©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence, Genomic Organization, and Expression of the Novel Homeobox Gene Hesx1(*)

(Received for publication, July 1, 1994; and in revised form, November 22, 1994)

Paul Q. Thomas (1)(§) Brett V. Johnson (1)(¶) Joy Rathjen (1) Peter D. Rathjen (1)(**)

From the Department of Biochemistry, University of Adelaide, Adelaide, S. A. 5005, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Extensive analyses of homeobox gene expression and function during murine embryogenesis have demonstrated that homeobox gene products are key components in the establishment of pattern formation and regional identity during development. In this paper we report the molecular characterization and expression of a novel murine homeobox sequence, Hesx1, isolated from pluripotent embryonic stem cells. Hesx1 is expressed as two transcripts of 1.0 and 1.2 kilobases which encode an identical 185 amino acid open reading frame. The transcripts differ in the 3`-untranslated region due to the differential utilization of a weak splice donor site located immediately downstream of the translation termination codon. The Hesx1 homeodomain shared 80% identity with the Xenopus homeoprotein XANF-1 and was less than 50% related to other homeodomain sequences. Hesx1 and XANF-1 therefore constitute the founder members of a new homeodomain class. Hesx1 expression was down-regulated during embryonic stem cell differentiation and was detected in tissue-specific RNA samples derived from the embryonic liver, and at lower levels in viscera, amnion, and yolk sac. Expression in adult mice was not detected. These sites of expression are consistent with a role for Hesx1 in the regulation of developmental decisions in the early mouse embryo and during fetal hematopoiesis.


INTRODUCTION

Homeobox genes encode a 60-amino-acid conserved DNA-binding domain, termed the homeodomain, which mediates specific interaction between homeodomain proteins and DNA. Specific residues within the homeodomain primary sequence are highly conserved and are critical for homeodomain stability and DNA interaction (reviewed in (1) ). Homeodomain sequences are divided into classes on the basis of additional sequence homology across the homeodomain(2, 3) . Homeobox genes within a class are usually greater than 70% homologous across the homeodomain, while sequence identity between classes is generally less than 50%.

Of the 60 or more murine homeobox genes which have been identified, the developmental role of the Hox genes is best understood. The 38 Hox genes, which belong to the Antennapedia sequence class, are arranged into four related clusters each containing about 10 genes which lie in the same transcriptional orientation(4) . Mutational analyses of Hox gene function during development, mainly using gene knockout strategies, have demonstrated that Hox genes coordinate pattern formation and specify regional identity in the paraxial mesoderm, hindbrain, and limbs (reviewed in (4) ). At the cellular level, it has been shown that overexpression of Hox 2.4 (Hoxb-7) in myeloid progenitor cells results in inhibition of differentiation(5) , supporting a role for homeobox genes in the specification of cellular identity. The remaining murine homeobox genes belong to a variety of homeodomain classes and are not located within genomic clusters. In most cases, a developmental role for these homeobox genes is implied by their spatially restricted and transient expression during embryogenesis(6, 7, 8, 9) . This has been supported by mutational analysis(10) .

Relatively little is known about expression and developmental function of homeobox genes in the pluripotent cells of the early murine embryo. Homeobox genes expressed by these cells are of particular interest because they are likely to regulate the complex developmental behavior of these cells. Cellular decisions such as proliferation, differentiation, and lineage specification must be coordinated precisely through the appropriate action of specific transcription factors during embryogenesis. Homeobox genes expressed by pluripotent cells may also provide molecular markers for the recognition of these cells in the murine embryo and in other mammalian species.

Embryonic stem (ES) (^1)cells, which are derived from the pluripotent cells of the early murine embryo(11, 12) , can be maintained in vitro in the undifferentiated state by culture in the presence of the cytokine DIA/LIF(13, 14, 15) , or directed to differentiate along alternative pathways by withdrawal of DIA/LIF or by chemical induction(16) . The ability of ES cells to reintegrate into the blastocyst and to contribute to all of the tissues in the developing embryo indicates that ES cells cultured in vitro retain pluripotence. In a previous study of homeobox gene expression in ES cells, we identified a novel homeobox sequence termed Hesx1 (formerly HES-1; 17). Here we report the isolation of Hesx1 cDNA and genomic clones and analyze the expression of this gene during ES cell differentiation and murine development.


