INTRODUCTION

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The study of tumor-associated chromosomal translocations has led to the identification of genes that play a key role in controlling cell growth and differentiation (26). In the t(8;21) of acute myeloid leukemia (AML), early work showed the breakpoints to fall within the coding sequence of two previously unknown genes, runx1 at 21q22 (previously named AML1/CBFA2) (21) and MTG8 at 8q22 (also named ETO/CDR) (20). The former is related to the runt gene of Drosophila melanogaster and, together with it, defines a novel family of DNA-binding transcription factors (14, 33). Mouse runx1 has been shown to be essential for definitive hemopoiesis (23, 37) and haploinsufficiency at runx1 in humans is associated with diserythropoiesis and an increased risk of AML (32). Another runx member (runx2) is essential for osteogenesis in mammals, as shown both by gene targeting (16, 24) and by its mutations in cases of human cleidocranial dysplasia (22).

Relatively little is known about MTG8/ETO. It belongs to a small, phylogenetically conserved family, consisting of three members in humans and mice (4, 7, 10, 15) and one member in D. melanogaster (9). The latter (nervy) was identified as a target of the homeotic gene Ubx, and its expression in embryogenesis is largely restricted to precursors of the central and peripheral nervous system. However, there is no known phenotype associated with its mutations. Sequence comparison has identified four regions conserved among all MTG8-like polypeptides (4, 15). The COOH-terminal of these (NHR4, for nervy homology region 4) has the potential to fold as a double zinc finger, although it does not bind DNA (F. Calabi, unpublished results). The most NH₂-proximal region (NHR1) is related to TAF_II, a class of molecules involved in transcription initiation by RNA polymerase II (1). The notion that MTG8 is implicated in gene transcription is strengthened by two additional observations: first, MTG8 products are primarily localized to the nucleus (7, 8, 18) and, second, they interact with corepressors such as N-CoR and mSin3, leading to the recruitment of histone deacetylase to the transcription complex (11, 19, 36). Based on Northern blotting analysis, MTG8 in the adult is expressed mainly in the central nervous system, lungs, heart, and testis (20, 40). However, the cellular functions in which MTG8 is involved are not known.

In order to gain an insight into the latter, we have introduced a targeted mutation at the mouse MTG8 locus. Its phenotype reveals a crucial role in the gastrointestinal system.

	MATERIALS AND METHODS

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Gene targeting. A mouse genomic library from strain 129/Sv in phage 2001 (38) was screened with an MTG8 probe spanning exons 2 to 4 (Table 1). Two overlapping clones (ESMM5 and ESMM9) were chosen for further manipulations. A targeting vector was assembled in pUC18, by subcloning an ~1.5-kb partial BglII/SstI fragment from ESMM5 spanning most of exon 2, and an ~7-kb SstI fragment from ESMM9 spanning exon 3. An Escherichia coli lacZ gene (from the KpnI site at nucleotide (nt) 624 to the XbaI site at nt 4158 in plasmid pSV--galactosidase [Promega]) was inserted, after blunting, at the internal SstI site, giving an in-frame fusion to exon 2. A 1.1-kb fragment encoding the neo^r gene (from a modified pMC1neo PolyA vector [34]) was inserted at the BamHI site at the 3' end of the lacZ gene, and a 2-kb fragment encoding the herpes simplex virus tk gene (38) was inserted at the 3' end of the mouse sequence (with respect to the MTG8 transcriptional orientation).

Histological analysis. Organ samples were removed immediately following sacrifice and fixed either in buffered formalin or in Bouin's solution overnight at room temperature, prior to embedding in paraffin and sectioning at 4 to 6 µm.

RNA analysis. Total RNA was extracted from fresh tissues by the guanidine-acid phenol method (6). For gene expression studies, probes (Table 1) were prepared by PCR amplification from mouse genomic DNA, cloned in M13 phage, and sequenced to confirm their identity.

Western blotting analysis. Tissue extracts were prepared by homogenizing fresh organs in 10 volumes of 10% sodium dodecyl sulfate (SDS)-10 mM EDTA-25 mM Tris-Cl (pH 6.8) using an Ultra-Turrax homogenizer (IKA). Approximately 150 µg of total protein was fractionated by SDS-polyacrylamide gel electrophoresis on 7.5% gels and electroblotted onto polyvinylidene difluoride membranes (Hybond-P; Amersham Pharmacia Biotech) in 192 mM glycine-25 mM Tris-20% methanol at 125 V for 2 h. Blots were probed with a rabbit polyclonal antiserum raised against the C-terminal 212 amino acids of MTG8 (PC283; Oncogene Research Products; final concentration, 2.5 µg/ml), followed by horseradish peroxidase-coupled anti-rabbit antiserum (Amersham Pharmacia Biotech; 1:40,000), and developed using the ECL-Plus system (Amersham Pharmacia Biotech).

