Pitx2 is a Critical Early Regulatory Gene in Normal Cecal Development

Abstract

Purpose

The murine cecum is a critical digestive structure. Morphogenesis of the cecum involves several key genes including Homeobox (Hox) d12. Ectopic expression of Hoxd12 has been shown to result in cecal agenesis and a down regulation of both Fibroblast growth factor 10 (Fgf10) and the Pituitary homeobox 2 gene (Pitx2). Homozygous null mutation of Fgf10 or its cognate receptor Fgfr2IIIb results in severe cecal defects where there is the initiation of mesodermal budding, but a failure of the endoderm to grow and extend into this structure. We examined the expression of Pitx2 in the cecum and hypothesized that homozygous null mutation of Pitx2 would result in cecal agenesis.

Methods

IACUC Approval was obtained for these studies. Whole mount in situ hybridizations for Pitx2 were performed on wild-type embryos between Embryonic days (E) 11.0 and E12.5. Pitx2 −/− and Fgfr2IIIb −/− embryos were generated from n/+ heterozygote breedings and harvested at E10.5, E11.5, and E13.5. Genotypes were confirmed by PCR. Morphology of Pitx2 −/− cecae were compared to those of wild-type littermates and Fgfr2IIIb −/− embryos at identical stages. Embryos were fixed overnight and photographed the following day.

Results

Pitx2 is expressed in the cecal mesoderm and endoderm as early as E11.0. Expression becomes increasingly more robust by E12.5. Homozygous null mutation of Pitx2 results in agenesis of the cecum. In contrast to Fgfr2IIIb −/− embryos, which demonstrate a persistent mesodermal bud as late as E18.5, no mesodermal bud is present in Pitx2 −/− embryos.

Conclusions

Our findings demonstrate that Pitx2 is a critical regulatory gene in cecal morphogenesis and suggest that Pitx2 is required for initiation of mesodermal budding and likely resides upstream of Fgf10-Fgfr2IIIb signaling in the normal development of this structure.

Introduction

The cecum is tubular gut structure that lies between the small intestine and colon on the antimesenteric side of the bowel. It is host to a number of gut microbes that assist in the digestion of complex carbohydrates (1, 2). Homozygous null mutations in specific genes such as Fgf9 result in both cecal agenesis as well as congenital short gut in mice (3, 4). This indicates that the process of lengthwise intestinal growth and cecal development are linked and that cecal development can serve as a model for studying molecular pathways involved in lengthwise intestinal growth. The morphogenesis of the cecum can be described in three general steps: budding, elongation, and arrest. A number of genes have been implicated in the formation of this structure. For example loss of Fgf9 expression in the endoderm results in cecal agenesis and also loss of Fgf10 expression within the cecal mesoderm (3). Loss of Fgf10 signaling via mutation in either Fgf10 or the gene for its cognate receptor Fgfr2IIIb in the endoderm results in a phenotype in which a mesodermal aggregation or bud forms on the antimesenteric side of the intestine but the endoderm fails to bud into this structure (5). These findings suggest that the budding phase of cecal development can be subdivided into additional steps. The first step would appear to be the induction of a mesodermal aggregation or bud which is dependent on Fgf9. During the formation of the mesodermal bud, Fgf10 is expressed in the mesoderm which appears to act upon its target receptor Fgfr2IIIb in the endoderm. Thereafter, the endoderm begins to bud into the aggregation of cecal mesoderm and then the elongation phase of the endodermal and mesodermal components of the cecum begins.

Pitx2 is a transcription factor that is expressed during cecal development (5). The role of this gene in cecal development and its tissue specific expression pattern (endoderm and mesoderm) has not been described. Over expression of the Hoxd12 has been shown result in a loss of Fgf10 and Pitx2 expression in the cecal region as well as cecal agenesis (6). This phenotype is very similar to that described for Fgf9 −/− mutants (3). To better understand the role of Pitx2 in the budding phase of cecal development we chose to characterize the tissue specific expression of Pitx2 during this phase and examined the morphology of the cecum in Pitx2 −/− mouse embryos. These embryos show severe abnormalities as they fail to undergo proper turning and abdominal wall closure and are unable to progress beyond E 14.5 in development (7, 8).

We hypothesized that Pitx2 would be expressed in both the endoderm and mesoderm of the cecum. Because Pitx2 expression has been observed in the mesodermal bud of the cecum in Fgf10 −/− and Fgfr2IIIb −/− mutants (5), we postulated that Pitx2 resides upstream of the Fgf10 signaling pathway and therefore the cecal agenesis observed due to over expression of Hoxd12 is a direct result of the loss of Pitx2 expression. Accordingly we predicted that Pitx2 −/− cecum would lack the mesodermal bud seen in Fgf10 −/− and Fgfr2IIIb −/− mutants.

