Loss of Hoxa5 gene function in mice perturbs intestinal maturation

Josée Aubin1, Pierre Chailler2, Daniel Ménard2, and Lucie Jeannotte1

1 Centre de Recherche en Cancérologie de l'Université Laval, Centre Hospitalier Universitaire de Québec, Pavillon de L'Hôtel-Dieu de Québec, Québec G1R 2J6; and 2 Groupe du Conseil de Recherches Médicales du Canada sur le Développement Fonctionnel et la Physiopathologie du Tube Digestif, Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).

To study the morphology and the different enzymatic activities along the proximodistal axis of the intestine, healthy wild-type and Hoxa5 homozygous mutant animals were killed at different time points after birth [postnatal day (D) 0, 4 wild-type and 3 mutant specimens; D6, 1 wild-type and 1 mutant specimen; D15, 5 wild-type and 5 mutant specimens; D17, 6 wild-type and 9 mutant specimens; D30, 11 wild-type and 6 mutant specimens]. The entire small intestine was removed, kept on ice, and divided in three equivalent portions, corresponding, respectively, to the duodenum (proximal one-third), the jejunum (middle one-third), and the ileum (distal one-third). For each, a portion of the midsection of the duodenum, jejunum, and ileum was dissected and fixed in cold 4% paraformaldehyde in phosphate-buffered saline followed by paraffin embedding. The rest of each specimen was immediately frozen in liquid nitrogen for enzymatic dosages. Embryonic gut specimens were harvested at E17.5 (2 wild-type and 2 mutant specimens) and processed as described above for histology.

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). gamma -Glutamyl transpeptidase (gamma -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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Hoxa5 expression in developing midgut. Sections of embryonic day (E) 9.5 (A-C), E10.5 (D-F), and E12.5 (G-I) mouse embryo and E17.5 (J-L) and D0 (M-O) midgut tissue were hybridized with either probe A (B, E, H, K, and N) or probe B (C, F, I, L, and O). Bright-field views of middle panels are shown on left panels (A, D, G, J, and M). At E9.5, E10.5, and E12.5, probe A detected a widespread distribution of Hoxa5 transcripts in midgut mesenchyme (B, E, and H). Onset of expression with probe B was observed at E10.5 and was transient in mesenchyme until E13.5 (C, F, and I). From E14.5 onward, probe A revealed Hoxa5 expression in myenteric plexus of enteric nervous system (ENS) as shown for E17.5 and D0 (K and N), whereas no signal was detected with probe B (L and O). e, Epithelium; lb, forelimb bud; m, muscular layer; me, mesenchyme; mg, midgut; nt, neural tube; s, submucosa. Bars, 100 µm.



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Fig. 2.   Postnatal ontogeny of enzymatic activities in small intestine of wild-type () and Hoxa5 homozygous mutant mice (triangle ). Enzymatic activity dosages were measured for sucrase, trehalase, maltase, lactase, and gamma -glutamyl transpeptidase (gamma -GTase) on duodenum (A), jejunum (B), and ileum segments (C) of specimens harvested at postnatal day (D) 0, D6, D15, D17, and D30. Results (U/g protein) were compiled according to genotype, and an ANOVA statistical analysis followed by a Tukey-Kramer test was performed. In Hoxa5 homozygous mutants, most of brush-border membrane hydrolytic activities at different levels of intestine displayed statistically significant differences at D15 and D17 compared with wild-type samples (black-triangle). At D15, activity of sucrase, trehalase, and maltase enzymes remained at newborn levels in Hoxa5 mutant specimens. By D30, all three enzymes were produced at wild-type levels. For lactase, at D15 jejunal activity in Hoxa5 mutants was significantly lowered than in wild-type specimens, whereas at D17, ileal lactase activity decreased more rapidly in mutants. For gamma -GTase, duodenal activity remained at newborn levels even in animals of postnatal D30 in Hoxa5 mutants. In contrast, jejunal and ileal activities were normal, except for a statistically significant decrease at D15.

Hybridization with probe B showed that the onset of expression of the larger transcripts did not occur until E10.5 in the midgut (Fig. 1, C and F). As for the 1.8-kb transcript, the larger forms were present in the mesenchymal layer (Fig. 1, F and I), and their mesenchymal expression stopped around E14.5 (data not shown). ENS never expressed the larger transcripts (Fig. 1, L and O). Consequently, from E14.5 onward, the sole Hoxa5 expression detected corresponded to the 1.8-kb transcript in ENS structures of the intestine (data not shown), showing that the larger transcripts displayed a more restricted spatial and temporal profile than the major 1.8-kb transcript during midgut morphogenesis.