	RESULTS

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Gene targeting of mouse MTG8. The human MTG8 locus consists of at least 13 exons spanning >87 kb (40; F.C., unpublished). We chose to target exon 2, since it represents the common splice acceptor for a number of alternative upstream exons (20; F. Calabi, unpublished data), and it is the exon to which 5' runx1 sequences are most frequently spliced in transcripts deriving from the t(8;21) (30, 35).

View larger version (20K): [in this window] [in a new window]   FIG. 1.    Generation and expression of the MTG8Ex2/lacZ allele. (A) Diagram of the wt locus, the targeting vector and the mutant locus. Thick black line and boxes, murine MTG8; white box, lacZ; dark gray box, neor gene; light gray box, tk gene; thick striped lines, promoters (arrows indicate transcriptional starts) and 3' UTR [lollipop symbols indicate poly(A) signals]; thin line, pUC18. The 5' and 3' bars indicate the positions of the probes flanking the targeted region and used in the Southern analysis. Relevant restriction sites are represented by capital letters (B, BamHI; H, HindIII; S, SstI, Sl, SalI). (B) Southern blotting analysis of wt (+/+) and MTG8Ex2/lacZ-targeted (+/) CCB ES cells. Restriction enzymes are as in panel A. The probes are described in Materials and Methods. The numbers on the left indicate the molecular size markers in kilobase pairs. (C) S1 protection analysis of brain RNA from wt (+/+) and MTG8 exon 2-null (/) pups with a probe spanning MTG8 exons 2 to 4 (Table 1). tRNA, negative control for probe hybridization. The top band corresponds to residual undigested full-length probe. The double filled arrows point to the band resulting from full protection of MTG8 sequences; the single filled arrow points to the band resulting from protection of exons 3 and 4 only. For reference purposes, protection by an actin probe added to the same hybridization mixture is shown at the bottom (single open arrow). The numbers on the left indicate the molecular size markers in nucleotides. (D) Western blotting analysis of brain from wt (+/+) and MTG8 exon 2-null (/) mice with an antiserum against the C-terminal domain of MTG8. No bands were visible with normal rabbit serum (data not shown). Asterisks mark species that are absent in the mutant. The numbers on the right indicate the molecular size markers in kilodaltons.

Reduced viability of MTG8 exon 2-targeted mice. The viability and fertility of MTG8^Ex2/lacZ heterozygous mice were essentially identical to those of wt controls. Upon mating, heterozygous mice gave birth to the three expected genotypes in Mendelian ratios. However, postnatal viability of homozygous mutant pups was greatly reduced (Fig. 2A). Moreover, nearly all of those that survived past the first 2 days showed significantly reduced size and usually died before reaching puberty. Of the few adults, females were fertile, despite the reduced size, while no progeny were ever obtained from the males, despite successful mating, as judged by the formation of a vaginal plug.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   (A) Viability of the offspring from crosses between MTG8Ex2/lacZ heterozygous mice. The cumulative percent survival over the first 2 weeks after birth of 153 newborn pups representing all accounted offspring of 17 matings is shown. All five MTG8 exon 2-targeted homozygous mice surviving past day 15 were severely growth retarded. (B) Midgut phenotype. An in situ view following sacrifice of a P0 homozygous mutant pup is shown. The defect extends from the distal portion of the duodenum to the rectum. The duodenal end is covered by an outgrowth of the mucosa, the rectal stump is surrounded by a prominent vascular network and the residual mesentery is hypervascularized. du, duodenum; ki, kidney; me, mesentery; re, rectum. (C) Abnormal gut structure in growth-impaired, MTG8 exon 2-targeted mice. A low-power view of hematoxylin and eosin-stained jejunal sections from mutant (/) and control (+/+) P29 mice is shown. Note in the mutant the reduction in gut wall thickness due primarily to disorganization of the villi; note also the dilation of the lumen.

Absence of the midgut in MTG8 exon 2-targeted mice. While there was no significant difference in size and general appearance among P₀/P₁ pups born of heterozygous crosses, a fraction showed a distinctive pallor and no milk in their stomach. Upon sacrifice, these pups revealed a striking phenotype, consisting in the absence of most of the intestine, spanning from the distal duodenum to the greater part of the colon (Fig. 2B). The missing segments largely correspond to the districts supplied by the superior mesenteric artery, i.e., the midgut. On this basis, we operationally refer to this phenotype as midgut (i.e., deletion of the midgut). It was never observed past P₁, likely causing early postnatal death.