Methods

Animals

IACUC approval for these studies was obtained from the University of Wisconsin School of Medicine and Public health (P.F.N. protocol # M02258, YS #10-05008). All animals were maintained in a clean facility with ad libitum access to fresh food and water, and kept on a 12 hour alternating light/dark cycle. Fgfr2IIIb −/− and Pitx2 −/− embryos were generated through traditional heterozygous +/n breedings (9, 10) of established lines. Genotyping of embryos for Fgfr2IIIb −/− and Pitx2 −/− alleles was performed as described previously (9, 10).

Morphologic studies

Embryos were harvested at either (E) 10.5, E11.0, E11.5, E12.0, E12.5, E13.5 or E18.5. The thorax was opened and the embryos were fixed overnight in 4% paraformaldehyde at 4°C. The intestines were dissected out and photographs of the cecal regions were obtained under a standard dissecting microscope.

Histological studies

E12.5 embryos were dissected free of the yolk sac and embryos were fixed overnight at 4°C in 4% paraformaldehyde. They were then dehydrated through a series of escalating Methanol/PBST washes, isopropyl alcohol and xylene. They were washed 3 times in paraffin at 65 °C in a vacuum oven and then embedded in paraffin. Sagittal sections were taken a 10μm thickness. Sections were stained for standard H&E and photographed under a standard light microscope.

Whole mount in situ hybridization

Samples were dehydrated through a series of escalating PBS-Tween Methanol steps then stored overnight at −20 °C. The following day they were rehydrated into PBS-Tween, treated with hydrogen peroxide and proteinase K, and in situ hybridization was performed at 70 °C with antisense probes for Pitx2 (11). Specimens were stained at 37°C, washed in PBS-Tween and fixed with 4% PFA. Photographs were taken under a dissecting light microscope.

Results

Morphogenesis of the cecum

We examined the morphogenesis of the cecum in detail. The cecum can be seen forming as early as E10.5 just distal to the bend in the intestinal tube (Figure 1A). This is a full day earlier than had been previously reported (5). As the intestine increases in length at E11.0, the mesoderm continues to thicken in the cecal region (Figure 1B). By E11.5 the endoderm can be seen extending into the mesodermal bud (Figure 1C). After this, the mesoderm and endoderm extend outward from the antimesenteric side of the intestine (Figure 1D and E). On sagittal section at E12.5, the endoderm near the distal tip of the cecum is cuboidal (Figure 1F, white arrow) where as that in the proximal region is stratified pseudo-columnar (black arrow). The basement membrane in the distal region is thinned compared to the proximal region indicating invasion of the endoderm bud into the mesoderm at this stage.

Figure 1.

A–E. Morphogenesis of the murine cecum. Whole mount photographs to the murine cecum as indicated by white arrows between E10.5 to E12.5.

F. A sagittal section through the cecum at E12.5. Black arrow indicates stratified pseudo-columnar epithelium and thick basement membrane of the proximal cecum. White arrow indicates distal cecum with cuboidal endoderm and a thinned basement membrane. Small intestine (s.i.), colon (co.).

Pitx2 expression during intestinal development

Examination of Pitx2 expression during intestinal development demonstrates that this gene is expressed in distinct regional patterns. In the small intestine its expression is restricted to the endoderm and is seen as early as E11.0 (Figure 2A). In the cecum, Pitx2 is expressed in both the endoderm and the mesoderm at E11.0 (Figure 2A). In contrast no expression is seen in the colon. These distinct regional patterns persist through at least E12.5 (Figures 2B and C). The expression in the mesoderm of the cecum is asymmetric in that there is more intense staining of the side of the cecum that is towards the intestine or rostral (white arrows). By E11.5 the endodermal bud can be seen extending into the mesoderm and the mesodermal staining for Pitx2 remains rostral (Figure 2B). The distribution of the mesoderm at this stage is also more rostral than caudal as well. At E12.5 in development, Pitx2 is robustly expressed throughout the entire cecum and discrepancies in rostral-caudal staining are absent (Figure 2C). The data indicate that Pitx2 is expressed within the endoderm and mesoderm of the developing cecum. In contrast, expression in the small intestine appears limited to the endoderm whereas in the colon expression is absent at all stages examined.

Figure 2.

Expression of Pitx2 in the early murine cecum. Whole mount in situ hybridization for Pitx2 at A. E11.0, B. E11.5 and C. E12.5. White arrows indicate rostral side of the cecum. Small intestine (s.i.), colon (co.) and intestinal mesentery (mese.).

