Expression of the Diabetes-Associated Gene TCF7L2 in Adult Mouse Brain

Abstract

Polymorphisms of the gene TCF7L2 (transcription factor 7-like 2) are strongly associated with the development and progression of type 2 diabetes. TCF7L2 is important in the development of peripheral organs such as adipocytes, pancreas, and the intestine. However, very little is known about its expression elsewhere. In this study we used in situ hybridization histochemistry to show that TCF7L2 has a unique expression pattern in the mouse brain. TCF7L2 is expressed in two distinct populations. First, it is highly ex pressed in thalamic and tectal structures. Additionally, TCF7L2 mRNA is expressed at moderate to low levels in specific cells of the hypothalamus, preoptic nucleus, and circumventricular organs. Collectively, these patterns of expression suggest that TCF7L2 has distinct functions within the brain, with a general role in the development and maintenance of thalamic and midbrain neurons, and then a distinct role in autonomic homeostasis.

TCF7L2 (transcription factor 7-like 2), also known as TCF-4, is a transcription factor and a key component of the canonical WNT signaling pathway (Huelsken and Birchmeier, 2001; Smith, 2007). Since its identification as a diabetes risk-conferring gene in 2006 (Grant et al., 2006), numerous population studies have shown that TCF7L2 variants increase the odds of developing diabetes by 30%–50% for each inherited allele (Hattersley, 2007). Thus, TCF7L2 is the most significant and consistent genetic diabetes risk factor identified to date, far exceeding the risk conveyed by previously established diabetes genes, such as peroxisome proliferator-activated receptor γ (PPARγ) (Altshuler et al., 2000) and β-cell inwardly K⁺ channel KIR6.2 (KCNJ11) (Gloyn et al., 2003). Carriers of TCF7L2 polymorphisms show decreased insulin secretion and proinsulin conversion, reduced incretin effects, impaired beta-cell function, and increased hepatic glucose production (Florez et al., 2006; Lyssenko et al., 2007; Schafer et al., 2007; Kirchhoff et al., 2008). Furthermore, carriers have an increased risk of progressing from impaired glucose tolerance to diabetes (Florez et al., 2006). Mice lacking TCF7L2 are neonatal lethal, and show abnormal intestine development and pituitary hyperplasia (Korinek et al., 1998; Brinkmeier et al., 2003; Brinkmeier et al., 2007).

Recent studies have highlighted the function of TCF7L2 in peripheral organs such as the pancreas, adipocytes, and intestine. Its role within the central nervous system, however, is largely unknown. One previous report (Murray et al., 2007) showed TCF7L2 expression in the primate thalamus, and a brief description of TCF7L2 in the mouse brain was reported in the online resource, the Allen Brain Atlas (Lein et al., 2007). Our data confirm the initial expression reports—we show that TCF7L2 is expressed densely in the tectum, thalamus, mid-brain, and in a highly cell specific manner in autonomic regions such as the hypothalamus, basal forebrain, preoptic area, brainstem, and circumventricular organs. In addition, we describe the colocalization of TCF7L2 within specific tyrosine hydroxylase (TH)- and choline acetyltransferase (ChAT)-expressing populations.

MATERIALS AND METHODS

Animals and histology

Adult male C57BL/6 mice (20–30 g, 6–8 weeks old, n = 6) (Jackson Laboratory, Bar Harbor, Maine) were housed with ad libitum access to both food and water in a light (12/12-hour on/off) and temperature (21.5–22.5°C) controlled environment. The animals and procedures used were in accordance with the guidelines and approval of the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committees. Mice were deeply anesthetized with an intraperitoneal injection of chloral hydrate (500 mg/kg) and transcardially perfused with 0.9% saline made with diethyl pyrocarbonate (DEPC)-treated water, followed by 10% neutral buffered formalin. Brains were removed, postfixed in 10% formalin for 4 hours at room temperature, cryoprotected in 20% sucrose made in DEPC-treated phosphate-buffered saline (PBS), pH 7.0 at 4°C, and sectioned coronally at 25 μM into five series on a freezing microtome. Tissue was stored at −20°C in an antifreeze solution (Simmons et al., 1989) until processed.