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 and gamma -GTase activities, a more complex situation was found. In wild-type samples, lactase declines at weaning (15). However, although in the duodenum of the Hoxa5 mutant the enzymatic activity was normal at all stages, it was lower in the jejunum at D15 and the ileum at D17 compared with wild type (Fig. 2). As well, postnatal development of gamma -GTase level is normally seen as a fall in activity after birth, followed by an increase to reach adult level before weaning in the duodenum and 3 wk after birth in the jejunum and the ileum (28). In the duodenum of Hoxa5 mutants, the gamma -GTase activity remained at newborn levels even in weaned animals of postnatal D30. In contrast, jejunal and ileal activity remained normal, although a statistically significant decrease was observed at D15 in these portions of the intestine. Thus, in contrast to what was observed with the disaccharidases, these two enzymes displayed an abnormal pattern in their temporal regulation that may not be attributed to an immature condition.

Morphological 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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Fig. 3.   Comparative morphology of duodenum, jejunum, and ileum during embryonic and postnatal development. Duodenum (A-D), jejunum (E-H), and ileum (I-L) samples from wild-type (A, C, E, G, I, and K) and Hoxa5 homozygous mutants (B, D, F, H, J, and L) were stained with hematoxylin and eosin. Histological analysis failed to unveil any difference in development and morphology of small intestine between wild-type and Hoxa5 homozygous specimens. At E17.5 (A and B, E and F, and I and J), no delay in formation of nascent villi or in remodeling of epithelium and no alteration in submucosal and muscular layers could be observed. As well at D15 (C and D, G and H, and K and L), no apparent difference was found in organization of crypt-villus unit, in intervillous region, and in length and number of villus formed. e, Epithelium; m, muscular layer; s, submucosa. Bars, 100 µm.

The differential enzymatic activities measured at weaning in Hoxa5 mutants prompted us to carry out a more detailed histochemical analysis. Staining of several postnatal specimens for representation of all intestinal cell lineages failed to show any differences at D0, D6, D15, and D30. As shown for D15 jejunum samples, enterocytes were morphologically identical in Hoxa5 wild-type and mutant specimens, and the number and the localization of Paneth cells at the base of the crypts were unaffected (Fig. 4, A and B). The presence of neutral- and acid-mucus producing cells, as assessed by PAS and alcian blue stainings, was also similar for both genotypes (Fig. 4, C-F), whereas the relative proportion of enteroendocrine cells and their distribution remained unchanged in Hoxa5 mutants (Fig. 4, G and H). Furthermore, neither the proliferation nor the apoptotic rates were altered in the Hoxa5 mutant intestinal epithelium (data not shown). Finally, given the expression pattern of the 1.8-kb transcript in the myenteric plexus, we examined the consequences of the loss of Hoxa5 function on these structures. Using c-ret as a marker, we did not detect any apparent anomaly of the ENS, because c-ret-expressing ganglions were as numerous in both wild-type and mutant mucosa (shown for E15.5; Fig. 4, I and J). Taken together, these results established that the loss of Hoxa5 function did not affect in a noticeable fashion the morphogenesis and the cellular organization of the small intestine.


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Fig. 4.   Comparative histology of small intestine and ENS of wild-type and Hoxa5 homozygous mutant mice. Sections of jejunum at D15 from wild-type (A, C, E, and G) and Hoxa5 homozygous mutant (B, D, F, and H) animals were stained for representation of different intestinal cell lineages: hematoxylin and eosin (enterocytes and Paneth cells, shown by solid arrowheads and open arrows, respectively; A and B), periodic acid/Schiff (goblet cells, open arrow heads; C and D), alcian blue (acid-mucus producing cells, open arrowheads; E and F), and Grimelius silver method (enteroendocrine cells, solid arrows; G and H). Histochemical analyses fail to show any apparent difference between wild-type and Hoxa5 homozygous specimens in number, distribution, and morphological appearance of the various cell types. Furthermore, expression studies using c-ret, a specific marker of ENS, on E15.5 wild-type (I) and Hoxa5 mutant (J) samples showed that loss of Hoxa5 function did not affect formation of myenteric plexus. Bars, 100 µm.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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 gamma -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aubin, J., M. Lemieux, M. Tremblay, R. Behringer, and L. Jeannotte. Transcriptional interferences at the Hoxa4/Hoxa5 locus: importance of correct Hoxa5 expression for the proper specification of the axial skeleton. Dev. Dyn. 212: 141-156, 1998[Medline].

2.   Aubin, J., M. Lemieux, M. Tremblay, J. Bérard, and L. Jeannotte. Early postnatal lethality in Hoxa-5 mutant mice is attributable to respiratory tract defects. Dev. Biol. 192: 432-445, 1997[Medline].

