey-Campos and Yaniv, 1991). For most of thesegenes, it was shown that the HNFl-binding site isessential for the transcriptional activity of the corre-sponding promoter (Godbout et al. 1986; De Simone etal. 1987; Courtois et al. 1987; Schorpp et al. 1988;Cereghini et al. 1988; Lichtsteiner and Schibler, 1989;Herbomel et al. 1989; Tranche et al. 1989; Toniatti et al.1990). Titration of the factor in liver nuclear extracts,using an excess of specific oligonucleotides containingan HNFl-binding site, abolishes in vitro transcription

from the albumin promoter (Cereghini et al. 1988). Inaddition, a highly efficient, liver-specific promoter canbe obtained with only a TATA box and an HNF1 site(Tronche et al. 1989; Ryffel et al. 1989; Maire et al.1989). Finally, spleen nuclear extracts that are inactivein transcription of the mouse albumin promoter in vitrocan be rendered active by addition of purified rat HNF1(Lichtsteiner and Schibler, 1989).

While HNF1 could be detected in liver or differen-tiated hepatoma cells expressing liver-specific func-tions, a distinct DNA-binding activity, named vAPF orvHNFl, was detected in dedifferentiated hepatomasand somatic hybrids that had lost the capacity to expressliver-specific markers (Cereghini et al. 1988; Baum-hueter et al. 1988). The DNA-binding properties ofHNF1 and vHNFl were shown to be indistinguishableby several criteria, thus suggesting a close relationshipbetween the two factors at least in their DNA-bindingdomain (Cereghini et al. 1988). These results and thecorrelation between the presence of HNF1 and thedifferentiated state led us to postulate a key role forHNF1 during hepatocyte differentiation (Cereghini etal. 1988).

Recently, HNF1 cDNA clones from rat liver and rathepatoma cells were independently obtained (Frain et

590 M. Blumenfeld and others

al. 1989; Baumhueter et al. 1990; Chouard et al. 1990).Sequence analysis showed that the protein is 628 aminoacids long and presents in its middle portion a clearhomology to the DNA-recognition helix of homeopro-teins (Affolter etal. 1990; Ottinger al. 1990; Kissinger etal. 1990). Alignment of the HNFl homeodomain withall reported homeodomain sequences (Scott et al. 1989)showed that the HNFl homeodomain is highly diver-gent, lacking conserved residues inside and outside thethird helix (Chouard et al. 1990). A better alignmentcan be obtained by the introduction of a loop of 24amino acids between helices 2 and 3, instead of thecanonical 3 amino acids in the turn (Finney, 1990;Baumhueter etal. 1990; Chouard etal. 1990; Nicosia etal. 1990). In addition, a weak homology with the POUA box of POU proteins (Herr et al. 1988) can be foundupstream of the HNFl homeodomain (Frain et al. 1989;Baumhueter et al. 1990; Chouard et al. 1990); however,both the POU-specific box and the POU homeobox aretoo poorly conserved to consider HNFl as a member ofthe POU family of transcription factors (Chouard et al.1990).

The functional relevance of the homeodomainhomology has been confirmed by deletion analysis. TheHNFl homeobox was shown to be necessary (Frain etal. 1989), but not sufficient, for specific DNA-binding invitro (Nicosia et al. 1990; Chouard et al. 1990).Additional sequences contained in the N-terminal halfof the polypeptide are also required, and they areinvolved in dimerization of HNFl, which is known tooccur in the presence or absence of its DNA target site(Chouard et al. 1990; Nicosia et al. 1990). Amino acidslocated C-terminal to the homeodomain are alsofunctionally important since deletion of this regionalmost totally abolished the capacity of HNFl toactivate transcription in vitro (Nicosia et al. 1990) aswell as in vivo (D. Sourdive, personal communication).The importance of the different HNFl domains isfurther supported by the observation that the humanand mouse HNFl protein sequences are, respectively,93 % and 99 % homologous to the rat counterpart(Bach et al. 1990; Kuo et al. 1990).