MATERIALS AND METHODS

Library Screening, Subcloning, and Sequencing of cDNA and Genomic Clones

Hesx1 cDNA clones were isolated from a ZAP II ES cDNA library derived from D3 ES cell RNA manufactured by Clontech Inc. Library screening was carried out using standard procedures (18) with a [P]dATP oligonucleotide-labeled DNA probe (Gigaprime kit, Bresatec Ltd.) derived from the Hesx1 partial homeobox fragment(17) . Third round duplicate positive clones were isolated and zapped into the pBluescript KS+ vector according to manufacturer's instructions. cDNA inserts were isolated, restricted with AluI, HaeIII, and RsaI (Pharmacia) and subcloned into pT7T319U (Pharmacia) for sequencing using the T7 sequencing kit (Pharmacia). The Hesx1 genomic clone was isolated from a BALB/C murine genomic library (Clontech) as above. Genomic regions containing exons were identified by Southern blot, subcloned, and sequenced.

Isolation of Genomic DNA and Southern Blot Analysis

MBL-5 ES cells (19) cultured on a 10-cm Petri dish (approximately 1.5 times 10^7 cells) were harvested by trypsination, washed in phosphate-buffered saline, and resuspended in 2 ml of TNM (30 mM Tris-HCl, pH 7.6, 150 mM NaCl, 15 mM MgCl(2), 0.4% Nonidet P-40). The cells were lysed by vigorous pipetting, incubated on ice for 5 min, and nuclei were pelleted by centrifugation at 3000 revolutions/min for 5 min. After removal of the supernatant, the pellet was resuspended in 700 µl of Tris-saline (25 mM Tris-HCl, pH 7.6, 75 mM NaCl, 24 mM EDTA). After the addition of 70 µl of 10% SDS and 0.2 mg of proteinase K (Boehringer Mannheim), samples were incubated at 37 °C for 2.5 h. Following two extractions with Tris-HCl-saturated phenol and two extractions with chloroform, genomic DNA was precipitated by adding one-tenth volume 2 M KCl, spooled, and resuspended overnight in 1 ml of water. Genomic DNA was digested with a 4-fold unit excess of restriction enzyme, electrophoresed on a 1% agarose gel, and blotted onto a Nytran nylon filter (Schleicher & Schuell) according to manufacturer's instructions. Filters were prehybridized in 40% deionized formamide, 1 M NaCl, 1% SDS, 10% polyethylene glycol, 50 mM Tris-HCl, pH 7.4, 5 times Denhardts, at 42 °C for at least 4 h. The genomic Southern blot was probed using the Hesx1 partial homeobox fragment (17) as described under library screening, washed at 1 times SSC, 0.1% SDS at 42 °C for 45 min, and exposed to Konica medical grade x-ray film at -80 °C with intensifying screen for several days.

Maintenance and Differentiation of ES Cells

Maintenance of MBL-5 ES cells (19) and ES cell differentiation by DIA/LIF withdrawal, exposure to retinoic acid (Sigma) or 3-methoxybenzamide (Sigma) was performed as described by Smith(20) . For dimethyl sulfoxide (Me(2)SO, BDH chemicals) induction, ES cells were seeded at 1000 cells/cm^2 in ES media (as above) containing 100 units/ml recombinant leukemia inhibitory factor (Amrad) and allowed to adhere overnight. On the following day the culture media was replaced with identical media plus 1.5% Me(2)SO. Me(2)SO induction was maintained for 72 h with daily media changes. Following induction, cells were cultured in media without LIF for a further 3-4 days before harvesting.

Isolation of Embryonic and Cellular RNA

Tissue samples from day 10.5, 12.5, and 16.5 post coitum embryos were isolated from CBA strain mice. RNA from these samples was isolated according to the method of Chomczynski and Sacchi(21) . Tissue-specific samples isolated from MF1 (outbred albino) day 14.5 post coitum embryos were a kind gift from Dr. Austin Smith and have been described previously(22) . RNA was isolated from cultured cells using the method of Edwards et al.(23) .