Growth impairment in MTG8 exon 2-targeted mice. Of the offspring of heterozygous crosses surviving past the first 48 h, a number showed impaired growth, becoming progressively more marked during the subsequent 2 weeks. Typically, pups were 30 to 50% the size of normal littermates in weight, albeit well proportioned and normally active, except for the most extreme cases. Mortality was high. The few surviving mice gradually recovered with age after puberty, while remaining of below-average weight.

View larger version (75K): [in this window] [in a new window]   FIG. 3.   Cell lineages and proliferation in the jejunum of growth-impaired MTG8 exon 2-targeted mice. Jejunal sections from the mice shown in Fig. 2 were stained for markers of differentiated and proliferating cells. Sucrase-isomaltase (S/I) is a brush border enzyme characteristic of enterocytes; UEAI lectin (UEAI) binds to -linked fucose residues in polysaccharides secreted by goblet and Paneth cells; -smooth muscle actin (-smAct) is present in the muscle layers of the gastrointestinal tract, as well as in vascular smooth muscle cells and myofibroblasts; -tubulin III (-tubIII) identifies ENS cells, and PCNA is an antigen associated with actively cycling cells. A positive reaction (brown color) was developed by the immunoperoxidase-diaminobenzidine technique, followed by counterstaining with Gill's hematoxylin. Despite the disruption of the gut architecture, the rates of cell differentiation and cell proliferation in the mutant are not significantly different from those in the control.

Mouse MTG8 is expressed in the mesoderm of the developing gut. While MTG8 has not been reported to be expressed in the adult gut, the phenotype of MTG8 exon 2-targeted mutants suggested it plays a key role in the gastrointestinal system. In order to test this hypothesis, MTG8 expression was studied during development, by staining heterozygous MTG8^Ex2/lacZ embryos for -galactosidase. Validation of the method was sought in preliminary experiments on adult tissues, in which the results obtained with the -galactosidase stain were found to match faithfully those obtained by RNA analysis in wt mice (data not shown).

View larger version (77K): [in this window] [in a new window]   FIG. 4.   MTG8 expression in the embryonic gut. Transverse sections through MTG8Ex2/lacZ-heterozygous and wt embryos at E10 and E14.5, stained for -galactosidase activity as a proxy for MTG8 expression, are shown. E14.5 sections were counterstained with eosin. MTG8 is expressed in the mesodermally, rather than in the endodermally, derived tissue and becomes restricted, at the later time point, to the outermost gut layers. fg, foregut; hg, hindgut; da, dorsal aorta.

Developmental origin of the gut phenotype in MTG8 exon 2-targeted mice. In order to define the developmental origin of the gut phenotype associated with the MTG8 exon 2-null allele, embryos from heterozygous crosses were collected between E9.5 and E17.5, corresponding to the stages at which most of the critical gut morphogenetic events occur. Compared to wt littermates, no significant difference was observed up to E15.5 although, at the latter time point, the size of the umbilical hernia appeared to be somewhat smaller than in controls and the complexity of the midgut loops was reduced (data not shown). However, a disruption of the villi similar to, albeit less extensive than, that seen in postnatal cases was clearly apparent at E17.5 (Fig. 5). This was associated with persistence of the umbilical hernia, normally disappearing entirely by E16.5. Although preliminary, the data indicate that the requirement for wt MTG8 in the gut starts in the late stages of prenatal development.

View larger version (102K): [in this window] [in a new window]   FIG. 5.   Developmental origin of the gut phenotype in MTG8 exon 2-targeted mice. Hematoxylin and eosin-stained transverse sections through the region of the umbilical hernia of MTG8 exon 2-targeted (/) and control (+/+) E17.5 embryos are shown. In the former, the double-headed arrow indicates the communication between the abdominal cavity and the umbilical hernia, which is still prominent, whereas it has normally disappeared at this stage in control littermates.