HNFl mRNA is absent in dedifferentiated hepatomacells or extinguished somatic hybrids (Baumhueter et al.1990; Cereghini et al. 1990). Nuclear run-on exper-iments have shown that HNFl is controlled at thetranscriptional level in these cases (Cereghini et al.1990). These results indicated that vHNFl was not theresult of a post-translational modification of HNFl buta protein encoded by another gene. Several cDNAclones were recently isolated on the basis of postulatedpartial homology with the HNFl homeodomain andcharacterized as coding for vHNFl (Rey-Campos et al.1991). Both HNFl and vHNFl were introduced intodifferent cell types, including dedifferentiated hepato-mas, by transfection with plasmids carrying the corre-sponding cDNAs under the control of the RousSarcoma Virus LTR promoter. With both factors, itresulted in the activation of a cotransfected CATconstruct driven by the albumin promoter, normallyinactive in these cell lines, although with variable

efficiencies (Rey-Campos et al. 1991; F. Tranche,personal communication). In addition, vHNFl ap-peared to be expressed along with HNFl in differen-tiated hepatoma cells, although at much lower rates(Rey-Campos etal. 1991). These results showed that themere molecular switch between HNFl and vHNFl,previously thought to be, respectively, a positive and anegative effector of the hepatocyte-specific gene ex-pression, was not sufficient to explain the change inactivity of genes like albumin, during dedifferentiation,extinction or reversion.

Considering that HNFl is a homeobox-containing.protein that is involved in the transcriptional control ofterminal differentiation markers, it was of interest toclarify its pattern of expression during development andto compare it to that of the genes that it is known toregulate. Interesting preliminary information was ob-tained about the distribution of the HNFl mRNAwithin subsets of adult organs by northern blot analysis(Frain et al. 1989; Chouard et al. 1990) and RNAaseprotection studies (Baumhueter et al. 1990; Kuo et al.1990). Positive signals were detected in rat or mouseliver, kidney and intestine as well as in mouse stomachand, to a lesser extent, in rat spleen and thymus.Among organs that were examined, lung, brain andheart appeared negative in both rat and mouse, as wellas skin, testis and thyroid in rat and spleen and ovary inmouse (Frain et al. 1989; Chouard et al. 1990;Baumhueter et al. 1990; Kuo et al. 1990).

Since strong signals were detected in various non-hepatic organs (mainly kidney and the gastrointestinaltract), we wondered whether it concerned distinct celllayers within them and wanted to complete the analysisby scoring the entire body using in situ hybridization. Inaddition, we wanted to ask whether HNFl wasexpressed at the initial steps in liver ontogeny andwhether its expression pattern would follow that of thealbumin gene. We also used gel retardation assays andwestern blot analysis to confirm the presence of genuineHNFl protein in kidney. Comparisons with the albumingene expression pattern, as well as with published dataabout the distribution of other known HNFl targets,indicate that this tissue-specific transcription factordepends on the cellular context for its forcing highlevels of gene expression. In addition, these compari-sons suggest that some HNFl-controlled gene transcrip-tion might occur in non-hepatic tissues during mousedevelopment.

Materials and methods

AnimalsFoetuses were obtained at days 10.5, 11.5, 15.5 and 16.5 ofgestation from F: C57BL/6xDBA/2 female mice which hadbeen mated to ¥x C57BL/6XDBA/2 males. The morningwhen a vaginal plug was found is day 0.

Tissue preparationImmediately after dissection, foetuses were fixed overnight at4°C in 4% paraformaldehyde in PBS. Dehydration throughgraded alcohols, paraffin embedding and preparation of 7 fan

HNF1 expression in developing liver, kidney and gut 591

thick embryo sections was performed as described by Sassoonet al. (1988).

Molecular probesA 490 bp Pvull fragment of rat HNF1 cDNA (nt 992 to 1482;Chouard et al. 1990) and a 550 bp Pstl fragment of rat albumincDNA clone pRSA57 (Sargent et al. 1979) were respectivelysubcloned in the Smal or Pstl sites of a T7/T3 promoter-containing pBS vector (Stratagene). Sense and antisenseRNA probes, labeled with SS-UTP (>1000Cimmor1,Amersham) to a specific activity of 1.2 or 1.6xl09disintsmkP'/ig"1, were generated from each cDNA insert usingbacteriophage T3 or T7 RNA polymerase (Pharmacia) asappropriate. Probes were hydrolyzed at pH 10 to an averagefragment length of 100 bp prior to hybridization (Sassoon etal. 1988).

In situ hybridizationBefore hybridization, slides were rehydrated through gradedalcohols, treated with 20 jig ml"1 proteinase K, fixed with 4 %paraformaldehyde/PBS, acetylated in 0.25% acetic anhy-dride in 0.1M triethanolamine, dehydrated through gradedalcohols and dried, as described in Sassoon et al. (1988).Hybridizations were carried out at 52 °C for 16 h with amixture containing O.CSng/il"1 (HNF1) or 0.06 ng/d"1