Antisense Riboprobes, RNase Protection Analysis, and Northern Analysis

Hesx1a- and Hesx1b-specific riboprobes for RNase protection were generated from the pHESa and pHESb plasmids, respectively. pHESa was prepared by ligating a 928-bp XmnI/ScaI restriction fragment (ScaI site at position 628) derived from the Hesx1a pBluescript clone (see library screening) into SmaI linearized pT7T319U vector. pHESb plasmid was constructed from a 926-bp ScaI/PstI fragment (PstI site at position 770), derived from the Hesx1b pBluescript clone, ligated into SmaI/PstI linearized pT7T319U. The XmnI and ScaI restriction sites in the Hesx1a and Hesx1b pBluescript clones, respectively, were due to the presence of artifactual 5` sequences generated during library construction. The Hesx1a (643 bp) and Hesx1b (761 bp) riboprobes were transcribed from EspI (position 26, Fig. 1A) linearized templates using T7 RNA polymerase. The Hesx1 riboprobe for Northern blot analysis was prepared from the pHESc construct, generated by ligating a 394-bp AluI fragment (+30 to +424, Fig. 1A) contained within the Hesx1a/Hesx1b common region. A 425-bp antisense riboprobe was generated from BamHI linearized pHESc plasmid using T3 RNA polymerase (Promega). Mouse glyceraldehyde-3-phosphate dehydrogenase was used as a loading control(32) . As variation in mouse glyceraldehyde-3-phosphate dehydrogenase expression levels in different tissues has been observed (45) , RNA samples were quantitated by spectrophotometry prior to RNase protection.


Figure 1: Nucleotide sequence of the Hesx1a (A) and Hesx1b cDNA clones. Nucleotide sequence common to the Hesx1a and Hesx1b clones spans nucleotide positions -65 to 560 and encodes a 185 amino acid open reading frame from position 1 to 558. The additional 13 nucleotides at the 5` end of the Hesx1b clone which contain an in frame stop codon (underlined) are shown in bold. Nucleotide sequence within the boxed region corresponds to the homeobox. Positions of introns within the genomic clone are indicated by arrows. The 3` unique sequences of the Hesx1b clone, which correspond to nucleotides downstream of position 560 in Hesx1a, are shown in B. The 12 consecutive CA repeats in the Hesx1b transcript are indicated by a bold underline. The Hesx1a and Hesx1b cDNA clones were sequenced in both directions.



The generation of radioactive riboprobes for RNase protection and Northern blot analysis was performed using the method of Krieg and Melton(24) . RNase protection and Northern blot probe transcription reactions contained 240 µCi of [alpha-]PrUTP and 60 µCi of [alpha-]PrUTP (Bresatec Ltd.), respectively. Unincorporated radioactive label was separated from reaction products by loading transcription products onto a Sephadex G-50 column and centrifugation at 3,200 revolutions/min for 4 min. RNase protection reactions were carried out using the protocol described by Krieg and Melton (24) except that 100,000 counts/min of single-stranded probe was added to each RNA sample. RNase digestion products were separated on a 7 M urea, 5% polyacrylamide gel and visualized using autoradiography by exposure to Konica medical grade x-ray film with intensifying screens at -80 °C for 4-10 days, or PhosphorImager analysis (Molecular Dynamics, ImageQuant software package). For Northern blot analysis, (A) RNA was isolated from MBL-5 ES cell cytoplasmic RNA using oligo (dT) cellulose beads (Sigma). Approximately 10 µg of (A) RNA was electrophoresed on a 1.3% agarose gel containing 1 times MOPS buffer (23 mM MOPS pH 7.0, 50 mM sodium acetate, 10 mM EDTA) and 1.1% formaldehyde at 6 V/cm gel length in 1 times MOPS buffer. After electrophoresis RNA was transferred to Nytran membrane (Schleicher & Schuell) by capillary blotting and the RNA immobilized by UV cross-linking and baking at 80 °C in vacuo for 2 h. Filters were prehybridized in a solution containing 5 times SSC, 60% formamide, 0.1% bovine serum albumin, 0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 20 mM sodium phosphate, pH 6.8, 1% SDS, 100 µg/ml sonicated salmon sperm DNA (Sigma), and 100 µg/ml denatured tRNA (Sigma) for 4-24 h at 65 °C. Filters were probed for 16 h at 68 °C and washed in 2 times SSC, 1% SDS at 50 °C for 3 times 15 min and then in 0.2 times SSC, 1% SDS at 75 °C for 1 h. Northern blot filters were exposed to Konica medical grade x-ray film with intensifying screens at -80 °C for 7 days.

