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Molecular and Cellular Biology, August 2001, p. 5658-5666, Vol. 21, No. 16
Institute of Child Health, London WCIN
1EH,1 and MRC Laboratory of Molecular
Biology, Cambridge CB2 2QH,2 United Kingdom
Received 7 February 2001/Returned for modification 2 April
2001/Accepted 23 May 2001
The MTG8 (ETO) locus is involved in a
reciprocal exchange with runx1 in the t(8;21) of acute
myeloid leukemia. It is a member of a small gene family encoding
transcriptional regulators that interact with corepressors and histone
deacetylase. However, the physiologic cellular processes controlled by
MTG8 are not known. In order to gain an insight into the
latter, we have generated mutant mice with an insertional inactivation
at the locus, which disrupts transcription of exon 2. The postnatal
viability of homozygous mutants was greatly reduced. In approximately
25% the midgut was missing, whereas practically all pups surviving
past the first 2 days showed severe growth impairment, which was likely
due to a gross disruption of the gut architecture. The latter phenotype could be traced back to late embryonic development. No difference in
gut cell differentiation or proliferation was found compared to
wild-type littermates. Levels of factors known to be involved in gut
morphogenesis were also unchanged. MTG8 is expressed in the
outermost layers of the developing gut from at least E9.5. Thus,
MTG8 plays a novel, essential role in the gastrointestinal system.
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 TAFII, 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.
Gene targeting.
A mouse genomic library from strain 129/Sv
in phage
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.16.5658-5666.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Gene Targeting Reveals a Crucial Role for
MTG8 in the Gut
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 neor 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).
TABLE 1.
Probes used in the present study
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.
Staining with hematoxylin and eosin, or Alcian blue was performed according to standard protocols. For immunohistochemistry, the following monoclonal antibodies were used as primary reagents: anti-PCNA (clone sc-56 [Santa Cruz Biotechnology], 1 µg/ml), anti-human sucrase-isomaltase (clone MGlu2 [12], culture supernatant diluted 1:64), anti-
smooth muscle actin (clone 1A4
[Sigma], ascitic fluid diluted 1:800), and anti- tubulin III
(clone SDL.3D10 [Sigma], ascitic fluid diluted 1:1,600). A
biotinylated horse anti-mouse immunoglobulin G (Vector Laboratories,
7.5 µg/ml) was used as a secondary reagent. Biotinylated Ulex
europaeus agglutinin I (UEAI) lectin was purchased from Vector
Laboratories and used at 7.5 µg/ml. Sections were deparaffinized,
rehydrated, treated with 3% H2O2 in methanol
for 10 min, heated to 95°C in a microwave oven for 10 min, and
blocked in 10% horse serum in phosphate-buffered saline (PBS) for 30 min. Antibodies were diluted in 10% horse serum in PBS and incubated
for 30 to 60 min at room temperature. The results were visualized with
the Vectastain Elite kit (Vector Laboratories), using diaminobenzidine
as the substrate, following the manufacturer's instructions. Sections
were lightly counterstained with Gill's hematoxylin.
Embryos were fixed in 4% paraformaldehyde in PBS for 3 to 12 h
and stained with X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) according to a
published protocol (29) prior to embedding and sectioning
as described above. Sections were counterstained with eosin.
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.
High-specific activity, single-stranded DNA probes were prepared according to standard procedures (28). Ca. 5 × 104 cpm were mixed with ~20 µg of total RNA in 20 µl of 50% formamide-0.5 M NaCl-1 mM Na2EDTA-25 mM PIPES (pH 6.8), heated at 50°C for 30 min, and then left to hybridize at 45°C for ~18 h. S1 digestion was with 25 U for 30 min at 37°C.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).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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).
Mouse genomic clones containing MTG8 exons 2 and 3 were isolated from an 129/Sv library. An E. coli lacZ coding sequence was inserted in frame in place of the 3' end of exon 2 and of the adjoining intron, in order simultaneously to disrupt MTG8 transcription and to enable tracking of exon 2 expression by assaying for-galactosidase activity.
neor and tk cassettes were further
inserted in order to allow selection of homologous recombinants
according to a standard strategy. The structure of the resulting
targeted allele, MTG8Ex2/lacZ, is illustrated in
Fig. 1A.
|
) 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 (Reduced viability of MTG8 exon 2-targeted mice.
The viability and fertility of MTG8Ex2/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.
|
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.
midgut phenotype to occur nearly exclusively
in homozygous mutant pups, at a frequency of ~25%. It can thus
account for most of the increased perinatal mortality of this class.
Much rarer cases were observed in heterozygotes (~1.3%), and none
were seen in wt mice.
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.
Nearly all affected mice were homozygous mutants. Conversely, all of the latter showed growth impairment. Thus, the phenotype was strongly associated with homozygosity for the MTG8 exon 2-null allele. Upon sacrifice, growth-impaired mice did not show any obvious anomaly outside the gastrointestinal tract. The intestine, while of reduced length compared to control littermates, was proportionate to the lower body weight. However, the gut histology was grossly abnormal, particularly at the level of the jejunum (Fig. 2C). The intestinal wall was thinner, largely due to a reduction in the length of the villi, which also looked highly disorganized, thicker, and fewer in numbers. Moreover, the lumen was often dilated, probably reflecting a reduced tone of the muscle layers. In order to investigate whether the abnormal architecture was associated with changes in cellular differentiation, sections from pathological and normal guts were stained for gut cell markers. Of the four main types of gut epithelial cells, enterocytes can be identified by the expression of sucrase-isomaltase and goblet and Paneth cells by a combination of Alcian blue and lectin UEAI staining. Contractile cells in the tunica muscularis, as well as in perivascular locations, express-smooth muscle actin, and ENS cells express -tubulin III.
As shown in Fig. 3,
all five cell types were present in
homozygous mutant guts, in proportions and locations that were not
significantly different from those of wt littermates.
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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 MTG8Ex2/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).
|
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.
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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
MTG8Ex2/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.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In order to investigate the function of the MTG8 locus,
we generated a mutant allele, MTG8Ex2/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 MTG8Ex2/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 MTG8Ex2/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.
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ACKNOWLEDGMENTS |
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We are particularly grateful to Terence Rabbitts for constant encouragement and strategic advice. We also thank Vania Cilli for help in the isolation of mouse MTG8 genomic clones, Dallas Swallow for the gift of the anti-human sucrase-isomaltase monoclonal antibody, Andy Copp and Patrizia Ferretti for comments, and the staff of the Royal Veterinary College, London, England, for expert mouse husbandry.
This work was supported by MRC PG9311737.
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FOOTNOTES |
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* Corresponding author. Mailing address: Developmental Biology Unit, The Institute of Child Health, 30 Guilford St., London WCIN 1EH, United Kingdom. Phone: 44-20-7813-8492. Fax: 44-20-7831-4366. E-mail: fcalabi{at}hgmp.mrc.ac.uk.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, August 2001, p. 5658-5666, Vol. 21, No. 16
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.16.5658-5666.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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