(albumin) RNA probe, 50% formamide, 10mM DTT, 0.3 MNaCl, 10mM Tris-HCl (pH7.4), 10mM NaH2PO4 (pH8.0),lmM EDTA, 10% dextran sulfate, l x Denhardt's solution(0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% BSA frac-tion V) and l m g m r 1 yeast tRNA. After hybridization,sections were thoroughly washed at a final stringency of 50 %formamide, 2xSSC at 65°C, treated with 20ngml"1 RNAaseA and 2/igmT1 RNAase Tl for 30min at 37 °C, rinsedthoroughly, washed in O.lxSSC for 15min at 37°C, dehy-drated in graded alcohols containing 0.3 M ammoniumacetate, air dried and covered with a Kodak NTB2 emulsion.Exposure times were 1 to 2 weeks for HNF1 probes and 3 to 7days for albumin. Slides were counter-stained with toluidineblue. Controls included hybridization of adjacent sectionswith sense RNA probes, and RNAase A treatment of sectionsprior to hybridization with antisense probes.

Nuclear extract preparation and gel retardation assaysNuclear extracts were prepared as described in Cereghini etal. (1987) for rat liver and kidney and according to Schreiberet al. (1989) for H5 cells. Nuclear extracts were preincubated,during lOmin at room temperature, with 10 ng of eitherunlabelled competitor oligonucleotide or 1 /A of a dilution ofanti-HNFl rH183 serum, 80 % (v/v) in TBST, containing ornot O.Smgmn1 of peptide rHp3 as specific competitorantigen. Gel retardation assays were then performed aspreviously described (Cereghini et al. 1988), using 1 ng of ^P-labelled PE56 (HNF1 site) and either 6/ig, 4.5 ng or 30 fig ofliver, kidney or H5 cell nuclear extracts, respectively, asindicated in Fig. 3. The H5 cell signals shown in the Fig. 3result from a 3-fold longer autoradiography exposure than forthe liver and kidney ones. Resolution of high molecularweight complexes was increased by running 16 cm nativepolyacrylamide gels for 7h at 4°C at 12 VcirT1.

Immune serums and western blot analysisThe peptide rHp3 containing the 21 amino acids from F541 toT561 in the rat HNF1 sequence (FTSDTEASSEPGLHEPSS-PAT, Chouard et al. 1990) was chemically synthesized andcovalently linked to Keyhole Limpet Haemocyanin (KLH)using glutaraldehyde (Ausubel et al. 1989). Various amountsof the partially linked KLH-rHp3 mixture were injected into a

Blanc-Bouscat rabbit as follows: day 1: 400 ng with completeFreund's adjuvant into popliteal lymph nodes; day 23: 400ngwith incomplete Freund's adjuvant into the subscapularcavity; day 31: 200ng in PBS intramuscularly; day 32: sameintravenously; day 46 bleeding and serum (rH183) prep-aration. Western blot analysis was performed according tostandard procedures (Harlow and Lane, 1988). Briefly, 0.6 ngand 0.45 ng of liver and kidney nuclear extracts, respectively,were separated by 10% SDS-PAGE and transferred tonitrocellulose before incubation with the first antibody andrevelation by an alkaline phosphatase-conjugated anti-rabbitserum (Promega). First antibody solution consisted in a 1:250dilution of rH183 in TBST (10 mM Tris-HCl pH8.0; 150 mMNaCl; 0.05% Tween 20) containing or not 4^gml~1 ofpeptide rHp3 as rat HNFl-specific competitor antigen (10-fold molar excess of peptidic antigen over specific antigen-binding sites, best estimated as 13 ^M in undiluted serum).Molecular weight markers were E. coli beta-galactosidase(116.5xlO3Mr), rabbit muscle phosphorylase-b(97.4x10^,-), bovine serum albumin (66.2xlO3Mr) and henegg white ovalbumin (45xlO3Mr), (Bio-Rad).

Resuns

HNF1 mRNA is detected early in embryogenesisA segment of rat HNF1 cDNA corresponding to the 3'end of the HNF1 coding region was chosen as a probe toavoid cross-hybridization with other homeoproteins, inparticular with vHNFl, a protein closely related toHNF1 in its DNA-binding domain but highly divergentin the region of our probe (see Materials and methodsand Rey-Campos et al. 1991). The HNF1 cDNA showsa striking conservation at the nucleotide level inmammals (Bach et al. 1990 and Kuo et al. 1990). Indeed,the sequence of our probe is 95 % homologous to thecorresponding segment in mouse cDNA (Kuo et al.1990), thus allowing us to use the rat probe in high-stringency analysis of mouse HNF1 transcripts. Thespecificity of our HNF1 probe was further confirmed bynorthern blot analysis and by competition experimentsin which full-length sense RNA, synthesized in vitrofrom HNF1 (Chouard et al. 1990) or vHNFl (Rey-Campos et al. 1991) cDNA clones, were added duringhybridization at a 10-fold molar excess with respect tothe HNF1 probe. While the HNF1 sense RNAcompletely abolished the signal over all the positiveorgans, hybridization was not affected by the vHNFlsense RNA (data not shown).

