1 Centre de Recherche en
Cancérologie de l'Université Laval, The Hox gene
family of transcription factors constitutes candidate regulators in the
molecular cascade of events that governs establishment of normal
terminal differentiation along the duodenum to colon axis. One member
of this family, Hoxa5, displays a
dynamic pattern of expression during gut development.
Hoxa5 transcripts are present in
midgut mesenchyme at the time of remodeling, supporting a role for this
gene in digestive tract specification. To study the role of
Hoxa5 in proper intestinal development
and maturation, we examined whether
Hoxa5 mutant mice exhibit any defect
in this process. We report here that even though
Hoxa5 is not required for midgut
morphogenesis, its loss of function perturbs the acquisition of adult
mode of digestion, which normally is temporally coordinated with the
process of spontaneous weaning. Impaired maturation of the digestive
tract might be related to altered specification of intestinal
epithelial cells. Our findings provide evidence that
Hoxa5 expression in the gut mesoderm
is important for the region-specific differentiation of the adjacent endoderm.
Hox gene expression; intestinal
enzymes; gut morphogenesis; regional specification; epithelial-mesenchymal interactions
IN VERTEBRATES, the gastrointestinal tract arises from
the association of the visceral endoderm with the splanchnic mesoderm into a closed tube during early embryonic development. After closure, morphological and functional regionalization progressively emerges, dictated by the cross talk of the epithelium with the underlying mesenchyme that will lead to intestinal maturation and differentiation (6, 20, 31, 33, 34). A developmentally defined molecular cascade of
events controls the regionalization in the form and function of the
gut, and ontogenetic modifications continue after birth up until
adulthood (27). Consequently, changes in gene expression happen as
cells differentiate along the crypt-to-villus axis, and each given
lineage will maintain its specificity in the differentiation program
occurring along the duodenal-to-colonic axis (13). In addition, a
temporal axis acting during and after gut morphogenesis will lead to
the acquisition of an adult mode of digestion at weaning age (15).
Among the hierarchy of molecular regulators that may control the
establishment and the maintenance of normal terminal differentiation along the gut, the Hox gene family of
transcription factors are putative candidates (6, 8, 18, 33, 34, 41).
In mammals, 39 Hox genes are organized
into four clusters. The genomic organization of the
Hox genes, including the relative
position of each gene within the complex, has been retained throughout
evolution (16). They are characterized by a colinearity rule in their
embryonic expression pattern and activation time along the
anteroposterior axis; Hox genes
localized at the 3'-end of a complex are expressed earlier and in
more anterior domains of the embryo than their 5'-counterparts
(22). In a similar way during gut ontogeny, Hox genes are also expressed in a
nested fashion along the rostrocaudal axis in a manner that reflects
their relative order in the complexes, and accumulating evidence
supports a role for Hox genes in the regional specification of the gut in vertebrates (4, 8, 21, 32-34, 41-43; Aubin and Jeannotte, unpublished data) as they do in
Drosophila (3). For instance,
misexpression of Hoxc8 as well as
overexpression of Hoxa4 lead,
respectively, to hamartomatous lesions and megacolon associated with
hypoganglionosis and abnormalities in the muscular layer (32, 38, 43).
One hypothesis is that Hox genes may participate in the control of positional information as well as cell
differentiation in the intestinal epithelium (8). The fact that
Hox genes are also expressed in a
graded fashion in adult intestine also supports their involvement in
the maintenance of positional information in a continuously
self-renewing epithelium (18).
The role of Hox genes in the
morphogenesis of several organ systems has been unveiled by the
analysis of Hox mutant mouse lines
(36). We have shown from the characterization of
Hoxa5 mutant mice that
Hoxa5 is essential for the proper
patterning of the cervicothoracic region of the axial skeleton, the
pectoral girdle, and the respiratory tract (1, 2, 19). The loss of
Hoxa5 function results in a high rate
of perinatal lethality due to dysmorphogenesis of the respiratory
tract, a consequence that can be linked to perturbed
epithelial-mesenchymal interactions during lung ontogeny (2). Four
transcripts are generated from the
Hoxa5 locus, and of these, the
shortest and most abundant one is highly expressed in the mesenchyme of
the structures affected by the Hoxa5
mutation (1, 2). The Hoxa5 gene
displays a dynamic pattern of expression during midgut development (5). Because it has been suggested that the developmental and positional information along the intestinal anteroposterior axis is fixed in the
fetus before endoderm cytodifferentiation (6, 8, 35), we have examined
the involvement of Hox genes in midgut development and maturation by testing the impact of the loss of Hoxa5 function on proper small
intestine morphogenesis and function in
Hoxa5 mutant mice. We report here that
even though the loss of Hoxa5 has no
observable effect on small intestine morphogenesis, it causes a delay
in acquisition of adult mode of digestion that is normally coordinated
with the process of spontaneous weaning.
