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Keywords:

  • brain;
  • development;
  • embryogenesis;
  • liver X receptor;
  • nuclear receptors

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

We characterized the expression pattern of the nuclear receptors liver X receptor (LXR) α and β during mouse embryonic development and in adulthood by in situ hybridization experiments. LXRα and LXRβ are detected in the liver starting at 11.5 days postcoitum. Later, LXRα expression remains high in organs involved in lipid homeostasis, such as liver, intestine, and brown adipose tissue, whereas LXRβ is more ubiquitously expressed and enriched in tissues of neuronal and endocrine origin. Anat Rec Part A 277A:312–316, 2004. © 2004 Wiley-Liss, Inc.

LXRα (NR1H3) and LXRβ (NR1H2) are nuclear receptors initially identified in the liver (Lu et al., 2001; Francis et al., 2003) that bind as heterodimers with retinoid X receptor (RXR) to direct DNA repeats separated by four nucleotides. LXRs are activated by a number of naturally occurring oxysterols and by synthetic nonsteroid compounds such as T0901317 and GW3965 (Janowski et al., 1996, 1999; Lehmann et al., 1997; Schultz et al., 2000; Collins et al., 2002). LXRs regulate genes involved in cholesterol homeostasis, including CYP7A1, ABCA1, CETP, LPL, and SREBP-1c, indicating that these nuclear receptors exert key metabolic functions (Lehmann et al., 1997; Lu et al., 2000; Luo and Tall, 2000; Venkateswaran et al., 2000; Luo et al., 2001). As a matter of fact, LXRα null mice display severe abnormalities in lipid and bile acid homeostasis (Peet et al., 1998). These studies have established the role of LXRα as a sterol sensor and metabolic regulator of cholesterol and lipid homeostasis. The exact function of LXRβ is, however, at present less clear. We characterize here the onset and pattern of expression of mLXRα and mLXRβ during embryonic development by in situ hybridizations in embryos between 9.5 to 16.5 days postcoitum (dpc) and in multiple adult tissues. Our data indicate that LXRβ could have important endocrine and neuronal functions.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

CD1 embryos from 9.5 to 16.5 dpc and C57B6 adult tissues were directly embedded in cryomatrix (Shandon, Pittsburgh, PA). In situ hybridizations were performed as described (Dolle and Duboule, 1989). In summary, mouse LXRα and LXRβ cDNAs were linearized by BamHI and HindIII, respectively. Antisense mLXRα and -β RNA were synthesized using T7 and T3 RNA polymerase (Promega, Madison, WI), respectively, and 35S-CTP as radiolabeled nucleotide. Sections were incubated in acetone for 20 min at 4°C and postfixed in 4% paraformaldehyde in PBS for 15 min at 4°C, washed twice with PBS. Tissues were then equilibrated with 0.1 M triethanolamine (TEA) in PBS for 5 min at room temperature (RT), acetylated in freshly prepared 0.25% acetic anhydride in 0.1 M TEA under vigorous shaking, washed twice with PBS, and incubated at 60°C for 10 min in 50% formamide in PBS. Cryosections were then dehydrated, air-dried for 1 hr at RT, and hybridized with the radiolabeled probes overnight at 52°C in a humidified chamber. Sections were washed in 5 × SSC at 55°C for 1 hr, equilibrated with 4 × SSC for 15 min at 37°C, incubated with 20 μg/ml of Rnase A for 30 min at 37°C, and washed with 4 × SSC 10 min at 37°C. Sections were then subjected to three high-stringency washes: 50% formamide for 1 hr at 55°C, 2 × SSC for 15 min at 55°C, followed by a 15-min wash in 0.1 × SSC at 55°C. Negative controls were performed in parallel using sense RNA probes for mLXRα and mLXRβ (data not shown).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

Earlier studies by Northern blotting have reported the presence of LXRα mRNA in 13.5 dpc whole embryos (Willy et al., 1995). In the present study, a more detailed analysis was performed by in situ hybridizations. In these experiments, LXRα mRNA expression begins at 11.5 dpc in hepatoblasts of the liver and remains exclusively expressed in this tissue until 13.5 dpc (Fig. 1A–D). Between 14.5 and 16.5 dpc, LXRα mRNA becomes also evident in brown adipose tissue, lung, small intestine, the submandibular gland, and the thyroid gland (Fig. 1C–H). The general expression pattern is consistent with a role for LXRα in lipid and bile acid homeostasis, since liver, small intestine, and brown adipose tissue are all involved in lipid metabolism. Although this nuclear receptor is expressed very early during embryonic development, LXRα null mice have no overt defects in organogenesis and tissue differentiation, suggesting that this receptor is redundant for developmental control.

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Figure 1. LXRα and LXRβ mRNA expression by in situ hybridizations of embryos from 11.5 to 16.5 dpc. AH: LXRα is expressed in liver (liv), thyroid gland (thyr), lung (lu), small intestine (int), submandibular gland (sug), and brown adipose tissue (bat). IP: In situ hybridizations of embryos demonstrate LXRβ expression in liver, trigeminal ganglion (tg), vestibulocochlear ganglion (co), olfactory epithelium (olf), medulla oblongata (mo), thyroid gland, thymus (thym), salivary gland (sg), adrenal (ad), dorsal root ganglion (rg), kidney (k), small intestine, bladder (b), brown adipose tissue (bat), and testis (t). Representative images of embryos of 11.5 dpc (A, B, I, J), 14.5 dpc (C, D, K, L), 15.5 dpc (E, F, M, N), and 16.5 dpc (G, H, O, P) are shown.