Expression of gut patterning factors in MTG8 exon 2-targeted mutants. Gut development is known to be controlled by a number of factors which, as in other systems, can be distinguished into two classes: signaling factors, mediating cellular interactions, and transcription factors, directly controlling gene activity. The former includes mesodermally derived Bmp4 and endodermally derived shh/ihh (39). Among the latter class, in addition to Hox gene products (Hoxd13), knockout experiments have revealed a crucial role for the D. melanogaster caudal homologue Cdx2 (encoded in the ParaHox cluster) (5), as well as for Fkh6 (belonging to the forkhead family) (13) and Nkx2-3 (a homeobox gene product) (25). Since MTG8 is expressed in the mesoderm of the primitive gut and since its mutation has dramatic consequences on the gut structure, we sought to determine their relationship to other gut patterning factors by examining its expression in MTG8 exon 2-targeted mutants. RNA was extracted from proximal and distal gut segments of growth-impaired MTG8^Ex2/lacZ-homozygous mice and wt littermates, and transcript levels were analyzed by nuclease protection. Representative results are shown in Fig. 6. Despite some occasional minor differences, there was no consistent change in RNA levels of Bmp2/4, Cdx1/2, Nkx2-3, or Fkh6 between the null mutant and the wt in either segment. Thus, the role of MTG8 in the gut is not mediated through one of the already-identified gut patterning factors.

View larger version (59K): [in this window] [in a new window]   FIG. 6.   Expression of gut morphogenetic factors in growth-impaired MTG8 exon 2-targeted mice. S1 protection analysis of RNA from proximal (je) and distal (co) gut segments from a P7 MTG8 exon 2-targeted homozygous pup (weight, 2.78 g) and a control (wt) littermate (weight, 5.98 g) was carried out. tRNA, negative control for probe hybridization. The probes are described in Table 1. The top band corresponds to residual undigested full-length probe. Filled arrows indicate the expected protected fragment for each probe. For reference purposes, protection by an actin probe added to the same hybridization mixture is shown at the bottom (white arrow). The numbers on the left indicate the molecular size markers in nucleotides. The apparent minor differences with some probes were inconsistent.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In order to investigate the function of the MTG8 locus, we generated a mutant allele, MTG8^Ex2/lacZ, in which part of exon 2 and of the downstream intron have been replaced by sequences encoding -galactosidase and neomycin phosphotransferase. The sequence encoded by exon 2, spanning 46 amino acids, is very close to the proposed alternative amino termini. While alternative splicing has been observed both 5' and 3', exon 2 has never been found missing either from wt MTG8 transcripts (20, 40; Calabi, unpublished) or from runx1/MTG8 fusion transcripts arising from the t(8;21) of AML (30, 35). This suggests that this exon plays a crucial function, although it does not encode any of the four regions (NHR1 to -4) that are conserved among all MTG8-like polypeptides, and database searches have yet to reveal any significant homology.

Lack of MTG8 polypeptides carrying exon 2-encoded sequences results in high mortality, either perinatally or associated with significant growth impairment during the first 2 weeks of life. Most of the early mortality is due to a massive defect in the gastrointestinal tract. An abnormal gut structure is also found in growth-impaired mice and is a plausible cause of the latter phenotype, given its likely effects on the absorption of nutrients. As in other cases of gene targeting, variable phenotypic penetrance and/or expression may be explained by genetic heterogeneity within the strain resulting from targeting and may indicate the existence of interacting genetic factors. Moreover, a gene dosage effect is suggested by the occurrence of a similar phenotype in heterozygous mice, albeit at a much reduced frequency.

Our results indicate that MTG8 has a crucial function in the gastrointestinal system. While no appreciable expression has been detected in the adult gut, our data show that the embryonic gut is, with the heart (data not shown), one of the main sites of expression at least from E9.5. The highest levels are found in the outer layers, in contrast to other factors so far found to be expressed in the developing gut, which are either endodermal (Cdx1 and -2, shh/ihh, Bmp2, and Tcf4) or primarily restricted to the subendodermal mesoderm (Nkx2-3, Fkh6, Gli1/Ptc, and Bmp4) (2, 13, 17, 25, 27). Intriguingly, the Drosophila homologue of MTG8 (nervy) was isolated as a downstream target of Ubx (9), which is known to play a role in gut patterning in the fruitfly (3). However, no phenotype is associated with nervy mutations, and the role of the latter in the fruitfly remains to be established, as is the potential existence of a Hox-MTG8 pathway in higher organisms.

The midgut phenotype shows some analogies to intestinal atresias in humans, which are generally believed to result from vascular accidents, although a genetic origin has been implicated in some cases (31). The extent of the defect largely coincides with the districts supplied by the superior mesenteric artery. Moreover, while the latter seems to be properly formed, there is vascular congestion over the proximal and particularly the distal gastrointestinal stumps, albeit with no evidence of necrosis. Unlike human cases, however, there is no proximal atresia, while a peculiar mucosal outgrowth extends from the duodenal end. There are no remnants of the missing gut segments, and the mesentery, albeit greatly shortened, shows no gaps. The contribution of MTG8 mutations to gut defects in humans remains to be investigated.