Sequence Comparison

The Hesx1 nucleotide and translated open reading frame sequences were compared to the GenBank, EMBL, PIR, and Swiss-Prot data bases using the FastA program(25) .


RESULTS

Hesx1 cDNA Analysis

Two Hesx1 transcripts of 1.2 and 1.0 kb were detected by Northern blot analysis of undifferentiated ES cell RNA (Fig. 2A). Five Hesx1 cDNA clones were isolated from an ES cell cDNA library derived from undifferentiated D3 ES cells (26) maintained in the presence of DIA/LIF. Sequence analysis revealed that four of these clones contained an 847-bp Hesx1 cDNA which included a 3`-poly(A) tail (Hesx1a, Fig. 1A) while the fifth Hesx1 cDNA clone (Hesx1b, Fig. 1B) contained 1117 bp of Hesx1 sequence. Hesx1a and Hesx1b were identical in sequence from the 5` end to position 560 bp, apart from 13 extra bases at the 5` end of the Hesx1b clone, but differed in sequence downstream of this point.


Figure 2: The Hesx1a and Hesx1b cDNAs are derived from the 1.0- and 1.2-kb Hesx1 transcripts, respectively. A, Northern blot analysis of approximately 10 µg of (A) MBL-5 ES cell RNA probed with an antisense Hesx1 riboprobe derived from the common Hesx1a and Hesx1b sequences. B and C, RNase protection analysis of 20 µg of undifferentiated MBL-5 ES cell RNA was carried out using antisense riboprobes spanning positions 26-628 and 26-770 of the Hesx1a (B) and Hesx1b (C) cDNAs, respectively. Hesx1 bands derived from a common transcript are connected by dashed lines. D, molecular origin of the Hesx1a and Hesx1b riboprobes. Wide boxes represent Hesx1 transcripts, and corresponding riboprobes are shown as narrow boxes above. 3` unique sequences are indicated by diagonal shading.



The relative sizes of the Hesx1a and Hesx1b clones suggested that they were derived from the 1.0- and 1.2-kb transcripts, respectively. This was confirmed by comparison of RNase protection products generated by antisense riboprobes derived from the 3` ends of the two cDNAs (Fig. 2). The most abundant Hesx1 transcript expressed by undifferentiated ES cells was the 1.0-kb transcript (Fig. 2A). RNase protection using an antisense riboprobe spanning positions 26-628 of the Hesx1a clone (Fig. 2, B and D) generated a more intense 602 bp band (resulting from protection across the entire probe), and a less intense 532 bp band (resulting from protection across the common cDNA sequence). The greater relative intensity of the 602 bp band indicated that the Hesx1a clone was derived from the more abundant 1.0-kb transcript. The reciprocal experiment, using a Hesx1b antisense probe (Fig. 2D), supported this conclusion (Fig. 2C). The Hesx1a and Hesx1b cDNAs therefore correspond to the 1.0- and 1.2-kb Hesx1 transcripts, respectively.

An identical 185 amino acid open reading frame, containing the Hesx1 homeodomain (amino acids 108-167), was identified in the Hesx1a and Hesx1b nucleotide sequences (boxed in Fig. 1A). The identity of the initiation codon for this open reading frame was confirmed by the presence of an upstream in-frame stop codon at position 7 in the Hesx1b transcript, underlined in Fig. 1A. This termination codon is thought to be common to both Hesx1 transcripts although the 5` end of the Hesx1a transcript was not identified in this analysis. The termination codon for the Hesx1 open reading frame was located immediately upstream of the sequence divergence between the Hesx1 transcripts. The alternative transcripts therefore encode identical proteins and differ only in the 3`-untranslated region. A (CA)(n) repeat sequence containing 12 direct dinucleotide repeats was located within the unique 3`-untranslated region of the Hesx1b sequence (Fig. 1B, positions 956-979 (bold underline)). (CA)(n) repeat sequences in which n > 10 are usually polymorphic with respect to the number of repeats and have been used to generate a genetic map of the murine genome on the basis of this variation in repeat sequence length(27) .

