Cloning and embryonic expression pattern of the mouse Onecut

transcription factor OC-2

Patrick Jacquemin*, Christophe E. Pierreux, Sebastien Fierens, Jonathan M. van Eyll,Frederic P. Lemaigre, Guy G. Rousseau

Hormone and Metabolic Research Unit, Institute of Cellular Pathology and Universite catholique de Louvain, 75 Avenue Hippocrate,

B-1200 Brussels, Belgium

Received 27 February 2003; received in revised form 25 March 2003; accepted 7 May 2003

Abstract

Onecut (OC) transcription factors are evolutionarily conserved proteins with important developmental functions. They contain a bipartite

DNA-binding domain composed of a single cut domain associated with a divergent homeodomain. The human genome contains three Onecut

paralogues, Hnf6 (also called Oc1), Oc2 and Oc3. We describe here the cloning of mouse (m) OC-2 and its expression pattern in the mouse

embryo. The mOc2 gene was localized on chromosome 18. Analysis of the mOC-2 amino acid sequence revealed overall identities of 67%

with mHNF-6 and of 56% with mOC-3, and the presence of functional domains delineated earlier in HNF-6. The sequence of the 153 residue-

long cut-homeodomain was very conserved, as it was 92% identical to that of mHNF-6 and 89% identical to that of mOC-3. In situ

hybridization showed expression of mOc2 in the developing nervous system and gut endoderm. Like Hnf6, Oc2 was expressed in developing

liver and pancreas. As many genes that are targeted by Onecut factors are recognized by both OC-2 and HNF-6, this overlap of expression

patterns may have functional implications.

q 2003 Elsevier B.V. All rights reserved.

Keywords: Mouse; Onecut; Transcription factors; Oc2; Hnf6; Endoderm; Liver; Pancreas; Duodenum; Nervous system

1. Results and discussion

Transcription factors containing a homeodomain are

evolutionarily conserved proteins that play key roles in

developmental processes. The identification (Lemaigre

et al., 1993) and characterization (Lemaigre et al., 1996)

of Hepatocyte Nuclear Factor (HNF)-6 in the rat led to the

discovery of the Onecut class (Lannoy et al., 1998) of

homeodomain transcription factors. The Onecut proteins

contain a bipartite DNA-binding domain consisting of a

single cut domain associated with a divergent homeodomain

in which residue 48 is a phenylalanine instead of the

canonical tryptophan and residue 50 is a methionine, an

amino acid never found at this conserved position in

classical homeodomains.

Onecut proteins have been identified not only in rodents

(Lemaigre et al., 1996; Rausa et al., 1997) and in

humans (Moller et al., 1999), but also in the nematode

Caenorhabditis elegans (Lannoy et al., 1998), the fly

Drosophila melanogaster (Nguyen et al., 2000), the

ascidian Halocynthia roretzi (Sasakura and Makabe,

2001), the sea urchin Strongylocentrotus purpuratus

(Oliveri et al., 2002), and the zebrafish Danio rerio (Hong

et al., 2002). The evolutionary relationships between cut-

homeobox genes have been analyzed (Burglin and Cassata,

2002). There is evidence for a developmental role of Onecut

proteins in the ascidian (Sasakura and Makabe, 2001) and

fly (Nguyen et al., 2000) nervous system. In the mouse,

inactivation of the gene coding for HNF-6, also called OC-1

(Onecut-1—Mouse Genome Informatics), has shown that

this factor is important for pancreas (Jacquemin et al., 2000,

2003) and liver (Clotman et al., 2002) development.

In addition to Hnf6, the human genome contains two

other Onecut genes called Oc2 and Oc3 (Jacquemin et al.,

1999; Vanhorenbeeck et al., 2002). In the mouse, HNF-6

and OC-3 have been cloned, but OC-2 has not. It was shown

that the adult patterns of expression of mouse (m) HNF-6

and mOC-3 are tissue-restricted and partially overlapping

(Landry et al., 1997; Rausa et al., 1997; Vanhorenbeeck

1567-133X/03/$ - see front matter q 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S1567-133X(03)00110-8

Gene Expression Patterns 3 (2003) 639–644

www.elsevier.com/locate/modgep

* Corresponding author. Tel.: þ32-2-764-7531; fax: þ32-2-764-7507.

E-mail address: jacquemin@horm.ucl.ac.be (P. Jacquemin).

et al., 2002). mHNF-6 and mOC-3 are both expressed in

brain and in organs derived from the endoderm, namely

liver and pancreas for HNF-6, gut and stomach for OC-3. As

a first step towards elucidating the role of OC-2 in

development, we have now characterized the coding

sequence and the pattern of expression of the mOc2 gene

in the embryo.