In situ hybridization histochemistry

Brain sections were processed for in situ hybridization histochemistry to determine TCF7L2 mRNA distribution. Sections were mounted onto SuperFrost plus slides (Fisher Scientific, Pittsburgh, PA), air-dried, and stored in desiccated boxes at −20°C. Prior to hybridization, sections were fixed in 4% formaldehyde in DEPC-treated PBS (pH 7.0) for 20 minutes, dehydrated in ethanol, cleared in xylenes for 15 minutes, and placed in prewarmed sodium citrate buffer (pH 6.0). Slides were then submitted to microwave treatment as described (Marcus et al., 2001; Kishi et al., 2003) for 10 minutes, dehydrated in ethanol, and air-dried. Probes for TCF7L2 were derived from PCR fragments amplified with iTaq DNA polymerase (Bio-Rad, Hercules, CA) from cDNA generated with SuperScript III First-Strand Synthesis System for reverse-transcription polymerase chain reaction (RT-PCR) (Invitrogen, Carlsbad, CA) from total mouse hypothalamic RNA (BD Biosciences, Palo Alto, CA). The PCR products were cloned with the TOPO TA Cloning Kit for Sequencing (Invitrogen). The probe spans nucleotides 640–1114 of GenBank accession number NM_009333. Antisense and sense ³⁵S-labeled probes were generated with MAXIscript In Vitro Transcription Kits (Ambion, Austin, TX). The nucleotide mixture was then digested with DNAase and the labeled probe was purified and collected using resin spin columns (GE Healthcare, Piscataway, NJ). The ³⁵S-labeled probes were diluted (10⁶ dpm/mL) in a hybridization solution containing 50% formamide, 10 mM Tris-HCl, pH 7.5 (Invitrogen), 1% sheared salmon sperm DNA (Sigma-Aldrich, St. Louis, MO), 5 mg tRNA (Invitrogen), 2.5% total yeast RNA (Sigma), 100 mM dithiothreitol, 10% dextran sulfate, 0.6 M NaCl, 0.5 mM EDTA (pH 8.0), 0.1% SDS, 0.1% sodium thiosulfate, and 1× Denhardt's solution (Sigma). Hybridization solution and a coverslip were applied to each slide and sections were incubated at 57°C for 12–16 hours. On the following day, sections were incubated in 0.002% RNase A (Roche Applied Bioscience, Indianapolis, IN) solution and submitted to stringency washes. Sections were then dehydrated and enclosed in x-ray film cassettes with Kodak BioMax MR-2 film (Carestream Molecular Imaging, New Haven, CT) for 3 days. Subsequently, the slides were dipped in Kodak NTB autoradiographic emulsion (Carestream Molecular Imaging), dried, and stored at 4°C for 15 days. Slides were developed with Dektol developer and Fixer (Kodak/Carestream Molecular Imaging), counterstained with thionin, dehydrated, cleared in xylenes, and coverslipped with Permaslip (Newcomer Supply, Middleton, WI). The density of hybridization signal was subjectively estimated based on the number of silver grains per brain nucleus or area. Only regions that displayed hybridization signal were described, but our estimates were based on a comparison of all areas throughout the brain. We used “+”(s) to comparatively classify the density of silver grains in each cell group: the areas that showed inconsistent signal between different runs/series received “+/−,” those with a slightly higher density of silver grains above background received “+,” those with moderate and high number of silver grains received “++” and “+++,” respectively, and the areas with the highest density of silver grains were designated “++++.” Control procedures included hybridization with sense probes and tissue pretreatment with RNase A (200 μg/mL). No specific hybridization was observed following either procedure.

Dual label in situ hybridization histochemistry/ immunohistochemistry

The protocol used for combined immunohistochemistry and in situ hybridization is a modification of previously described protocols (Chan et al., 1995; Chan and Sawchenko, 1995; Kelly and Watts, 1996; Elias et al., 1998; Yamamoto et al., 2002, 2003; Liu et al., 2003). Free-floating tissue was rinsed with DEPC-treated PBS, pH 7.0, for 1 hour before pretreatment with 0.1% sodium borohydride (Sigma) in DEPC-PBS for 15 minutes. After washing in DEPC-PBS, tissue was briefly rinsed in 0.1M TEA, pH 8.0, and incubated for 10 minutes in 0.25% acetic anhydride in 0.1M TEA. The tissue was then rinsed in DEPC-treated 2× sodium chloride/sodium citrate (SSC) before hybridization. cRNA probes were diluted to 10⁶ cpm/mL in 50% formamide, 10 mM Tris-HCl, pH 7.5 (Invitrogen), 1% sheared salmon sperm DNA (Sigma-Aldrich), 5 mg tRNA (Invitrogen), 2.5% total yeast RNA (Sigma), 100 mM dithiothreitol, 10% dextran sulfate, 0.6 M NaCl, 0.5 mM EDTA (pH 8.0), 0.1% SDS, 0.1% sodium thiosulfate, and 1× Denhardt's solution (Sigma) and applied to the tissue. Sections were incubated overnight at 57°C. The next day the tissue was rinsed four times in 4× SSC before being incubated in 0.002% RNase A (Roche Applied Bioscience) diluted in 0.5M NaCl, 40 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA, pH 8.0 (RNase buffer) for 30 minutes at 37°C. Sections were then washed in RNase buffer for another 30 minutes at 37°C and rinsed in 2× SSC at room temperature. Tissue was then submitted to stringency washes in 2× SSC at 50°C, 0.2× SSC at 55°C, and 0.2× SSC at 60°C for 1 hour each; an additional 0.1× SSC wash at 55°C for 30 minutes was also performed. Tissue sections were then processed for immunohistochemistry of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), β-endorphin (β-End), melanin-concentrating hormone (MCH), and orexin (ORX). The tissue was incubated in goat primary antiserum anti-ChAT (1:5,000, Millipore, Billerica, MA), rabbit primary antiserum anti-TH (1:20,000, Millipore), anti-β-End (1:10,000, Phoenix Pharmaceuticals, Burlingame, CA), anti-MCH (1:10,000, Phoenix Pharmaceuticals), or anti-ORX (1:10,000, Phoenix Pharmaceuticals) overnight at room temperature with gentle agitation. The sections were next incubated in biotinylated donkey antigoat IgG (Jackson Laboratories, Bar Harbor ME; 1:1,000 for ChAT) or biotinylated donkey antirabbit IgG (Jackson Laboratories; 1:1,000 for TH, MCH, and ORX) for 1 hour at room temperature, diluted in 0.25% Triton X-100 in PBS. Next, tissue was incubated in avidin-biotin complex from the Vectastain Universal Elite ABC Kit (1:500 in PBS, Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. After rinsing, the sections were incubated in 0.05% diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01% hydrogen peroxide dissolved in PBS. The reaction was terminated after 3–5 minutes with successive rinses in PBS. The tissue sections were mounted onto SuperFrost plus slides (Fisher Scientific), and after air-drying, the slides were placed in x-ray film cassettes with Kodak Biomax MR-2 film (Carestream Molecular Imaging). Slides were next dipped in Kodak NTB autoradiographic emulsion (Carestream Molecular Imaging), stored at 4°C, and, 2 weeks later, developed with Dektol developer and Fixer (Kodak/Carestream Molecular Imaging). Slides were then dehydrated, cleared in xylenes, and cover-slipped with Permaslip (Newcomer Supply).