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Like LXRα mRNA, LXRβ mRNA is also detected from 11.5 dpc on (Fig. 1I–J). Its expression is initially confined to the liver (hepatocytes, Fig. 1I–J), but it is also expressed early on in the retina, olfactory epithelium, vestibulocochlear and trigeminal ganglions (data not shown). At 14.5 dpc, LXRβ expression persists in these tissues (Fig. 1K–L). At 15.5 dpc, LXRβ becomes strongly expressed in the thymus, thyroid gland, kidney, adrenal, small intestine, and dorsal root ganglion (Figs. 1M and N and 2A), whereas lower levels are detected in lung (data not shown), bladder, trigeminal ganglion, salivary gland, and medulla oblongata (Fig. 1M and N). At 16.5 dpc, LXRβ gene expression is detected in two additional tissues, the testis (seminiferous tubules; Figs. 1O and P and 2A) and brown adipose tissue (Fig. 1O and P). The presence of high levels of LXRβ in several endocrine tissues, such as the thyroid gland, the adrenal cortex, and the testis, may point to a role for this nuclear receptor in the morphogenesis or physiology of these tissues.

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Figure 2. LXR mRNA expression in embryonic organs. A: LXRβ is strongly expressed in small intestine and kidney, as well as in steroidogenic organs such as adrenals and testis. B: The expression of LXRβ is detected in transversal sections of the 16.5 dpc embryonic brain. Signals were observed in several nuclei of the midbrain (b, mid) and medulla oblongata (d and f, mo), as indicated by arrowheads. LXRβ is also expressed in the telencephalon (tel), the neopallial cortex (neo), the retina (ret), trigeminal ganglions (tg), as well as olfactory epithelium and lobes (olf).

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LXRα and LXRβ null animals are viable and fertile (Peet et al., 1998; Alberti et al., 2001). This suggests that these two nuclear receptors are not fundamental for embryonic development, which is in contrast to their principal heterodimerization partner RXRα whose deletion leads to death early during mouse ontogenesis due to placental abnormalities (Wendling et al., 1999). The more severe developmental phenotype of the RXRα null mice is explained by the fact that it alters signaling by numerous nuclear receptors.

In contrast to LXRα mRNA levels, which tend to remain high during development, LXRβ mRNA levels in the liver seem to decrease in the later stage of development (compare expression at 11.5, 14.5, 15.5, and 16.5 dpc; Fig. 1J, L, N, and P). These differences may reflect the relative importance of LXRα in liver function in the adult (Peet et al., 1998). Consistent with this lower expression of LXRβ in the liver, a study in LXRβ KO animals demonstrated that this nuclear receptor does not have the same predominant role as LXRα in liver cholesterol metabolism (Alberti et al., 2001).

During development, a marked expression of LXRβ is detected in a subset of neuronal structures, including the retina, the medulla oblongata, and the trigeminal, vestibulocochlear, and dorsal root ganglions (Fig. 1L, N, and P). We therefore studied the expression of LXRβ in more detail in the brain of 16.5 dpc embryos (Fig. 2B). LXRβ expression was detected in the telencephalon, the neopallial cortex, the olfactory lobe and its epithelium, the retina, the trigeminal ganglion, as well as in several nuclei of the midbrain and medulla oblongata (Fig. 2B).

We also analyzed LXRα and LXRβ expression in adult mice. In the adult spleen, these nuclear receptors show distinct profiles, with low expression of LXRα in the red pulp, which contains many blood elements (erythrocytes, platelets, monocytes, macrophages, lymphocytes, plasma cells, and granulocytes), and high mRNA levels of LXRβ in the Malpighian corpuscules of the spleen (white pulp), which contains principally macrophages and a few lymphocytes (Fig. 3A). However, even if their overall expression pattern in the spleen seems separated, their cellular expression might be overlapping, since both white and red pulp contain macrophages. These data are consistent with recent reports describing a role for LXRs in inflammation (Tangirala et al., 2002; Joseph et al., 2003) and suggest complementary functions for LXRα and -β in the immune response system. As observed in embryos, LXRβ (but not LXRα) is also detected in the three layers of the adrenal cortex, i.e., the zona glomerulosa, fasiculata, and reticularis, the sertoli cells of the testis, the medulla, the corona radiata, and membrana granulosa of the Grafiaan follicle, and the corpus luteum of the ovary (Fig. 3C).

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Figure 3. LXR expression in adult tissues. A: The adult spleen expresses both LXRα and -β, with low expression for LXRα in the red pulp (white arrowhead) and high expression for LXRβ in the white pulp (black arrowhead). B: LXRβ is expressed in the cortical region of the adrenal (a and b, indicated by arrows), the sertoli cells of the testis (c–f), the medulla (med), the corona radiata (cr) and granulosa membrana (gm) of the Grafiaan follicle (GF), and the corpus luteum (CL) of the ovary (g and h). The black box in c corresponds to the enlarged region in e and f.

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In summary, the mRNAs for both LXRα and -β are initially detected in the liver at 11.5 dpc. Later in development, expression patterns differ, with a more ubiquitous expression for LXRβ mRNA, with particular enrichment in endocrine, neuronal and immunological structures, and a more restricted expression pattern for LXRα, with highest levels of expression in tissues involved in metabolism. The distinct expression patterns of both receptors during development and adulthood emphasizes the necessity to characterize in more detail the functions of both LXRα and -β. This would be particularly relevant for LXRβ since no clear role for this nuclear receptor is yet established. Potential implications of LXRβ in steroid hormone synthesis, immune defense, and the nervous system are worth pursuing, since several steroidogenic organs, lymphoid organs, and particular brain nuclei express high levels of LXRβ mRNA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

We thank Thomas Ding, Gordon Francis, Estelle Heitz, David Mangelsdorf, Maryam Rastegar, and Marius Teletin for providing reagents, technical assistance, and advice. Supported by a fellowship of the Ligue Nationale Contre le Cancer (to J.-S.A.).

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED
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