A mouse liver cDNA library was screened with a probe

obtained with degenerate PCR primers corresponding to

human OC-2 nucleotide sequences. Positive clones lacked

the 50 sequence. This sequence was obtained by screening a

mouse PAC library with a PCR-made mOC-2 probe. This

yielded a fully coding OC-2 sequence (GenBank accession

number AY242995) containing an ORF of 486 amino acids

(Fig. 1) starting at an ATG in a Kozak consensus preceded

by a stop codon 60 nucleotides upstream. The alignment of

this amino acid sequence with those of the two other mouse

Onecut proteins showed a greater overall identity with

mHNF-6 (67%) than with mOC-3 (56%). The DNA-binding

domain of mOC-2 was more similar to that of mHNF-6 (141

identical residues out of 153) than to that of mOC-3 (136

identities). In the cut domain, the LSDLL motif known to

mediate coactivator recruitment in HNF-6 (Lannoy et al.,

2000) and conserved in mOC-3 was also present in mOC-2.

Other regions outside these domains were strongly con-

served in the three Onecut factors, including the TP box

which, in HNF-6, contribute to transcriptional activity

(Lannoy et al., 2000). mOC-2 also contained a polyhistidine

tract and a serine-rich C-terminus, like mHNF-6, and a

polyglycine tract, like mOC-3.

In the mouse, the Hnf6 gene is located on chromosome 9

(Rausa et al., 1997) and the Oc3 gene on chromosome 10

(Vanhorenbeeck et al., 2002). By screening the mouse

genome maps (http://www.ensembl.org), we found that the

Oc2 gene is located on chromosome 18 band D3, in a region

which is syntenic to the region of human chromosome 18

(18q21.2) on which hOc2 is located.

We first determined Oc2 expression using RT-PCR on

whole mouse embryo cDNAs. Oc2 expression became

detectable at the 6-somite stage, around embryonic day (E)

8.0 and increased starting at the 16-somite stage (data not

shown). We then investigated the regional pattern of Oc2

gene expression by whole-mount in situ hybridization. At

E8.25 (9-somite stage), no labeling was detectable (data not

shown). From E8.5 on (Fig. 2 A–D), expression of the Oc2

gene was found in the developing nervous system and in the

foregut– midgut endoderm. Expression in the neural

epithelium was first observed in the rostral half of the

mesencephalon (Fig. 2 A). Later on, the neural tube was

strongly labeled (Fig. 2 C, D), as were cells migrating from

the neural crest (data not shown). Whole-mount pictures at

E10.5 (Fig. 2 E) showed Oc2 expression in the dorsal root

ganglia. On sections at E12.5 (Fig. 2 F, G) and E15. 5

(Fig. 2 H) Oc2 expression was seen in distinct regions of

Fig. 1. Amino acid sequence of mOC-2 and its alignment on the sequence of mOC-1 (mHNF-6) and mOC-3. Residues that are identical at a given position in

two proteins are on a shaded background; those that are identical in the three proteins are on a black background. The TP box, the cut domain and the

homeodomain are overlined. The LSDLL sequence in the cut domain is underlined and the Onecut-specific F48M50 dyad of the homeodomain is indicated by

asterisks.

P. Jacquemin et al. / Gene Expression Patterns 3 (2003) 639–644640

the encephalon, in the dorsal root ganglia and in the ventral

horn of the spinal cord. This expression pattern is quite

similar to that of Hnf6 at these stages (Landry et al., 1997;

Rausa et al., 1997). Interestingly, the thalamus expressed

Oc2 at E12.5 (Fig. 2 F), but was no longer labeled at E15.5

(Fig. 2 H).

At E9.0 and E9.5 (Fig. 2 B, C), Oc2 expression examined

by whole-mount in situ hybridization was detected in the

foregut–midgut endoderm and in the hepatic and pancreatic

buds. We therefore examined by in situ hybridization Oc2

expression in this region starting in 14-somite (around E8.5)

embryos, when distinct portions of the endoderm become

Fig. 2. Expression of the Oc2 gene in the developing nervous system and foregut-midgut endoderm as detected by whole-mount in situ hybridization with the

digoxigenin-labeled Oc2 mRNA probe (A–E), and on sections by in situ hybridization with the 35S-labeled Oc2 mRNA probe (F–H). (A) Oc2 expression in

the brain of a E8.5 embryo (13-somite stage). Labeling is observed in the mesencephalon (arrow). (B) Lateral view of a E9.0 (18-somite stage) embryo showing