When 10.5 days post coitum (pc) mouse embryosections were hybridized with the FfNFl probe, apositive signal was detected over the developing liverand the visceral endoderm of the yolk sac (Fig. 1A, B).No positive signal was detected elsewhere even after 1month's exposure (data not shown). In contrast, at thesame stage, albumin expression was evident only for theliver primordia (Fig. 1D-F), in agreement with thereported results that mouse albumin mRNA expressionis 10-fold lower in yolk sac than in liver (Sellem et al.1984).

HNF1 mRNA is expressed at high levels in thekidney, gut and liver of mouse embryosBy day 11.5 pc, the body plan of the adult mouse is

592 M. Blumenfeld and others

Fig. 1. Expression of HNFl andalbumin mRNA in the 10.5-day-oldmouse embryo. In situ hybridizationwas performed as described in Materialsand methods on parasagittal sections(note the section shown in D-F is farfrom the sagittal plane).(A, B) Hybridization with HNFlantisense probe, bright-field and dark-field photographs respectively; (C) dark-field micrograph of an adjacent sectionhybridized with HNFl sense probe;(D-F) hybridization with albuminantisense probe; (D and E) bright-fieldand dark-field photographs,respectively; (F), a higher magnificationof E. L, liver; YS, yolk sac. Arrows inB, E and F indicate positivehybridization signal. All micrographswere taken at x25 magnification,except for F (x200).

already delineated (Rugh, 1990). At this embryonicstage, HNFl labelling was visible not only in the livercells and the yolk sac but also in the primary intestinal

loops, while albumin mRNA was again only detected inthe liver (data not shown).

The presence of HNFl transcripts in tissues other

HNFl expression in developing liver, kidney and gut 593

* •. .^ft-'f^'^'.'^JSifc

Fig. 2. Expression of HNFl and albumin genes in 15.5-day-old mouse embryos. Hybridization with the HNFl antisense(A, B, E and F), sense (C) or albumin (D) probes were performed as detailed in Materials and methods. (A, B) HNFlantisense probe, bright-field and dark-field photographs, respectively; (C) dark-field micrograph of an adjacent sectionhybridized with HNFl sense probe; (D) albumin probe, bright-field photograph; the liver tissue is black, due to intensesilver grain labelling; (E, F) HNFl antisense probe, bright-field and dark-field micrographs, respectively, showing a detailof a small intestine portion from the section depicted in A and B. k, kidney, Li, large intestine; si, small intestine;dd, distal duodenum, pd, proximal duodenum; L, liver; p, pancreas; r, rectum; s, stomach; sp, spleen.

than liver was even more evident for 15.5 day embryos.As shown in Fig. 2A and B, HNFl hybridization wasdetected in liver as well as in gut and kidney. This isconsistent with the RNAase protection data obtainedwith the adult rat and mouse (Baumhueter et al. 1990;Kuo et al. 1990). However, Kuo et al. (1990) reportedsome RNAase protection of total RNA prepared fromadult mouse stomach, whereas the portions of thedigestive tract that appeared to express HNFl tran-

scripts at day 15.5 pc were limited to distal duodenum,small and large intestine and rectum. Stomach, proxi-mal duodenum and pancreas were negative at this stage(Fig. 2B).

The in situ hybridization shows that positive cellscorrespond to the epithelial layer of the lumen ofintestine (Fig. 2B, E and F), while in kidney, thelabelling was exclusively cortical and mostly confined tothe boundary with medulla (Fig. 2B). Although the

594 M. Blumenfeld and others

histology of the sample was not good enough for firmconclusions to be drawn, hybridization in the kidneyseemed to be mostly associated with collecting tubules,while glomeruli and mesenchymal cells were negative.Attempts to obtain better conservation of the kidneymorphology by cryo-sectioning failed.

A high background was systematically detected withthe sense RNA probe in the pancreas, liver and spleenfrom 15.5 day embryos (Fig. 2C). The signal in thepancreas might indeed be due to a specific cross-hybridization of our sense probe, the significance ofwhich was not investigated. In the case of liver andspleen, the background was probably due to the usualnonspecific adsorption of RNA probes to erythrocytes,since at this stage both organs are actively hematopoi-etic (Rugh, 1990). However, the hybridization signalwith the antisense probe was above the background,although less clearly in spleen than in liver. Still, wesuggest that the spleen might contain low amounts ofHNF1 mRNA at this stage (compare Fig. 2B and C).Finally, no positive signal was observed in any othertissues examined, including brain (results not shown).Similar patterns of hybridization were observed inembryos of 16.5 days (data not shown).

