The divergent homeobox gene Hex, also known as Prh, was first identified in a screen for homeobox genes expressed in hematopoietic cells (Bedford et al., 1993; Crompton et al., 1992; Hromas et al., 1993). In addition to the homeodomain, Hex contains a region near the N-terminus that is proline-rich and a C-terminus that is highly acidic. These regions may serve to regulate the transcription of target genes (Crompton et al., 1992). At the amino acid level, the homeodomain of Hex is 55% homologous to Hlx/HB24 and Hox11, two other divergent murine homeobox genes and only 47% homologous to Antp. Hex has been mapped to chromosome 10 in the human (Hromas et al., 1993) and to chromosome 19 in the mouse (Ghosh et al., 1999), both regions that are outside the known genomic clusters of mammalian homeobox genes. Hex homologues have been cloned from chick (Crompton et al., 1992), mouse (Bedford et al., 1993), frog (Newman et al., 1997), zebrafish (Ho et al., 1999), rat (Tanaka et al., 1999), and human (Crompton et al., 1992; Hromas et al., 1993), indicating that the gene is conserved throughout evolution.
Recent studies on the expression of Hex in early mouse embryogenesis suggest that Hex may be involved in anterior-posterior patterning and early vascular development (Thomas et al., 1998). Hex expression, as determined by in situ hybridization during early development in the chick, frog, and mouse, shows similar domains of expression (Keng et al., 1998; Newman et al., 1997; Thomas et al., 1998; Yatskievych et al., 1999). The function of Hex during development and in the adult animal remains unknown. In an effort to begin to understand the role of Hex during organogenesis, we now report the expression of Hex in the mouse from E10.0–adult, including expression in the thymus, pancreas, gallbladder, and bile duct, organs where expression has not been previously demonstrated in situ. Our results, along with those of others, suggest that Hex is functioning from very early in embryogenesis through adulthood and, therefore, is likely to be involved in the regulation of genes at multiple developmental stages, including mature tissues and cell types.
RESULTS AND DISCUSSION
Expression of Hex in E10.0–E16.5 Embryos
At E10.0, Hex transcripts are present in the liver, thyroid, thymus, gallbladder, and pancreas (Fig. 1A–F). Figure 1B is a frontal view of the pharyngeal region of the embryo with the anterior structures (heart and liver) removed showing strong expression in the developing thyroid and third pharyngeal pouch, from which the thymus develops. Sections through this region show that the expression in the thyroid (Fig. 1C) is in all cells, with perhaps enhanced expression in the outer layer of cells. In the third pharyngeal pouch (Fig. 1D), Hex transcripts are present only in the endodermally derived cells of the pouch and not in surrounding mesoderm. For the first time, transcripts are seen in the gallbladder and in the dorsal pancreas (Fig. 1E). A transverse section through the region of the developing liver (Fig. 1F) reveals that expression in the developing liver is seen only in the endodermal cells that are budding into the mesoderm of the septum transversum and not in the mesoderm itself.
At E13.5, Hex transcripts are present in the thyroid, thymus, liver, pancreas, and in the lung (Fig. 1G–J). Expression in the thyroid and thymus appears ubiquitous. In the liver, expression is seen throughout the entire hepatic parenchyma (Fig. 1H), whereas expression in the pancreas and extrahepatic bile ducts is confined to the epithelium (Fig. 1H and I). Hex is present in both the mesenchyme and the epithelium of the E13.5 lung (Fig. 1J), although expression is highest in the distal lung epithelium as assessed by whole-mount in situ hybridization (data not shown). By E16.5, expression in all organs appears to decrease from early ages (Fig. 1K–N). Expression in the thyroid and pancreas remains high with lower, but still easily detectable, levels present in the liver and lung. Thymic expression is just barely detectable (Fig. 1L). In the thyroid and pancreas, expression is highest in the epithelial cells lining the thyroid follicles and pancreatic ducts (Fig. 1K and M), and expression in the liver and lung continues in both epithelium and mesenchyme (Fig. 1N).
Expression of Hex in E18.5 and Adult Tissues
At E18.5, Hex expression persists in the thyroid gland and, as is true at E16.5, is most abundant in the epithelium lining the thyroid follicles (Fig. 2A). Abundant transcripts are also present in the epithelium of the extrahepatic bile ducts (Fig. 2B), whereas expression in the lung (Fig. 2C) and liver (data not shown) is markedly decreased compared with E16.5. Expression can no longer be detected in the thymus (Fig. 2D) and pancreas (data not shown).