Sequence comparison showed that the Hesx1 homeodomain shares 80% identity with the Xenopus homeobox gene XANF-1 (28) but was not >50% related to other homeodomain sequences (Fig. 3A). This identifies Hesx1 and XANF-1 as founding members of a novel homeodomain class as has previously been suggested from analysis of partial homeodomain sequences(17) . Comparison between the entire Hesx1 and partial XANF-1 open reading frames revealed additional structural similarities. Hesx1 and XANF-1 are identical at the 8 upstream and 6 downstream amino acids which flank the homeodomain and terminate 15 amino acids downstream of the homeodomain (Fig. 3B). Apart from the sequence identity within and flanking the homeodomain, a stretch of 34 amino acid residues at the amino terminus of the Hesx1 open reading frame shares 56% homology with XANF-1 (Fig. 3B). Little homology was detected in the remaining regions of the Hesx1 and XANF-1 primary sequences. The similarities in primary structure suggest that the Hesx1 and XANF-1 genes may have evolved from a common ancestral gene.



Figure 3: Sequence comparison of the Hesx1 homeodomain. A, the positions of the three alpha-helices and homeodomain consensus sequence (2) are shown at the top of the figure. Consensus Hesx1 residues are shown in bold. Amino acid residues which are conserved in the Hesx1, XANF-1 (28) and Antennapedia(29, 30) homeodomains are indicated by dashes. The percentage homology with Hesx1 is shown on the right. B, comparison of the Hesx1 and XANF-1 open reading frames. Positions of identity are indicated by vertical lines, and homeodomain sequences are shown in boldface. Numbers on the right refer to amino acid positions in the Hesx1 (Fig. 1) and XANF-1 open reading frames.



Hesx1 Gene Structure

A Southern blot (Fig. 4) of murine genomic DNA isolated from MBL-5 embryonic stem cells (19) was probed with the Hesx1 partial homeobox fragment(17) , and a restriction map was deduced for this region (Fig. 5A). The absence of multiple bands in the genomic Southern blot indicated that homeobox genes closely related to Hesx1 were not present in the murine genome.


Figure 4: Hesx1 genomic Southern blot. Ten µg of murine genomic DNA isolated from MBL-5 ES cells was digested with PstI/BamHI (P/B) and EcoRI/BamHI (E/B) and probed with the Hesx1 partial homeodomain sequence(17) .




Figure 5: A, Hesx1 genomic restriction map and intron/exon structure. The restriction map was deduced from Southern blot analysis of murine genomic DNA and the 16-kb Hesx1 genomic clone. Genomic regions which contained exons were subcloned and sequenced and are indicated by arrows. Boxed regions represent Hesx1 exons. The 5` boundary of exon I has not been identified and is represented by the dashed line. The length of each intron is indicated in italics in parentheses. Light shading represents the Hesx1 open reading frame and darker shading the homeobox region. The alternative splice site, which is located within exon IV, is indicated by an asterisk. The exact positions of intron/exon boundaries are shown in Fig. 1. H = HindIII, E = EcoRI, P = PstI. B, derivation of the 1.0-kb (Hesx1a) and 1.2-kb (Hesx1b) transcripts. The 1.2-kb (Hesx1b) transcript results from splicing between the weak splice donor site (asterisk) in exon IV to the alternative downstream exon V. The 1.0-kb (Hesx1a) transcript terminates within exon IV.