Thus, some minor discrepancies appear betweenprevious data on the adult and our results on the non-hepatic embryonic tissues. Indeed, Baumhueter et al.(1990) detected a low signal in the adult rat spleen but itwas not confirmed in mouse (Kuo et al. 1990); theseauthors also detected the FfNFl transcript in thymus,whereas no thymus expression was detectable by in situhybridization, even at day 16.5 pc when the organ waseasily recognizable (data not shown). Thus, it is likelythat the HNF1 expression undergo some minordevelopmental and species-specific regulation.

Finally, as for earlier stages, albumin expression wasdetected only in the liver from 15.5 and 16.5 dayembryos, even after one month of autoradiographicexposure (Fig. 2D and data not shown).

Kidney HNFl is indistiguishable from the liver proteinThe fact that HNF1 mRNA was expressed at rathersimilar levels in the kidney, gut and liver of mouseembryos, raised the question of whether the proteinwas actually synthesized in the corresponding tissues.To test this issue, we prepared nuclear extracts fromthose organs in the adult. In nuclear extracts fromkidney, we detected a DNA-binding activity specific tothe HNFl-binding site (Fig. 3: lanes 6, 9 and 10).Among the different complexes that could still corre-spond to related activities distinct from the genuineHNF1, two were displaced by incubation of the extractswith an anti-FfNFl polyclonal serum (Fig. 3: lanes 6and 7). The specificity of this supershift effect wasverified by antigenic competition using the syntheticpeptide against which the antibodies were raised(Fig. 3: lane 8). The efficiency of the test was verified onthe liver HNF1 complex (Fig. 3: lanes 1, 2 and 3). Itsspecificity as regards to the HNFl-related factor calledvHNFl (Cereghini et al. 1988; Courtois et al. 1988) wastested on extracts from the H5 dedifferentiated

hepatoma cell line that contains vHNFl and not HNF1(Cereghini et al. 1990; Fig. 3: lanes 11-13). Theseresults show that the HNF1 mRNA is efficientlytranslated in the kidney. In addition, as describedelsewhere (Rey-Campos et al. 1991), the difference inthe DNA-binding activities on the FfNFl site, betweenliver and kidney can be fully explained by a much higherlevel of the vHNFl protein in the latter. Indeed, theFfNFl-DNA complexes involve dimers of the factor(Chouard et al. 1990) and vHNFl also forms homo-dimers as well as heterodimers with HNFl on DNA(Rey-Campos et al. 1991; Fig. 3). In accordance withthe high levels of mRNA that we observed in thekidney, the HNFl-specific DNA-binding activity on therat albumin proximal element (PE56, Cereghini et al.1988) seemed very high in kidney, as compared to liver.

Since the presence of high amounts of the FfNFlprotein in an organ where the albumin gene is silent waspuzzling, we wondered whether the absence of HNF1-promoted transcription could be attributed to modifi-cation of the FfNFl protein. In an attempt to addressthis question, we performed western blot analysis withkidney extracts using the HNFl-specific immune serumdescribed above. In this type of experiment, we usuallyobserved the liver HNFl as a broad band, illustrating amarked heterogeneity in molecular weight, comparableto silver-stained purified material (Chouard et al. 1990andT.C, unpublished results). This pattern is probablydue to the high degree of glycosylation of this protein(Lichtsteiner and Schibler 1989; T.C. unpublishedresults). Indeed, the HNFl protein produced from thecDNA in a cell-free system gave rise to a sharp band

i which co-migrates with the fastest species of hetero-'genous native HNFl (Chouard et al. 1990). In thekidney, HNFl showed the same heterogeneity ob-served in liver (Fig. 4). This pattern was found in allHNFl-producing mammalian cell types analysed so far,including various cell lines transfected with an exogen-ous HNFl gene (T.C, unpublished results).

Our attempts to prepare total or nuclear extractsfrom the intestine failed to produce any detectableHNFl protein, whether tested by band-shift assay orwestern blot analysis. In control experiments, whereintestine was mixed with liver prior to homogenization,in the presence of different protease inhibitors, we alsofailed to detect the liver HNFl (data not shown). Weconcluded that, even if it was present in the intestine,the HNFl protein could not be detected in extracts bythese methods, probably because of high proteolyticactivity. We were able to detect low DNA-bindingactivity with HNFl specificity in cell lines originatedfrom colon carcinoma. The band-shift pattern in Cacohuman cells was qualitatively comparable to thatobserved in kidney but we could not verify the presenceof genuine HNFl in those cells, since our anti-rat HNFlserum did not recognize the human protein (T.C.unpublished results). Rat TRb cells, derived from apartially dedifferentiated colon carcinoma (FrancoisMartin, personal communcation) gave rise to an activityqualitatively similar to that observed in the H5 cells andnot specifically altered by the anti-rat HNFl immune

HNF1 expression in developing liver, kidney and gut 595

Nuclear extract

K-HNF1 serumHNF1 peptidePE56 oligonucl.DS34 oligonucl.