Hex expression persists in the adult animal and, as shown in Figure 2E–G, is seen in the multiple cell types in the lung, thyroid, and liver. In adult lung, expression of Hex appears relatively uniform (Fig. 2E), present in both the epithelial and mesenchymal compartments of the lung. Hex transcripts appear to be present in the epithelium of alveoli and bronchi. In the adult thyroid, Hex is highly expressed in the epithelial cells lining the thyroid follicles (Fig. 2F). No expression is seen in the adjacent cartilage or muscle. In the liver, like the lung, Hex transcripts appear ubiquitously expressed (Fig. 2G). However, expression in intrahepatic bile ducts appears to be higher than in the surrounding hepatic parenchyma, consistent with findings at earlier ages showing that Hex expression in the hepatobiliary tissue is highest in cells of the biliary tract. There is no significant expression of Hex messenger ribonucleic acid (mRNA) in adult pancreas or thymus (data not shown).
Hex Expression and Development of Foregut-Derived Organs
The divergent homeobox gene Hex is expressed in the mouse beginning at E4.5 in the primitive endoderm of the blastocyst and by E5.5 is expressed asymmetrically in the embryo as evidenced by expression in the cells of the anterior visceral endoderm (AVE) (Thomas et al., 1998). The expression of Hex in the AVE precedes primitive streak formation by at least 12 hr. There is an increasing body of evidence that suggests that the AVE affects anterior patterning of the embryo (reviewed in Beddington and Robertson, 1998; Beddington and Robertson, 1999). Recent evidence in the frog shows that the development of the anterior endoderm and the endodermal expression of Xhex are modulated both by elements of the Wnt/β-catenin signaling pathway and by TGF-β signaling pathway (Jones et al., 1999; Zorn et al., 1999). As yet, however, the specific role that Hex plays in anterior-posterior patterning and in endoderm development remains unknown. Here, we show that Hex is expressed throughout embryogenesis and in adult tissues, primarily in tissues that are derived from the foregut—the thymus, thyroid, lung, liver, gallbladder, and pancreas. Maintenance of Hex expression in a subset of these organs in the adult animal indicates that Hex is important not only during embryonic development but also for the ongoing function of mature cell types and organs.
Previous reports of Hex expression in the mouse embryo (Thomas et al., 1998; Keng et al., 1998), in frog (Newman et al., 1997), and in chick (Yatskievych et al., 1999) have focused on earlier stages of development and are consistent with a potential role for Hex in development of anterior structures, vasculature, hematopoietic cells, and in foregut endoderm. We have extended these studies to the period of organogenesis through adulthood. We have confirmed the expression of Hex in the anterior foregut endoderm adjacent to the developing heart at E8.5 (data not shown). This endodermal tissue forms the hepatic diverticulum, which interacts with the precardiac mesoderm to begin formation of the liver (reviewed in Zaret, 1996). Approximately 24 hr later, the hepatic endoderm, which has moved toward the midsection of the embryo and is no longer in direct contact with the cardiac mesoderm, begins migrating into the mesoderm of the surrounding septum transversum in a cordlike fashion. We show, at this stage of liver development, that Hex expression is confined to the invading endodermal cells and is not expressed in the surrounding mesoderm. Also at this age (E10.0), strong Hex expression is present in the gallbladder primordium, a structure that develops from the nearby posterior endoderm. At later ages and in the adult, Hex expression persists in most cell types in the liver, with highest expression seen in the bile ducts (both intrahepatic and extrahepatic) and gallbladder. The strong expression of Hex in the bile duct epithelium that persists in the adult animal suggests that Hex may be important for bile acid transport. A consensus-binding site for the Hex homeodomain has been proposed by others (Crompton et al., 1992), and this core-binding site is present in the promoter of the ntcp gene, a bile acid transporter (Karpen et al., 1996). We recently observed that Hex transactivates the ntcp promoter in vivo (Denson et al., 2000), making the ntcp promoter the first known target of Hex in vitro. Functional analysis of Hex in vitro using fusion-protein expression vectors of GAL4 DNA-binding domain and Hex suggests that Hex can also function as a repressor (Tanaka et al., 1999). Further investigation in vivo is needed to definitively determine the downstream targets of Hex and whether it acts as a repressor, activator, or both.
From E10.0 to adult, the organ that appears to express Hex at the highest level is the thyroid. The thyroid follicular cells originate by invagination of pharyngeal endoderm beginning at E8–8.5 of mouse development (Ericson and Frederiksson, 1990). Shortly thereafter, the thyroid primordium migrates distally, reaching its final destination anterior to the trachea by E13–14. Several homeobox genes are known to be expressed in the thyroid and have been shown, by targeted mutagenesis, to be essential for normal thyroid development. These include TTF-1 (Guazzi et al., 1990; Kimura et al., 1996; Lazzaro et al., 1991; Manley and Capecchi, 1995), Pax8 (Mansouri et al., 1998; Mansouri et al., 1999; Mansouri et al., 1999), and Hoxa3 (Manley and Capecchi, 1995). Mutations of the Pax8 gene have also been shown to be present in humans with the thyroid dysgenesis (Macchia et al., 1998). Hex expression in the thyroid gland begins at E8.5 and continues into the adult animal. In the adult thyroid, a high-degree expression is seen in the follicular cells. This pattern of expression is similar to that of TTF-1. Hex is not expressed in the parathyroid gland in the embryo or adult. The specific role that Hex plays in thyroid development and thyroid specific gene expression remains to be delineated, although on the basis of these data some role for Hex in thyroid development is likely.