Oc2 expression in the brain as well as in the foregut–midgut endoderm (box). Expression in the mesencephalon is broader than at E8.5. In the endoderm,

expression is found in the hepatic bud (hb) and in the region of the prospective dorsal pancreatic bud (p) (inset). (C) Lateral view of a E9.5 embryo showing Oc2

expression along the cephalocaudal axis of the nervous system and in the foregut–midgut endoderm (box). Labeling of the otic vesicle (ov) and of the hindgut

is due to trapping of the probe and is therefore nonspecific. (D) Dorsal view of the same embryo as in panel C showing Oc2 expression in the neural tube. (E)

Dorsal view of a E10.5 embryo showing Oc2 expression in the neural tube and dorsal root ganglia. (F) Sagittal section of a E12.5 embryo showing Oc2

expression in the thalamus (t), septum (s), optic chiasma (oc), hindbrain and dorsal root ganglia. (G) Transverse section through the spinal cord of a E12.5

embryo showing Oc2 expression in the ventral horn (vh) and dorsal root ganglia (dg). (H) Sagittal section of the encephalon of a E15.5 embryo showing Oc2

expression in the cerebellum (c), mamillary body (mb), and optic chiasma and suprachiasmatic nucleus (triangle). The thalamus (t) is not labeled at this stage.

P. Jacquemin et al. / Gene Expression Patterns 3 (2003) 639–644 641

specified to a hepatic fate or to a pancreatic fate. Expression

of the transcription factor Pdx-1, a marker of the

prepancreatic endoderm and of the ventral and dorsal

pancreatic buds (Offield et al., 1996; Ahlgren et al., 1996)

was determined by immunohistochemistry on adjacent

sections. As shown in Fig. 3 A, Oc2 expression in the

endoderm included the hepatic bud as well as the Pdx-1-

labeled territories (Fig. 3 B) that will give rise to the dorsal

and ventral pancreatic buds. This pattern of expression of

Oc2 in the foregut–midgut endoderm is indistinguishable

from that of Hnf6 (Burke and Oliver, 2002; Jacquemin et al.,

2003). The hepatic and pancreatic expression of Oc2 was

also found in 20-somite (around E9.0) embryos (Fig. 3 C, D

and E, F) when these organs bud out of the endoderm.

Sections at E10.5 (Fig. 3 G) showed Oc2 expression in the

pancreatic buds, the duodenum and the liver parenchyma.

At E12.5, strong labeling was observed in the duodenum

(Fig. 3 H) and antrum of the stomach (data not shown). Oc2

expression in the liver was no longer observed by in situ

hybridization; however, it remained detectable at low levels

by RT-PCR until birth (data not shown). Labeling of the

pancreas was decreased at E12.5 as compared to earlier

Fig. 3. Expression of the Oc2 gene in the developing foregut–midgut endoderm at the embryonic stages indicated. (A, C, E, G, H) In situ hybridization on

sections with the digoxigenin-labeled OC-2 mRNA probe showing expression of Oc2 in the ventral endoderm (ve), dorsal endoderm (de), hepatic bud (hb),

dorsal pancreas (dp), ventral pancreas (vp), liver parenchyma (lp) and duodenum (d). c, colon (not labeled). (B, D, F) Immunohistochemistry on sections with

the anti-Pdx-1 antibody. A and B, C and D, E and F are paired adjacent sections. The Pdx-1 marker allows localization of the prospective ventral and dorsal

pancreas in the endoderm (B) as well as of the dorsal pancreatic bud (D) and ventral pancreatic bud (F).

P. Jacquemin et al. / Gene Expression Patterns 3 (2003) 639–644642

stages (Fig. 3). To refine the latter observation we measured

Oc2 expression by RT-PCR in the pancreas from E12.5 until

birth and compared Oc2 expression to that of Hnf6 and of

Oc3. As shown in Fig. 4, Oc2 gene expression was biphasic

in the pancreas, with a decrease (or extinction in some RNA

samples, data not shown) after E12.5, and increased

expression after E15.5. The temporal expression patterns

of Hnf6 and Oc3 in developing pancreas differed from that

of Oc2. High expression of Hnf6 was observed until E16.5

and then expression decreased, whereas Oc3 was expressed

at a low level at E12.5 and E13.5, but was no longer

expressed thereafter.