Supershiftedspecies

HNF1/HNF1

Liver

i i

i i

II

I +

i i

+ +

i +

i i

+ I

II

Kidney

II

I +

i i

+

i

+

+

t i

+

II

I

i i

i i

— Gel origin —

N" M HNF1/HNF1HNFl/vHNFl

vHNFl/vHNFl

10 11 12 13 14 IS

Fig. 3. Kidney contains the HNF1 protein in addition to other HNFl-related DNA-binding activities. Gel retardationassays were performed using an excess of labeled PE56a oligonucleotide (which contains the rat albumin HNFl-bindingsite: Cereghini et al. 1988; see Materials and methods) and 6/ig, 4.5/zg or 30/vg of liver (lanes 1-5), kidney (lanes 6-10) orH5 cell (lanes 11-15) nuclear extracts, respectively. The specificity of the DNA-protein interactions was verified bycompetition with a 10-fold excess of unlabeled oligonucleotides, encompassing either the wild-type HNFl-binding site(PE56a: lanes 4, 9 and 14) or a mutated site (DS34; Cereghini et al. 1988: lanes 5, 10 and 15). For immunological detectionof HNF1, the extracts were preincubated with an HNFl-specific immune serum (rH183) before mixing with the DNAprobe (lanes 2, 7 and 12). The specificity of the serum was tested by adsorption of the antibodies on a 10-fold molar excessof the HNF1 rHp3 peptide (lanes 3, 8 and 13). The main complex observed in liver is formed by the HNF1 homodimer(Chouard et al. 1990) and is indicated by an arrow to the left side of the gel. The complex observed in H5 cells is due tothe vHNFl PE56-binding protein (Cereghini et al. 1988) which was recently characterized as forming homodimers as wellas heterodimers with HNF1 on their common DNA-binding site (Rey-Campos et al. 1991). The vHNFl homodimers thatare detected in H5 cells and kidney, as well as the HNFl/vHNFl heterodimers detected in kidney and to a lesser extent inliver are indicated by arrows according to Rey-Campos et al. (1991). The H5 complexes were resolved on the same gel asfor the tissue nuclear extracts, in the same conditions of migration (see Materials and methods).

serum (data not shown). Our failure to identifyunambiguously an intestinal form of the HNF1 proteinis probably due to technical constraints rather than tothe absence of the protein in this tissue.

Discussion

Molecular cloning of many mammalian transcriptionfactors during recent years has led to a betterunderstanding of transcriptional control mechanismsunderlying the differentiation processes (for a review,see Mitchell and Tjian, 1989). For example, the case ofthe leucine-zipper containing C/EBP protein and thehelix-loop-helix containing MyoD factor are particu-larly enlightening. C/EBP was first characterized by itsspecific DNA-binding activity in liver extracts and hasnow been shown to control terminal adipogenesis, alsoantagonizing cell growth (Umek et al. 1991). MyoD wasfirst identified as capable of forcing undifferentiatedprecursor cells to enter the myogenic differentiationpathway and is now well characterized as a transcrip-tional factor (Weintraub et al. 1991).

Another group of tissue-specific transcription factors,isolated by biochemical approaches, happened to

contain an homeodomain (Affolter et al. 1990) of newtype in addition to specific conserved residues, whichdefined the POU-protein family (Ruvkun and Finney,1991). One member of this family, the pituitary-specificPit-l/GHF-1 factor, was found containing a mutatedhomeodomain in mouse dwarf mutants in which severalcell types of the pituitary gland are affected (Li et al.1990). Thus, the reverse genetic approach confirmedthe role played by the homeobox-containing genes intranscriptional control of development, including ter-minal differentiation processes.