Generation of mice, genotyping, and tissue collection.
The establishment of the Hoxa5 mutant
mouse line in an inbred 129/SvEv congenic background has been described
by Aubin et al. (1). Heterozygous
Hoxa5 mice were intercrossed to
generate specimens of all three possible genotypes. Embryonic age was
estimated by considering the morning of the day of the vaginal plug as
embryonic day (E) 0.5. Pups were separated from their mother at
3-4 wk of age. Animals were genotyped by Southern blot analysis of
yolk sac or tail DNA as previously reported (19).
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
RNA in situ hybridization analyses. The RNA in situ hybridization protocol was based on that described by Jaffe et al. (17). The following murine fragments were used as templates for synthesizing 35S-UTP-labeled riboprobes: a 850-bp Bgl II-Hind III genomic fragment containing the 3'-untranslated region of the second exon of the Hoxa5 gene (probe A), a 603-bp Bgl II-Xho I genomic fragment in the intergenic region between Hoxa5 and Hoxa6 genes (probe B), and a 2.8-kb cDNA fragment of the murine c-ret protooncogene (provided by F. Costantini). In situ hybridization experiments were performed on sections from E9.5, E10.5, and E12.5 embryos and intestine from E15.5, E17.5, D0, D15, and D30 wild-type specimens. The c-ret probe was also used on E15.5 gut sections of Hoxa5 homozygous samples.
Enzymatic activity dosages.
Enzymatic dosages were performed on duodenum, jejunum, and ileum of
postnatal specimens harvested at different intervals. Enzymatic
activities of sucrase, maltase, lactase, and trehalase were assayed
using the method of Dahlqvist as modified by Lloyd and Whelan (24).
-Glutamyl transpeptidase (
-GTase) activity was estimated
following the procedure of Naftalin et al. (29) and protein content
according to Lowry et al. (25). Specific activities were expressed in
international units (µmol substrate hydrolyzed/min) per gram of
proteins and were compiled according to the genotype. With regard to
the significance of enzymatic activity variations between wild-type and
mutant animals, ANOVA analysis was performed to determine whether an
overall difference existed over time. This was followed by individual
Tukey-Kramer posttests to determine significant variations at selected
ages. The minimal significance was fixed at
P < 0.05.
Histological analyses. Sections (6 µm) of the different portions of the small intestine were stained according to standard histochemical procedures to identify the different cell types: hematoxylin and eosin (enterocytes and Paneth cells), periodic acid/Schiff (PAS; goblet cells), alcian blue (acid-mucus producing cells), and Grimelius silver method (enteroendocrine cells).
Corticosterone dosage. Serum was collected from D6 (1 wild-type and 1 mutant specimen), D17 (4 wild-type and 5 mutant specimens), and D30 (10 wild-type and 8 mutant specimens) mice and stored frozen until dosage analyses. Estimation of plasma corticosterone was performed by radioimmunoassay as described by Grose and Lebel (14).
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RESULTS |
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Hoxa5 expression pattern during midgut embryonic and postnatal development. It was known that Hoxa5 transcripts are detectable in the gut at midgestation during embryonic development (5, 12) and in adult gut (18, 19). To determine the contribution of the different transcripts and how the temporal expression pattern of Hoxa5 evolves during midgut development and maturation, we characterized its profile of expression at different stages of embryonic and postnatal development by in situ hybridization. The Hoxa5 locus produces four transcripts, a major 1.8-kb transcript and three minor ones of 4.9, 9.5, and 11 kb (19). Because all skeletal and respiratory tract defects in the Hoxa5 homozygous mutant mice are confined to the specific domain of expression of the 1.8-kb Hoxa5 transcript (1, 2), differential expression studies were performed to evaluate the possible role of the short Hoxa5 transcript in gut specification and maturation. We carried out in situ hybridization experiments using probe A, corresponding to exon 2 of the Hoxa5 gene, which recognizes all four transcripts. On near adjacent sections, a probe derived from the Hoxa5-Hoxa6 intergenic region, specific for the three larger transcripts, was utilized (probe B; Jeannotte, unpublished data). According to this procedure, structures that hybridize with probe A but not with probe B must specifically express the 1.8-kb transcript.