3.   Bienz, M. Homeotic genes and positional signalling in the Drosophila. Trends Genet. 10: 22-26, 1994[Medline].

4.   Boulet, A. M., and M. R. Capecchi. Targeted disruption of Hoxc-4 causes esophageal defects and vertebral transformations. Dev. Biol. 177: 232-249, 1996[Medline].

5.   Dony, C., and P. Gruss. Specific expression of the Hox1.3 homeo box gene in murine embryonic structures originating from or induced by the mesoderm. EMBO J. 6: 2965-2975, 1987[Abstract].

6.   Duluc, I., J.-N. Freund, C. Leberquier, and M. Kedinger. Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development. J. Cell Biol. 126: 211-221, 1994[Abstract].

7.   Duluc, I., B. Jost, and J.-N. Freund. Multiple levels of control of the stage- and region-specific expression of rat intestinal lactase. J. Cell Biol. 123: 1577-1586, 1993[Abstract].

8.   Duluc, I., O. Lorentz, C. Fritsch, C. Leberquier, M. Kedinger, and J.-N. Freund. Changing intestinal connective tissue interactions alters homeobox gene expression in epithelial cells. J. Cell Sci. 110: 1317-1324, 1997[Abstract/Free Full Text].

9.   Duprey, P., K. Chowdhury, G. R. Dressler, R. Balling, D. Simon, J.-L. Guenet, and P. Gruss. A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine. Genes Dev. 2: 1647-1654, 1988[Abstract].

10.   Durbec, P. L., L. B. Larsson-Blomberg, A. Schuchardt, F. Costantini, and V. Pachnis. Common origin and developmental dependence on c-ret subsets of enteric and sympathetic neuroblasts. Development 122: 349-358, 1996[Abstract/Free Full Text].

11.   Gao, X., T. Sedwick, Y. B. Shi, and T. Evans. Distinct functions are implicated for the GATA-4, -5, and -6 transcription factors in the regulation of intestine epithelial cell differentiation. Mol. Cell. Biol. 18: 2901-2911, 1998[Abstract/Free Full Text].

12.   Gaunt, S. J., P. L. Coletta, D. Pravtcheva, and P. Sharpe. Mouse Hox-3.4: homeobox sequence and embryonic expression patterns compared with other members of the Hox gene network. Development 109: 329-339, 1990[Abstract].

13.   Gordon, J. I., and M. L. Hermiston. Differentiation and self-renewal in the mouse gastrointestinal epithelium. Curr. Biol. 6: 795-803, 1994.

14.   Grose, J. H., and M. Lebel. Radioimmunoassay for plasma corticosterone. Clin. Biochem. 11: 32-33, 1978[Medline].

15.   Henning, S. J., D. C. Rubin, and R. J. Shulman. Ontogeny of the intestinal mucosa. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 571-610.

16.   Holland, P. W. H., and J. Garcia-Fernàndez. Hox genes and chordate evolution. Dev. Biol. 173: 382-395, 1996[Medline].

17.   Jaffe, L., L. Jeannotte, E. K. Bikoff, and E. J. Robertson. Analysis of beta 2-microglobulin in the developing mouse embryo and placenta. J. Immunol. 145: 3474-3483, 1990[Abstract/Free Full Text].

18.   James, R., and J. Kazenwadel. Homeobox gene expression in the intestinal epithelium of adult mice. J. Biol. Chem. 266: 3246-3251, 1991[Abstract/Free Full Text].

19.   Jeannotte, L., M. Lemieux, J. Charron, F. Poirier, and E. J. Robertson. Specification of axial identity in the mouse: role of the Hoxa-5 (Hox1.3) gene. Genes Dev. 7: 2085-2096, 1993[Abstract].

20.   Kaestner, K. H., D. G. Silberg, P. G. Traber, and G. Shütz. The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation. Genes Dev. 11: 1583-1595, 1997[Abstract].

21.   Kondo, T., P. Dollé, J. Zakany, and D. Duboule. Function of posterior HoxD genes in the morphogenesis of the anal sphincter. Development 122: 2651-2659, 1996[Abstract/Free Full Text].

22.   Krumlauf, R. Hox genes in vertebrate development. Cell 78: 191-201, 1994[Medline].

23.   Larochelle, C., M. Tremblay, D. Bernier, J. Aubin, and L. Jeannotte. Multiple cis-acting regulatory regions are required for the restricted spatio-temporal Hoxa5 gene expression. Dev. Dyn. 214: 127-140, 1999[Medline].