In the same spirit, we have previously characterized aliver-specific transcription factor, APF, as a nuclearprotein that interacts with critical promoter sequencesfrom the rat albumin gene as well as from several otherliver-restricted genes (Cereghini et al. 1988; Tronche etal. 1989; Herbomel et al. 1989). APF was later purified,its corresponding cDNA was isolated and sequenced,and shown to be identical to the independently clonedfactors LFB1 and HNF1 (Chouard et al. 1990; Frain etal. 1989; Baumhueter et al. 1990). Several structuralfeatures of HNF1 led us and others to classify thisprotein as the prototype of a novel subclass oftranscription factors distantly related to homeoproteins

596 M. Blumenfeld and others

HNFl peptideNucl. extr. K L K L

HNFl— 116 —- 97 -— 66 —

— 45 —

1 2 3 4

Fig. 4. The kidney and liver HNFl are indistinguishable byimmuno-detection. 0.6 fig and 0.45 ̂ g of liver (lanes 2, 4)and kidney (lanes 1, 3) nuclear extracts were resolved ontwo identical 10 % SDS-polyacrylamide gels beforeimmuno-detection. The first blot was developed using theHNFl-specific rH183 immune serum (diluted 1:250 inTBST; lanes 1 and 2) and the second one using the sameserum in presence of a 10-fold molar excess of the HNFlrHp3 peptide as a specific competitor antigen (lanes 3, 4;see Materials and methods). The second antibody wascoupled to the alkaline phosphatase. Molecular weightmarkers are indicated in xlO3 MT between the two blots.Polypeptides corresponding to HNFl are indicated: notethe heterogeneity in molecular weight that is characteristicof HNFl purified from liver or immunodetected in cellstransfected with HNFl-expressing vectors (see text).

(Frain etal. 1989; Finney, 1990; Baumhueter etal. 1990;Chouard et al. 1990). Preliminary RNAase protectiondata on tissue distribution of the HNFl mRNA in adultorgans revealed unexpectedly strong signals in non-hepatic tissues (Baumhueter et al. 1990; Kuo et al.1990). Nevertheless, an overview of the entire body, aswell as the information about the HNFl expressionduring the early steps of organogenesis were stilllacking. In addition, quantitative data, coming fromorgan extractions, appeared to us as misleading in theabsence of histological information. Therefore, itseemed necessary to perform an in situ hybridizationstudy of the HNFl expression pattern during mousedevelopment.

The present report shows that the HNFl mRNA isalready transcribed by day 10 pc in post-gastrulationembryos, in cells that have begun to differentiate intomore-commited cell types, as in the liver primordium.At day 15.5 pc, the HNFl transcription is detected notonly in liver but also in kidney and gut. These resultsare consistent with RNAase protection data in adult ratand mouse, although some discrepancies appear in non-hepatic tissues such as stomach, spleen and thymussuggesting that the HNFl gene transcription mightundergo some weak tissue-, developmental- andspecies-specific variations (see the result section). Inaddition, the intensity of the in situ hybridization signalssuggests that the level of HNFl RNA in the positive

cells from gut and kidney at days 15.5 or 16.5 is at leastsimilar if not higher than that of the liver cells.

The detection of the HNFl mRNA in non-hepatictissues did not prove that the HNFl protein was actuallysynthesized in such organs. Kidney nuclear extractswere shown to contain high amounts of true HNFlprotein in addition to related DNA-binding activitiesdue to the presence of vHNFl (see the result sectionand Rey-Campos etal. 1991). The kidney HNFl proteinwas indistinguishable from the liver one by immuno-detection and as active in DNA-binding. In contrast, wefailed to detect HNFl in nuclear extracts from intestine.However, we believe it to be due to high proteaseactivity, rather than an hypothetical tissue-specific post-transcriptional regulation. This is further suggested bythe detection of HNFl-like DNA-binding activity insome colon carcinoma cells (see the result section).

Since these results strongly suggested that high levelsof authentic HNFl transcription factor might be activein tissues other than liver during mouse development,we wondered whether this was consistent with theexpression pattern of genes that it is known to control.In the case of albumin, in agreement with publishedresults, we observed high levels of its transcript in theliver as soon as it begins to bud from primitive gut; incontrast, its expression in tissues expressing high levelsof HNFl mRNA, like visceral endoderm of the yolk sacat day 10.5 or 11.5 pc, was very low; in the 15.5 day pcgut or kidney, the albumin gene transcription wasundetectable even after one month exposure while itsexpression in liver was obvious after only 1-2 days ofautoradiography. The situation is not different in theadult rat, where albumin remains barely detectable inkidney (Nahon et al. 1988; Poliard et al. 1988). Similarresults have been reported for other HNFl-dependentgenes such as rat alpha-fetoprotein (Poliard et al. 1988;Nahon et al. 1988), mouse alpha-1-antitrypsin (Rutheret al. 1987; Koopman et al. 1989), rat pyruvate kinase(Guder and Ross, 1984), or rat alpha- and beta-fibrinogen (Baumhueter et al. 1990). One exception isaldolase B for which relatively high levels wereobserved in kidney although still lower than in liver(Ruppert et al. 1990). Thus, the general rule for knownHNFl target genes remains a marked specificity ofexpression in hepatocytes.