Loss of Pitx2 expression results in an absence of a mesodermal cecal bud at E13.5

We next examined the morphology of cecal development in Pitx2 −/− embryos. We compared this morphology to that of wild-type embryos and Fgfr2IIIb −/− embryos which have a defect in cecal development, but do form a mesodermal bud (5). At E 13.5, the wild type cecum has extended and the distribution of the mesoderm continues to be asymmetric with more being distributed on the rostral side (Figure 3A). By E18.5, the cecum has lengthened substantially and the asymmetric distribution of mesoderm is absent although the cecum curves rostrally towards the small intestine (Figure 3D). At E13.5, the Fgfr2IIIb −/− embryo has a mesodermal bud (Figure 3B). This bud persists until term, yet no endoderm is present and the bud has elongated only minimally (Figure 3E). Similar to the other specimens, the Pitx2 −/− embryo at E13.5 has a clear angulation of the intestine at the boundary between the small intestine and colon. In contrast, however, there is no significant accumulation of the mesoderm in this area and no endodermal budding (Figure 3C, white arrow). These findings indicate that Pitx2 is required for in the induction and development of the mesodermal bud of the cecum.

Figure 3.

Pitx2 −/− embryos fail to develop a cecum. A. Wild-type cecum at E13.5 B. Fgfr2IIIb −/− cecum at E13.5. C. Pitx2 −/− intestine demonstrating a lack of a cecum at E13.5. D. Wild-type cecum at E18.5. exhibiting loss of asymmetric distribution of mesoderm and rostral looping of the cecum E. Fgfr2IIIb −/− cecum at E18.5 demonstrating the persistence of a mesodermal bud without elongation. Small intestine (s.i.), colon (co.) and cecum (ce.).

Discussion

Our findings demonstrate that Pitx2 is a critical regulatory gene in cecal morphogenesis. Expression of Pitx2 in the cecum is observed in both the endoderm and mesoderm which is a unique staining pattern compared to the remainder of the intestine. Our data also suggests that Pitx2 is required for initiation of mesodermal budding which appears to be a prerequisite for budding and extension of the endoderm into this structure.

Disruptions in Fgf10 signaling either through mutation of the Fgf10 gene or the gene for its receptor (Fgfr2IIIb) result in the formation of a cecal mesodermal aggregation or bud and expression of Pitx2 within this structure (5) however subsequent endodermal budding fails to occur after this. Our observation that Pitx2 −/− embryos fail to form a mesodermal cecal bud suggests that Pitx2 resides upstream of Fgf10-Fgfr2IIIb signaling in the normal development of this structure.

We have observed that the expression of Pitx2 in the cecal region in both the endoderm and mesoderm is unique to this part of the intestine. In contrast Pitx2 expression in the small intestine is limited to the endoderm and Pitx2 is to be absent from the colon. The cecal region has previously been described as having a unique permissivity to Fgf gene expression (3). This underscores the likelihood that Pitx2 regulates Fgf signaling. However, at least 2 Fgfs are in involved in the morphogenesis of the cecum: Fgf9 and Fgf10 (3, 5). Pitx2 could have a role in regulating both of these genes. For example, expression of Fgf10 in the mesoderm appears to depend on the formation of a mesodermal bud (3). And mesodermally expressed Pitx2 could be a key regulator of Fgf10 expression after formation of the mesodermal bud. Pitx2 is also expressed in the endoderm of the cecum as is Fgf9 (3). Since mutations in either Pitx2 or Fgf9 results in an identical phenotype of cecal agenesis with failure of the mesoderm to form a bud, it is possible that endodermal Pitx2 is regulating Fgf9 expression and mesodermal bud formation as well. We are undertaking studies to examine these possibilities using a tissue specific Cre strategy.

Examination of other structures in development also suggests a link between Pitx2 and Fgf10. For example In fact Pitx2 −/− embryos have massive abdominal wall defects. We have previously reported that mutations in gene encoding the cognate receptor isoform for Fgf10, Fgfr2IIIb, result in with mouse omphalocele (12). The greater severity of abdominal wall defect in Pitx2 −/− embryos compared compound Fgfr1/Fgfr2 embryos suggest that as in the cecum Pitx2 resides upstream in regulating Fgf10 signaling. We predict based the expression pattern of Pitx2 in the mesoderm that as a transcription factor is critical in regulating Fgf10 levels.

The cecum is a good model for studying lengthwise intestinal growth. In fact it appears to share many of the temporal and physical growth characteristics of the small intestine. Growth of the cecum begins with the budding phase which corresponds to the beginning of looping morphogenesis of the small intestine (E10.5): the most rapid phase of lengthwise intestinal growth. Similar to the small intestine, the cecum appears to bend or loop. Additionally mutations in genes that disrupt cecal development such as Fgf9 also result in foreshortened intestine (3, 4). However, one of the major advantages of this organ as a model for intestinal growth is that it can be manipulated by in vitro culture, enabling one to study the affects of various exogenously applied growth factors (5). In future studies, we intend to employ this strategy to further delineate the role of Fgfs in the development of this organ and in the mechanisms of linear intestinal growth. It would be better to mention communication between mesoderm and endoderm since Fgfr2b mutant develop mesoderm but no endoderm. These interaction would be very interesting.