To allow further characterization of the Hesx1 locus, and to identify the genomic origin of the Hesx1 transcripts, a BALB/C murine genomic library was screened and a 16-kb Hesx1 genomic clone was isolated. The genomic structure of the Hesx1 locus deduced by comparison of the Hesx1 cDNA and genomic sequences is shown in Fig. 5A. Three introns common to both the Hesx1a and Hesx1b transcripts were identified within the Hesx1 genomic sequence. Each of these was surrounded by conserved splice acceptor and splice donor sequences (Table 1) as defined by Mount(31) . Two of these introns were located within the homeobox itself. Although the presence of a single intron within a homeobox is not uncommon, few examples of two introns in this region have been reported, and Hesx1 is the first mammalian homeobox gene for which this has been described.



The molecular origin of the alternative Hesx1 transcripts could also be deduced from the genomic sequence. The 3` end of the Hesx1a transcript was contiguous with the Hesx1 open reading frame, while production of Hesx1b required a splicing event between a poor consensus donor splice site (Fig. 5C, Table 1) located at the point of Hesx1a/Hesx1b divergence within exon IV, and an alternative exon V (Fig. 5). Thus, the alternative Hesx1 transcripts are derived from differential utilization of this weak donor splice site. The biological roles of the alternative transcripts, which differ only in the 3`-untranslated regions, are unknown. Southern blot and sequence analysis indicated that exon V was located 663 bp downstream of the alternative splice site and that the entire 3` end of the Hesx1b cDNA was contained within this exon, although the 3` end of this transcript has not yet been identified. The 5` end of the Hesx1 transcripts has not been determined.

Hesx1 Expression in ES Cells and Differentiated Derivatives

Hesx1 expression was characterized in uninduced ES cells (maintained in the presence of DIA/LIF), and in ES cells induced to differentiate by withdrawal of DIA/LIF from the culture medium (spontaneous induction) or by exposure to the chemical inducers retinoic acid, 3-methoxybenzamide, or dimethyl sulfoxide. In our hands, retinoic acid is the most efficient of the differentiation agents and generates two terminally differentiated cell types, large fibroblast-like cells and variable amounts of refractile parietal yolk sac-like cells. Spontaneous, Me(2)SO and 3-methoxybenzamide induction resulted in nests of stem cells surrounded by a range of differentiated cells. The residual stem cells are thought to be maintained by feedback expression of DIA/LIF from the surrounding differentiated cells(32) .

RNase protection on uninduced and induced ES cell populations (Fig. 6) was carried out using the Hesx1a riboprobe derived from the pHESa construct (see ``Materials and Methods''). Hesx1 was expressed at highest levels in uninduced ES cells and was down-regulated during ES cell differentiation. Terminal differentiation of ES cells with retinoic acid resulted in massive down-regulation of Hesx1 expression, while ES cell differentiation induced by Me(2)SO or 3-methoxybenzamide, or by withdrawal of DIA/LIF, caused a reduction in Hesx1 expression. Residual Hesx1 expression in these cultures is thought to result from incomplete differentiation and the consequent persistence of stem cells within the differentiated population. (^2)Some variation in the profile of Hesx1 transcription during ES cell differentiation has been observed (data not shown) which may be due to variable differentiation within different cultures. In each case, however, there is a down-regulation of Hesx1 transcription with cell differentiation. The ratio of the 1.2- and 1.0-kb Hesx1 transcripts was the same in the uninduced and the spontaneous, 3-methoxybenzamide- and Me(2)SO-induced ES cells, with the 1.0-kb transcript the more abundant mRNA species.


Figure 6: Hesx1 expression in undifferentiated and differentiated ES cells. RNase protection analysis of Hesx1 transcripts expressed by undifferentiated and differentiated ES cells. Protections were carried out on 20 µg of total RNA using the Hesx1a antisense riboprobe (Fig. 2) and a murine glyceraldehyde-3-phosphate dehydrogenase (mGAP) loading control. ES, undifferentiated ES cells; Sp, differentiation induced by LIF withdrawal; low RA and high RA, induction with 2 and 10 µM retinoic acid, respectively; MB, induction with 3-methoxybenzamide; DM, induction with dimethyl sulfoxide.