We conclude from this work that OC-2 displays a tissue-

restricted and stage-specific pattern of expression which is

typical of transcription factors involved in the control of

developmental processes. The high amino acid sequence

similarity of the functional domains of HNF-6 with

corresponding conserved regions in OC-2 and OC-3, and

the fact that these three transcription factors can bind to the

same DNA control elements (Jacquemin et al., 1999;

Vanhorenbeeck et al., 2002), suggest similar modes of

action on transcription. The expression patterns of HNF-6

and of OC-2 overlap during pancreas and liver develop-

ment. However, the genes controlled by HNF-6 are not all

controlled by OC-2 (Jacquemin et al., 1999; Lannoy et al.,

2002). The lack of complete functional redundancy of

HNF-6 and OC-2 is illustrated by the fact that Hnf6 2 /2

mice have developmental defects of the pancreas (Jacque-

min et al., 2000; 2003) and liver (Clotman et al., 2002).

Analysis of Oc2 knockout mice should clarify the role of

OC-2 in embryogenesis.

2. Materials and methods

2.1. Cloning of the coding sequence of the mouse Oc2 gene

A 110-bp Oc2 probe was obtained by PCR using the

degenerate homeodomain primers HOM1 and HOM2

(Jacquemin et al., 1999) and a human OC-2 cDNA clone

as template. This probe was used to screen a mouse liver

cDNA library, prepared in lZAP (Stratagene), by hybrid-

ization at 42 8C in 6 £ SSC and 50% formamide. Filters

were washed at 50 8C in 2 £ SSC, and positive clones were

isolated according to the supplier’s instructions. All these

clones lacked the 50-end of the coding sequence, most

probably because of the high G and C content of this region.

To obtain the sequence of the 50 end, the mouse RPCI21

PAC library (Roswell Park Cancer Institute) was screened

with a probe containing a mOc2 sequence located upstream

of the cut domain. This probe was obtained by PCR using

primers mOC-2S (50-GCCACGCCGCTGGGCAAC-30) and

mOC2AS (50-CAGCTGCCCGGACGTGGC-30) and a

mOc2 cDNA as template. Positive clones were obtained.

A Pst I–Pst I fragment of 5 kb was excised from one of

these clones and subcloned in pBlueScript. This fragment

was sequenced on both strands to obtain a fully coding OC-2

sequence. The amino acid sequences of mHNF-6 (mOC-1),

mOC-2 and mOC-3 were aligned by combining results

given by the Match-Box server (Depiereux et al., 1997) and

ClustalW (Thompson et al., 1994) alignment programs.

2.2. In situ hybridization and immunohistochemistry

In situ hybridization on whole-mount embryos and on

sections were performed as described (Landry et al., 1997;

Jacquemin et al., 1999) using a 1.4 kb-long 35S-labeled or

digoxigenin-labeled Oc2 probe. This probe spans nucleo-

tides 439 to 1664 of the mOc2 sequence (nucleotide

numbering as in GenBank accession number AY242995).

Labeling was detected with NBT/BCIP (Roche) for whole-

mount hybridization and with the TSA Biotin System

(NEN) for hybridization on sections. Pdx-1 was detected by

immunohistochemistry as described (Jacquemin et al.,

2000).

2.3. RNA purification and RT-PCR analysis

RNA was isolated with the Tripure RNA Isolation

reagent (Roche) from single pancreata dissected at different

stages. Total RNA (0.5 or 1 mg) was reverse transcribed and

amplified as described (Clotman et al., 2002). The number

of PCR cycles was 36 for Oc2, 34 for Hnf6, 40 for Oc3, and

26 for actin. Primer sequences were 50-ATGCCGGTCT-

CAGGGGACTCTC-30 and 50-GGCGAAGAGTGTTCGGC

GTTGGAG-30 for Oc2, 50-TTCCAGCGCATGTCGGCGC

TC-30 and 50-GGTACTAGTCCGTGGTTCTTC-30 for

Hnf6, 50-CCTGTGCCGCTCGCAGGGCACG-30 and

50-CTTGAAGATGGCAATCAGCGTG-30 for Oc3, and

50-TCCTGAGCGCAAGTACTCTGT-30 and 50-CTGATC-

CACATCTGCTGGAAG-30 for actin.

Acknowledgements

The authors thank C. Wright for a generous gift of

anti-Pdx-1 antibody, F. Clotman and other members of

Fig. 4. Temporal expression pattern of the Onecut genes in developing

pancreas. The expression of Hnf6, Oc2, Oc3 and Actin was detected by

RT-PCR at the embryonic (E) and postnatal (P) days indicated.

P. Jacquemin et al. / Gene Expression Patterns 3 (2003) 639–644 643

the laboratory for discussions, and J-F. Cornut and S. Cordi

for technical help. 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 No.

GR0303/2000-M), and from the Fund for Scientific Medical

Research (Belgium). P.J. is Research Associate of the

F.N.R.S., C.E.P. is Senior Research Assistant of the

F.N.R.S., and J.v.E. holds a fellowship from the F.R.I.A.

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