24.   Lloyd, S., and W. Whelan. An improved method for enzymatic determination of glucose on the presence of maltose. Anal. Biochem. 30: 467-470, 1969[Medline].

25.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement using Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

26.   McDonald, M. C., and S. J. Henning. Synergistic effects of thyroxine and dexamethasone on enzyme ontogeny in rat small intestine. Pediatr. Res. 32: 306-311, 1992[Abstract].

27.   Ménard, D., and R. Calvert. Fetal and postnatal development of the small and large intestine: patterns and regulation. In: Growth of the Gastrointestinal Tract: Gastrointestinal Hormones and Growth Factors, edited by J. Morisset, and T. E. Solomon. Boca Raton, FL: CRC, 1991, p. 159-174.

28.   Ménard, D., C. Malo, and R. Calvert. Development of gamma -glutamyl transpeptidase activity in the mouse small intestine: influence of cortisone and thyroxine. Biol. Neonate 40: 70-77, 1981[Medline].

29.   Naftalin, L., M. Sexton, J. F. Whitaker, and D. Tracey. A routine procedure for estimating gamma -glutamyl transpeptidase activity. Clin. Chim. Acta 26: 293-296, 1969[Medline].

30.   Pachnis, V., B. Mankoo, and F. Costantini. Expression of c-ret proto-oncogene during mouse embryogenesis. Development 119: 1005-1017, 1993[Abstract/Free Full Text].

31.   Plateroti, M., D. C. Rubin, I. Duluc, R. Singh, D. Foltzer-Jourdainne, J.-N. Freund, and M. Kedinger. Subepithelial fibroblast cell lines from different levels of gut axis display regional characteristics. Am. J. Physiol. 274 (Cell Physiol. 43): C945-C954, 1998.

32.   Pollock, R. A., G. Jay, and C. J. Bieberich. Altering the boundaries of Hox3.1 expression: evidence for antipodal gene regulation. Cell 71: 911-923, 1992[Medline].

33.   Roberts, D. J., R. L. Johnson, A. C. Burke, C. E. Nelson, B. A. Morgan, and C. Tabin. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 121: 3163-3174, 1995[Abstract/Free Full Text].

34.   Roberts, D. J., D. M. Smith, D. J. Goff, and C. Tabin. Epithelial-mesenchymal signaling during the regionalization of the chick gut. Development 125: 2791-2801, 1998[Abstract/Free Full Text].

35.   Rubin, D. C., E. Swietlicki, K. A. Roth, and J. I. Gordon. Use of fetal intestinal isografts from normal and transgenic mice to study the programming of positional information along the duodenal-to-colonic axis. J. Biol. Chem. 267: 15122-15133, 1992[Abstract/Free Full Text].

36.   Stein, S., R. Fritsch, L. Lemaire, and M. Kessel. Checklist: vertebrate homeobox genes. Mech. Dev. 55: 91-108, 1996[Medline].

37.   Suh, E., and P. G. Traber. An intestine-specific homeobox gene regulates proliferation and differentiation. Mol. Cell. Biol. 16: 619-625, 1996[Abstract].

38.   Tennyson, V. M., M. D. Gershon, P. R. Wade, D. A. Crotty, and D. J. Wolgemuth. Fetal development of the enteric nervous system of transgenic mice that overexpress the Hoxa-4 gene. Dev. Dyn. 211: 269-291, 1998[Medline].

39.   Traber, P. G., and D. G. Silberg. Intestine-specific gene transcription. Annu. Rev. Physiol. 58: 275-297, 1996[Medline].

40.   Yeh, K. Y., and F. Moog. Influence of the thyroid and adrenal glands on the growth of the intestine of the suckling rat, and on the development of intestinal alkaline phosphatase and disaccharidase activities. J. Exp. Zool. 200: 337-348, 1977[Medline].

41.   Yokouchi, Y., J.-I. Sakiyama, and A. Kuroiwa. Coordinated expression of Abd-B subfamily genes of the HoxA cluster in the developing digestive tract of chick embryo. Dev. Biol. 169: 76-89, 1995[Medline].

42.   Warot, X., C. Fromental-Ramain, V. Fraulob, P. Chambon, and P. Dollé. Gene-dosage dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts. Development 124: 4781-4791, 1997[Abstract/Free Full Text].

43.   Wolgemuth, D. J., R. R. Behringer, M. P. Mostoller, R. L. Brinster, and R. D. Palmiter. Transgenic mice overexpressing the mouse homeobox-containing gene Hox-1.4 exhibit abnormal gut development. Nature 337: 464-467, 1989[Medline].


Am J Physiol Cell Physiol 277(5):C965-C973
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