With probe A, we observed Hoxa5 expression as early as E9.0 in the gut mesenchyme (data not shown). At E9.5, Hoxa5 transcripts were also present in the midgut (Fig. 1B). They first displayed a sparse mesenchymal distribution as shown for E9.5, E10.5, and E12.5 (Fig. 1, B, E, and H, respectively). Then, around E14.5, they became expressed in a dotlike fashion (shown for E17.5, Fig. 1K). This pattern has previously been attributed to the development of the longitudinal muscle layer of gut wall (5). We rather suggest that the signal corresponds to the plexus of the enteric nervous system (ENS) because of the overlapping c-ret expression, an ENS marker (as shown on Fig. 4I for E15.5; Ref. 30). After birth, Hoxa5-expressing myenteric plexus were still found along the duodenal-to-colonic axis, and their number increased cephalocaudally (Fig. 1N; not shown).
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Enzymatic activities in wild-type and Hoxa5 homozygous mutant intestine. At birth, mammals must be able to digest all of the components of the milk, and at the period of weaning, coordinated changes occur to allow production of enzymes required for the digestion of solid food. For instance, lactase displays high activity during the suckling period and then declines, whereas sucrase, trehalase, and maltase activities appear or rise at weaning. The dynamic of changes in the enzymatic profile of the small intestine in wild-type and Hoxa5 homozygous mutant samples is summarized in Fig. 2. In Hoxa5 homozygous mutants, most of the brush-border membrane hydrolytic activities measured in different segments of the intestine at D15 and D17 displayed statistically significant differences compared with wild-type samples. Hence, at D15, the activity of sucrase, trehalase, and maltase enzymes were still at newborn levels in the duodenum, jejunum, and ileum of Hoxa5 homozygous mutants. For example, sucrase activity was fivefold higher in normal specimens than in Hoxa5 mutants at the level of the duodenum, sevenfold in jejunum, and eightfold in ileum. High ratios were also observed for trehalase (3-fold) and maltase (4-, 3-, and 2-fold) in the same respective segments. Disaccharidase activities all increased at D17 in both mutant and wild-type specimens. However, Hoxa5 mutant levels remained below that of wild-type animals, although the statistical significance was reduced (P < 0.096) for ileal sucrase. By D30, these enzymes generally approached wild-type levels, suggesting that functional maturation of the small intestine was retarded in Hoxa5 mutants.
In the case of lactase andMorphological analysis of the small intestine from
Hoxa5 mutant mice.
Fetal gut development is characterized by a progressive cephalocaudal
wave of cytodifferentiation. Invagination of the mesenchymal layer into
the undifferentiated endoderm accompanies the conversion of the
pseudostratified epithelium to a monolayer of columnar cells, which
forms primordial villi separated by the intervillous epithelium that
will give rise to the crypts. Morphogenesis of the mouse intestine is
not completed until the third postnatal week (13). At birth,
rudimentary crypts can be observed in the intervillous epithelium.
Remodeling of the intervillous region continues from D0 to D14, and the
pathway of epithelial cell migration up the villus does not become
fully organized until D7-D14. During the third postnatal week,
villi lengthen as cell rate production exceeds cell loss to achieve an
equilibrium at D28. As mutation of the
Hoxa5 gene resulted in impaired
digestive function of the small intestine at weaning, we tested whether
abnormal morphogenesis could underlie the altered function. We
performed a detailed comparative histological analysis of the structure
on tissue sections from the duodenum, jejunum, and ileum of wild-type
and Hoxa5 homozygous mutants collected
at different stages of embryonic and postnatal development. During
embryonic development, as shown for E17.5 in Fig.
3, no morphological alterations or delay in
the organization of nascent villi units could be observed between
Hoxa5 mutant and control samples in
the duodenum, jejunum, and ileum regions (Fig. 3,
A and
B, E
and F, and
I and
J). For postnatal stages, hematoxylin and eosin-stained sections showed well-developed villi in
the duodenum, jejunum, and ileum of mice of all genotypes (Fig. 3,
C and
D, G
and H, and
K and
L). No apparent difference was observed in the number of villus formed, their length, and in the
intervillous region, indicating that no detectable morphological anomaly resulted from the loss of
Hoxa5 function.