The milder phenotype associated with the MTG8 exon 2 knockout has some superficial analogies with those recently described in other mice with targeted disruption of genes involved in gut development. Both Fkh6- and Nkx2-3-null embryos show delayed formation and slower growth of villi (13, 25). In both cases the changes are apparent from the time of the initial transition from pseudostratified to columnar gut epithelium, coincide with alterations in the proliferative compartment, and correlate with a reduction in the levels of Bmp2 and -4 mRNA, suggesting that they are mediated via a common signaling pathway. In Tcf4- and ihh-null mice (17, 27), which die at or shortly after birth, there is a substantial decrease in the size of the villi associated with a reduction or, respectively, a nearly complete absence of proliferating stem cells. Similarly to these other null mutants, the milder gut phenotype of MTG8 exon 2-targeted mice shows disorganization of the villi, which coexists with largely normal differentiation of gut cell lineages and is most pronounced in the proximal intestine (i.e., the jejunum). However, early midgut morphogenetic events (i.e., the formation of epithelial ridges) are not affected (data not shown), and cell proliferation is not reduced. Further proof that the MTG8 function in gut development and/or differentiation is independent of previously identified pathways is provided by the analysis of patterns of gene expression in the mutants: Bmp2/4, Cdx1/2, Nkx2-3, and Fkh6 mRNA levels are essentially unchanged in the MTG8 exon 2 knockout.

We hypothesize that the two distinct phenotypes of MTG8 exon 2 mutant mice represent different degrees of severity of the same condition, resulting from the lack of a single MTG8-controlled function. Such function is unlikely to be required for primary gut morphogenesis, since the gut was fully formed in a majority of mutants, and no gut anomaly was found in homozygous mutant embryos up to E15.5. Primary canalization of the gut tube in the midgut phenotype is also indicated by the finding of meconium in the rectal stump (data not shown) and by the absence of concomitant abdominal wall defects indicative of a failure in the process of embryonic folding or ventral midline fusion.

We suggest that MTG8 is required for the maintenance of a normal gut structure from late embryonic development, since pathologic changes can be clearly detected in the mutants by E17.5. This function may be related to the blood supply of the midgut, leading in the most extreme cases to a complete regression (midgut) and in less severe cases to dysplasia (causing malabsorption). Rescue of the latter phenotype may occur due to the postnatal triggering of compensatory mechanisms, similar to what has been reported in other knockouts. This hypothesis would be consistent with the localization of MTG8 expression to the outermost layers of the gut, containing the main submucosal vascular plexuses.

In addition to the gastrointestinal defects, sterility was consistently observed in the few male null mutants surviving into adulthood. While the basis of this phenotype remains to be clarified, X-Gal staining in MTG8^Ex2/lacZ heterozygotes shows MTG8 to be mostly expressed by Leydig cells in the adult testis. This suggests that male sterility in homozygotes, despite apparently normal testis size and morphology, is due to hormonal insufficiency. Hind limb paresis and/or paralysis was rarely observed in adult mutant mice. By X-Gal staining in heterozygotes, we have been unable to detect MTG8 expression in the spinal cord, peripheral nerves, or skeletal muscles, and the cause of this phenotype remains to be investigated. In contrast, insertional inactivation of MTG8 exon 2 is phenotypically silent in the brain, lung, or heart, all major sites of expression. Histological examination has also so far failed to reveal any abnormality (data not shown). Thus, the function of MTG8 in these organs is likely to be at least potentially redundant, and its absence may be compensated for by an increase in alternative isoforms and/or by MTG8 paralogues.

Finally, our data do not support a role for MTG8 in haemopoiesis. Upon X-Gal staining, no significant expression of the MTG8^Ex2/lacZ allele was found either in embryos or in the main hemopoietic lineages of adult mice (data not shown). The bone marrow Ly-6A/E⁺ subpopulation, containing hemopoietic stem cells, also scored negative. Moreover, no hemopoietic defect was observed in homozygous mutant mice. These data contrast with the report of MTG8 expression in human CD34⁺ cells (8). Apart from possible species-specific differences, the latter results may have rather been due to cross-reacting products encoded by MTG8 paralogues, which are known to be expressed in hemopoietic cells (4, 7, 10). We conclude that the role (if any) of MTG8 in leukemia may be at least partly related to its abnormal expression in hemopoietic precursors.