Several homeobox genes, members of both the Hox and divergent classes of murine homeobox genes, are expressed in the embryonic lung (Bogue et al., 1994; Cardoso, 1995; Lazzaro et al., 1991; Volpe et al., 1997; Wall et al., 1992). Studies of mice with null mutations in Hoxa-5 and TTF-1 show distinct roles of these genes in lung development (Aubin et al., 1997; Kimura et al., 1996). In addition, levels of homeobox gene expression in the lung are altered by factors and pathological conditions known to affect lung development and morphogenesis, such as retinoic acid (Bogue et al., 1994; Cardoso et al., 1996), dexamethasone and growth factors (Chinoy et al., 1998), and maternal diabetes (Jacobs et al., 1998). Hex expression in the lung does not begin when the lung buds form from the foregut at E9.5. Rather, it begins approximately 2 days later at E11.5 (data not shown). By E13.5, moderate expression is seen in the embryonic lung in both the epithelial and mesenchymal compartments. Hex expression in the epithelium of the large bronchi appears to be low or nonexistent. Expression in the lung decreases at E16.5, as it appears to do in other organs except the thyroid, and then is seen at higher levels in the adult lung. These data suggest that Hex expression is not necessary for the initial lung buds to form from the trachea at E9.5–10 but that it may be necessary for later stages of lung morphogenesis and for the regulation of genes expressed in mature lung.
Hex expression in the dorsal pancreas was first detected at approximately E10. This is the stage when the dorsal pancreatic bud forms as an outpouching from the gut endoderm (reviewed in Edlund, 1998; Slack, 1995). We did not detect expression of Hex in the pancreatic gut endoderm before this stage, unlike the expression of the divergent homeobox gene Pdx-1 (Edlund, 1998). Hex expression was high in the pancreatic primordium at E13.5 and at E16.5 and was confined to the epithelium of the ducts. It is interesting that Hex transcripts can no longer be detected in the pancreas at E18.5 and in the adult, suggesting that Hex has a specific function only during pancreatic organogenesis. Likewise, in the thymus, Hex is abundantly expressed at the early stages of development and by E18.5, Hex RNA can no longer be detected in thymic tissue. In addition, expression in the thymus does not appear to be confined to a particular cell type.
In summary, we have shown that the divergent homeobox gene Hex is expressed throughout embryogenesis and in the adult mouse in several organs of derived from foregut endoderm. Specifically, we have shown, for the first time, detailed expression of Hex in the thymus, pancreas, gallbladder, and biliary tract early in development. We have established the pattern of Hex expression in the adult liver, lung, and thyroid. These results, along with those of others, indicate that Hex is likely to regulate genes involved in multiple stages of the murine life cycle, from establishment of the A-P axis, through organogenesis, to the maintenance of mature organ function.
Whole-mount in situ hybridization and histology.
Timed-pregnant cd-1 mice were used to obtain embryos for analysis. The morning after mating was considered day 0.5 of gestation. Embryos were dissected in phosphate-buffered saline (PBS) and fixed for 1 hr in 4% paraformaldehyde/PBS at 4°C. In situ hybridization was performed according to Wilkinson (Wilkinson, 1992). Treatment time with proteinase K was varied according to the stage of the embryo. Digoxigenin-labeled antisense and sense Hex probes were generated according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN) from full-length mouse Hex, which included approximately 500 bp of 5′ untranslated sequence. For histology, stained embryos were cryoprotected in 30% sucrose for 24 hr, frozen in an isopentane bath in TissueTek, and serially sectioned.
In situ hybridization on embryo and tissue sections.
Embryos and tissues were collected and fixed for 24 hr in 4% paraformaldehyde/PBS. They were cryoprotected in 30% sucrose, frozen in Tissue Tek in an isopentane bath, and serially sectioned (10 μm) in a Jung cryostat. Full-length Hex antisense and sense probes were labeled with 33P-UTP. The tissues were processed according to Hogan (Hogan et al., 1986) and developed after 4–7 days. Bright- and dark-field images were captured, pseudocolored, and merged by using Adobe Photoshop.
We thank Feisha Zhao and Chris Wilson for excellent technical assistance.