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Hormonal status of Hoxa5 mutant mice. The role of glucocorticoid and thyroid hormones as regulators of intestinal development has been extensively studied and convincingly demonstrated for glucocorticoids (15). In the mouse, corticosterone is the major glucocorticoid hormone. Circulating levels of glucocorticoids surge just before enzymatic changes during the third postnatal week. Hormonal manipulation, such as precocious administration of glucocorticoid, induces premature changes whereas deprivation by adrenalectomy leads to a dramatic reduction in the rate of intestinal maturation. To test if corticosterone levels were normal in Hoxa5 homozygous mutants compared with wild-type littermates, measures of the seric levels of this hormone were performed at D6, D17, and D30. Although corticosterone levels were slightly reduced, no statistically significant differences were observed between wild-type and Hoxa5 mutant plasma levels, indicating that glucocorticoid levels in Hoxa5 homozygous mutants at weaning and adult ages were within normal range (data not shown).
Acquisition of adult-type intestine enzymatic levels in rodents is known to depend, at least in part, on thyroid hormones increasing the systemic release of glucocorticoids from adrenal glands (15, 40). Moreover, thyroid hormones might act synergistically with glucocorticoids to promote intestinal maturation (26). The levels of thyroid hormones in the serum of Hoxa5 mutants were identical to that of wild-type littermates at D15 and in adult animals (D. Meunier, J. Aubin, and L. Jeannotte, unpublished data). Taken together, these results indicated that the hormonal status of the Hoxa5 mutant mice resembled that of their wild-type littermates. ![]() |
DISCUSSION |
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The involvement of Hox genes in gut patterning has been supported by several studies. Their nested expression pattern along the duodenal-to-colonic axis, which reflects the genomic organization within a complex, is consistent with the existence of a Hox code conferring specification to the intestinal epithelium (21, 33, 41, 42). For instance, Hoxa13/Hoxd13 double-mutant embryos show virtually no development of the caudal part of the hindgut (42). Experimental evidence has also revealed the involvement of Hoxa5 in the morphogenesis of the stomach and proximal colon (Aubin and Jeannotte, unpublished data). The hypothesis of a preponderant role of Hox genes in epithelial specification has been challenged in the gut of the chicken where it was shown that ectopic expression of a 5'-Hox gene normally expressed in the colon in the mesenchyme of a more proximal part of the gut will induce development of an epithelium showing colonic characteristics (34). As results accumulate for the role of Hox genes in the patterning of the gut, their involvement in the proper differentiation and maintenance of positional information along the intestinal tract remains elusive.
In the present study, we used Hoxa5 mutant mice to assess the role of this gene in the morphogenesis and maturation of the small intestine. We first demonstrated that the Hoxa5 gene shows a dynamic spatial and temporal expression profile during midgut morphogenesis, rendering likely its participation in gut development. We previously reported that all the skeletal and respiratory tract defects observed in Hoxa5 homozygous mutant mice were confined to the 1.8-kb Hoxa5 transcript distinct domain of expression, suggesting that this transcript is responsible for the specific Hoxa5 function (1). In the gut, the 1.8-kb Hoxa5 transcript is the sole one detected in the mesenchyme at early stages (Fig. 1B), whereas the larger forms are expressed from E10.5. Hoxa5 was also previously reported to be specifically expressed in the gut mesenchyme at embryonic stages (5). When remodeling of the epithelium begins around E14.5, mesenchymal expression of all Hoxa5 transcripts ceases. Concomitantly, expression of the 1.8-kb transcript becomes restricted to the ENS in the midgut, and later on in the hindgut (Fig. 1; data not shown). The spatial restriction of the 1.8-kb Hoxa5 transcript in the ENS coincides with the coalescence of the neural crest cell derivatives generating the myenteric plexus (10). Interestingly, some ENS precursors originate from the vagal neural crest where only the short Hoxa5 transcript is expressed (23). The larger transcripts, whose transcription initiation sites are located upstream of that of the short transcript (Jeannotte, unpublished data), displayed a temporal profile that reflects the colinear relationship existing between the Hox complex organization and the expression of Hox genes. Their more restricted spatiotemporal pattern of expression also suggests that their contribution might be limited in patterning of the midgut.