In this context, our results show that large amounts ofHNFl protein, as demonstrated in the kidney, are notsufficient to force the high expression levels of itsknown target genes. Thus, even though target geneexpression in hepatocytes is strongly dependent on theoccupation of HNFl sites located in their regulatoryregions (Schorpp et al. 1988; Cereghini et al. 1988;Monaci et al. 1988), their proper regulation mustinvolve some other factors. Such co-factors might firstact positively, either at the level of chromatin organiz-ation of the various loci or at the level of interactionwith the transcriptional apparatus itself. Indeed, recentresults indicated a cell-type dependence of HNFltranscriptional activity on co-transfected albumin orbeta-fibrinogen promoter derivatives (F. Tranche,personal communication; Kuo et al. 1990). Moreover,

HNF1 expression in developing liver, kidney and gut 597

HNF1 was not able to turn on the endogenous albumingene of several cell types when transiently expressed (F.Tronche and G. Giriffo, personal communication).Second, other factors might act negatively on theexpression of specific HNF1 target genes in theappropriate tissues. For example, a 'silencer' elementwas characterized as part of the -10.5 kb albuminenhancer (Herbst et al. 1990 and references therein)and a so-called 'repressor' segment was recentlylocalized between the promoter and the first enhancerof the alpha-fetoprotein gene and shown to be able toswitch off its expression specifically after birth (Vacherand Tilghman, 1990).

Nevertheless, several genes known to contain func-tional HNFl-binding site(s), are expressed in non-he'patic organs which contain HNF1, at levels fre-quently above the background observed in othertissues. Indeed, it is not surprising to find a co-expression in liver and gut of many genes sincehepatocytes and epithelial cells of the intestine arederived from the embryonic foregut. Interestingly, thisis the case for alphafetoprotein, for which mutations inan HNFl-binding site had a dramatic effect ontranscription in gut or Caco-2 cells, even more than inliver (Tyner et al. 1990; Vacher and Tilghman, 1990). Incontrast, it is puzzling to observe co-expression in theseendoderm-derived tissues and in kidney which isderived from mesoderm. In situ hybridization dataavailable for several HNF1 target genes reveal lowexpression in kidney tubular structures, the samestructures that are suggested to express the HNF1mRNA by in situ hybridization. Albumin and alpha-fetoprotein were found in rat foetuses in all developingtubular cells, while post-natally they were mostlyassociated with the distal convoluted tubules, thecollecting ducts and the loops of Henle (Poliard et al.1988). For mouse alpha-1-antitrypsin, while no hybrid-ization was observed in the metanephros of 14.5 dayspcor later embryos, adult mice showed a positive signalprobably associated with the distal convoluted tubulesor the loops of Henle (Koopman et al. 1989).

Actually, the presence of high levels of HNF1 indeveloping non-hepatic tissues suggests the existence ofnot-yet-identified target genes that would be controlledby this factor. Gene disruption or inactivation of HNF1in different cell lines should help identify such genes.Obvious candidates for such genes are those co-expressed in kidney and liver or intestine: this is thecase of different glycosidases and peptidases which areexpressed in both the brush borders of the smallintestine and kidney proximal convoluted tubuli(Semenza, 1986); similarly, the gluconeogenic enzymes,phosphoenolpyruvatecarboxykinase (PEPCK), fruc-tose-1,6-biphosphatase and glucose-6-phosphatase, aswell as the positive regulatory factor alf (implicated inan albino lethal deletion), are expressed in liver and inthe kidney proximal tubules (Guder and Ross, 1984;Ruppert et al. 1990). Practically, the mechanisms bywhich derivatives of different embryonic layers such asendoderm (liver and gut) and mesoderm (kidney)follow common expression patterns remain to be

elucidated. Analysing the determinants of the regu-lation of regulators such as HNF1 and its relatives mightenlighten these questions, in terms of both develop-ment and evolution of the regulatory processes.

We are grateful to B. David-Watine for the gift of nuclearextracts, to Franck Bourgade for advice in rabbit immuneserum preparation, to J. Rey-Campos and S. Cereghini formaking available the vHNFl cDNA clone, to J. Gaillard, G.Lyons and M.-O. Ott for valuable discussions, to J. Ham,Curtis Pfarr and M. Weiss for critical reading of themanuscript and to J. Ars for typing the text. This work wassupported by grants from INSERM, ARC, LNFCC and theFRMF. M. B. was supported by a fellowship from the ARC.

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(Accepted 17 June 1991)