Tissue-specific Analysis of Hesx1 Expression

Embryonic expression of Hesx1 was investigated by RNase protection analysis of tissue specific RNA samples isolated from adult, day 14.5 post coitum and day 16 post coitum embryos, and total embryonic RNA derived from day 10.5, day 12.5, and day 16 post coitum embryos (Fig. 7, A and B). Hesx1 expression was not detected in total RNA isolated from day 10.5 and day 12.5 post coitum embryos and was expressed at low levels in the day 16 post coitum embryo. Of the tissues examined in the day 14.5 and day 16 post coitum embryo (Fig. 7B), Hesx1 expression was highest in the embryonic liver and was detected at lower levels in the viscera. Hesx1 expression was also detected at low levels in the amnion and yolk sac which form part of the extra-embryonic tissue. Hesx1 expression in the embryonic liver was at similar levels to those seen in undifferentiated ES cells. In situ hybridization will be required to ascertain whether this reflects ES level expression in the majority of embryonic liver cells or elevated expression levels in a subpopulation of liver cells. Hesx1 expression was not detected in adult liver (Fig. 7A) or in adult kidney, heart, lung, muscle, or spleen (data not shown).


Figure 7: Hesx1 expression in embryonic and adult tissues. A, embryonic samples were isolated from day 14.5 post coitum MF1 (outbred albino) embryos. Ca, calvaria; Vi, viscera; Am, amnion; Gu, gut; Lu, lung; Li, liver; YS, yolk sac; He, heart; Br, brain. ES, MBL-5 ES cells; A Li, adult liver. Each reaction contained 10 µg of RNA. B, total embryonic RNA was isolated from day 10.5, 12.5, and 16 post coitum CBA embryos. Embryonic samples from day 16 post coitum embryos are abbreviated as follows: Liv, liver; Ki, kidney; He, heart; Lu, lung; Lim, limbs; In, intestine; Br, brain; and Sk, skin. ES, MBL-5 ES cells. Each protection reaction contained 20 µg of RNA. RNase protections were carried out as described in Fig. 6. Hesx1a* indicates the Hesx1a transcript from the polymorphic allele (see text).



In all embryonic tissues in which Hesx1 expression was detected, the lower 532 bp band corresponding to the Hesx1b transcript was absent and a smaller protected product of approximately 490 bp (Hesx1a*) was detected. Expression of the 490 bp band results from a Hesx1 sequence polymorphism in MF1 and CBA strain mice which is not present in the inbred C129 strain mice from which the ES cells are derived. The absence of a 532 bp band in these samples showed that the 1.2-kb transcript (Hesx1b) was not expressed, indicating that expression of the two Hesx1 transcripts is regulated differentially in different cell types.


DISCUSSION

Analysis of the murine homeobox gene Hesx1 revealed that the gene is expressed as two distinct transcripts of 1.0 and 1.2 kb which can be visualized by Northern blot. cDNA clones corresponding to these transcripts, and a 16-kb Hesx1 genomic clone, were characterized, and the molecular difference between the Hesx1 transcripts was shown to arise from differential usage of a poor consensus splice site located immediately downstream of the open reading frame termination codon. The alternative regulation of these transcripts in murine ES cells and embryonic tissues suggests that there may be alternative biological roles for the different transcripts which encode identical proteins. It is interesting that we were only able to detect expression of the 1.2-kb Hesx1 transcript in undifferentiated ES cells and not in middle to late stages of murine embryogenesis.

Comparison between the Hesx1 and partial XANF-1 open reading frames revealed 47% homology across the entire Hesx1 open reading frame including 80% identity within the homeodomain, suggesting that these genes may be sequence homologues. The regionalized homology between Hesx1 and XANF-1 may reflect the evolutionary conservation of protein sequences which have a functional role, in particular DNA binding activity within the homeodomain. Murine and Xenopus homologues of the goosecoid(6, 33) and Nkx2.5(34, 35) homeobox genes have been isolated which have 78 and 62% identity, respectively. Evidence based on gene expression and function suggests that the goosecoid and Nkx-2.5 homologues fulfill similar roles in these different species during development. There is insufficient experimental data regarding Hesx1 and XANF-1 expression and function to determine whether these related genes might play similar developmental roles during murine and Xenopus embryogenesis.