Although we did not find Hoxa5 to be expressed outside the ENS after birth, James and Kazenwadel (18) reported the presence of Hoxa5 transcripts in intestinal crypts of adults. Their study was performed by RNase protection assay on isolated crypts. The discrepancy between their results and ours could be attributed to the methodologies used. Although Hoxa5 may be expressed at levels undetectable by in situ hybridization in cells of the crypt, clearly the major site of expression in fetus and adult midgut is in the ENS.
Our data show that the morphogenesis of the small intestine is not impaired in Hoxa5 homozygous mutants; rather, it seems that the absence of Hoxa5 function causes a delay in functional maturation of intestinal epithelial cells, as judged by the alteration of brush-border hydrolase activities around weaning. The specific activities of disaccharidases (sucrase, trehalase, maltase), which normally increase 10- to 20-fold between birth and D17, are significantly lowered in Hoxa5 mutant mice during this period. In fact, enzyme levels remained very low up until D15 in mutant specimens. With the consideration that these changes occur late during development, a major point to be resolved is whether abnormal intestinal function at weaning age is due to a secondary effect related to the hormonal status of the Hoxa5 mutant weanlings. The fact that the corticosterone and thyroid hormone measures show no statistically significant differences does not support this hypothesis. Furthermore, surviving Hoxa5 mutant pups appear healthy with no sign of stress that would suggest that their nutritional or metabolic state is affected.
Hence, it is possible that altered maturation is caused by a primary
defect related to incorrect epithelial cell specification. Moreover,
lactase and -GTase profiles are not consistent with a secondary
hormonal deficiency but rather favor the hypothesis of a primary
defect. For instance, it was previously shown that jejunal and ileal
lactases are not subject to the same hormonal constraint as duodenal
lactase (26). In Hoxa5 mutants, the
lactase activity in the median and distal part of the gut stays lower or decreases more rapidly than in wild-type samples, in contrast to
what would have been expected on the basis of normal ontogenic decline
(Fig. 2). Analogously, the deficient duodenal
-GTase activity at D30
also suggests a functional anomaly related to improper cell
specification. It was proposed that the ontogenic decrease of lactase
activity is dependent on posttranslational regulation, and changes at
weaning have to be considered as a redifferentiation rather then
maturation of the intestinal epithelium when the animals adapt to the
adult mode of digestion (7, 35). Furthermore, it was shown by grafting
experiments that temporal and positional information needed for
intestinal ontogeny up to the postweaning stage result from an
intrinsic program fixed in mammalian fetuses before endoderm
cytodifferentiation (6). Indeed, as early as E14, the onset and
modification of expression of lactase enzyme pattern is already
determined. Thus the fact that Hoxa5
transcripts are present at the time of remodeling of the midgut
epithelium may reflect the direct participation of the
Hoxa5 gene product in ontogenetic changes.
In conclusion, the present study provides evidence that the Hoxa5 gene may be a candidate transcription factor involved in the ordered genetic cascade responsible for intestinal epithelial cell differentiation. It has been suggested that homeobox genes are potential upstream regulators of epithelial-mesenchymal interactions in the gut system (31), and our study supports this view. Other transcription factors, such as Cdx, Fkh6, and GATA family members, are known to contribute to early specification of intestinal segments (11, 20, 37, 39). Although Cdx and GATA genes show an epithelial-specific expression profile (9, 11), Fkh6 and Hoxa5 expression is restricted to the mesenchyme. Therefore, as for lung morphogenesis (2), Hoxa5 may direct the complex program of enterocyte differentiation and functional maturation through epithelial-mesenchymal interactions. Finally, although it has been established that Hox genes are involved in organ morphogenesis, our findings unveil their potential role in organ function as well.
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ACKNOWLEDGEMENTS |
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We thank Dr. Rashmi Kothary for helpful comments on the manuscript, Dr. Marcel Lebel and Claude Villeneuve for corticosterone dosage, Dr. Frank Costantini for providing the c-ret probe, and Lina Corriveau for helpful technical expertise.
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FOOTNOTES |
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This work was supported by grants from the Medical Research Council of Canada (to L. Jeannotte and D. Ménard). L. Jeannotte is a Scholar of the Fonds de la Recherche en Santé du Québec, and J. Aubin holds a studentship from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. Jeannotte, Centre de Recherche de L'Hôtel-Dieu de Québec, 9 rue McMahon, Quebec, Canada G1R 2J6 (E-mail: lucie.jeannotte{at}crhdq.ulaval.ca).
Received 26 October 1998; accepted in final form 9 July 1999.
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