The presence of introns within the homeobox is a feature of many homeobox genes although it is interesting that introns are not found in the numerous homeobox sequences belonging to the Antennapedia class. The Hesx1 genomic structure is unusual in that it contains two introns within the homeobox. This is a relatively rare arrangement which has not been previously described in vertebrates. The position of the first intron is shared with the homeobox gene mec1, while the location of the downstream intron, within helix 3 of the homeobox, is shared with a variety of homeobox genes(36) . The regular occurrence of introns within the homeobox in a variety of species and genes, and the lack of conservation of the intron position, appear to conflict with the domain theory of intron-exon organization(37) .

Analysis of Hesx1 levels in embryonic tissues indicated that Hesx1 was expressed during 14.5 days and 16 days post coitum of embryogenesis and at highest levels in the embryonic liver. During embryogenesis the liver first becomes apparent around 11.5-12 days post coitum, and replaces the yolk-sac as the principal site of hematopoiesis with concomitant establishment of the definitive red blood cell lineage(38) . The hematopoietic function of the liver continues until about 18 days post coitum, when the spleen and subsequently the bone marrow take over this role. The restricted Hesx1 expression in embryonic liver, coupled with the absence of Hesx1 expression in adult mouse liver which does not have an hematopoietic function, suggest that Hesx1 might be expressed by cells of the hematopoietic lineages during embryogenesis. The levels of Hesx1 expression within the day 16.5 post coitum embryonic liver were similar to the levels expressed by undifferentiated ES cells in vitro. This could reflect generalized expression in all liver cells or elevated Hesx1 expression levels in a subpopulation of embryonic liver cells such as specific hematopoietic lineages. It will therefore be of interest to establish the expression pattern and function of Hesx1 during hematopoiesis. The lower levels of Hesx1 expression detected in the amnion and yolk sac may be due to the presence of hematopoietic stem cells which are believed to migrate from the wall of the yolk sac to the liver to establish hematopoietic foci(38) . Hesx1 expression was also detected at low levels in the viscera which is comprised of the embryonic tissues which remain after removal of the major organs. The detection of Hesx1 expression in the viscera may be due to the presence of hematopoietic cells within these remaining tissues or may indicate additional sites of Hesx1 expression in the embryo.

The expression pattern of Hesx1 in pre-implantation and early post-implantation murine embryos was not analyzed in this study. However, RNase protection analysis showed that Hesx1 was expressed at highest levels in undifferentiated ES cells and was down-regulated during ES cell differentiation. This is a relatively unusual expression pattern for a homeobox gene during ES cell differentiation in vitro since most homeobox genes that have been identified are either up-regulated during stem cell differentiation or unaffected by differentiation(39, 40) . This presumably reflects the isolation of these genes from terminally differentiated cell types or post-implantation embryos. The expression pattern of Hesx1, and related expression patterns such as Oct-4(41) , Oct-6(42) , Hex(43) , Hesx4 and MmoxB(44) ,^2 indicates the existence of a distinct group of homeobox genes that are down-regulated during pluripotent stem cell differentiation in vitro. These genes may play important roles in the early decisions of stem cell renewal, differentiation, and lineage determination in the early embryo.

The restricted expression of Hesx1, and the established role of homeobox genes in the determination of regional identity, indicate that this gene may be of importance in murine embryogenesis through the regulation of developmental decisions during early embryogenesis and fetal hematopoiesis. Resolution of the biological action of Hesx1 awaits detailed analysis of the sites of Hesx1 expression in the embryo and functional genetic analyses in the mouse.


FOOTNOTES

*
This work was supported by an Australian Research Council Grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X80040[GenBank].

§
Recipient of an Australian Postgraduate Research Award.

Present address: Dept. of Obstetrics and Gynaecology, Flinders Medical Centre, Flinders Dr., Bedford Park, S.A. 5042, Australia.

**
To whom correspondence should be addressed. Tel.: 61-83035354; Fax: 61-83034348.

(^1)
The abbreviations used are: ES, embryonic stem; bp, base pair(s); MOPS, 3-[N-morpholino]propanesulfonic acid; kb, kilobase(s).

(^2)
P. Q. Thomas and P. D. Rathjen, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Austin Smith for helpful discussions.


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