Cell counts were obtained in regions that showed colocalization of TCF7L2 mRNA with TH or ChAT antisera, including the medial septal nucleus, horizontal limb of the nucleus of diagonal band, substantia innominata, ventral pallidum, magnocellular preoptic nucleus, arcuate nucleus, A13 dopaminergic cells of the incerto-hypothalamic area, dorsal medial hypothalamus, subventricular zone of the hypothalamus, periaqueductal gray, and compact part of the substantia nigra. In general, we counted cells from one representative section for each nucleus, except in the arcuate nucleus and horizontal limb of the nucleus of diagonal band, which have long rostro-to-caudal distributions, and were analyzed at two levels that were spaced over 300 μm apart. The atlas levels, as described in Paxinos and Franklin (2001), are given in Tables 3 and 4. Double-labeled neurons were quantified using 10× or 20× objectives under brightfield illumination (Carl Zeiss Axioskop 2 microscope). We considered cells to be double-labeled if the silver grains overlying identified cell bodies were three times or more above background, and if the pattern of the overlying silver grains conformed to the shape of the DAB-stained cell. Cells were analyzed in three separate mice (n = 3) and counts are presented as the mean ± standard error of the mean (SEM). These data were corrected for double counting by applying Abercrombie's formula, N = n(T/T+D), where N = the corrected cell count, n = the observed number of cells, T = section thickness (25 μm), and D = the diameter of the nucleus (Guillery, 2002). For each brain nucleus or area with double-labeled cells, we examined adjacent thionin-stained sections to measure and calculate the mean nuclear diameter from 9–16 random cells that had clear and defined nuclei and nucleoli, and applied this value to Abercrombie's formula. Stereological techniques were not used as the staining techniques did not penetrate the entire thickness of the sections.

TABLE 3.

Double-Labeled ChAT+TCF7L2 Neurons in the Basal Forebrain

Regions	Atlas level	Total ChAT	Doubles	% Doubles/total ChAT
MS	23	55.9 ± 5.7	0.7 ± 0.4	1.3 ± 0.7
HDB (1)	26	32.1 ± 0.9	2.6 ± 0.5	8.3 ± 1.9
HDB (2)	30	49.2 ± 5.9	3.9 ± 0.4	7.9 ± 0.5
SI/VP	34	39.4 ± 1.9	2.1 ± 0	5.4 ± 0.3
MCPO	34	12.7 ± 0.7	3.9 ± 0.3	31.1 ± 3.1

Values represent estimates of mean counts of cells ± SEM (n=3). Immunoperoxidase reaction was used for choline acetyl transferase (ChAT) and ³⁵S-labeled riboprobe for TCF7L2. HDB1, anterior level of the horizontal limb of the nucleus of diagonal band; HDB2: posterior level of the HDB; MCPO, magnocellular preoptic nucleus; MS, medial septal nucleus; SI, substantia innominata; VP, ventral pallidum. The atlas level designations correspond to those described by Paxinos and Franklin (2001). No dual-labeled neurons were observed in any other brain region containing cholinergic neurons.

TABLE 4.

Double-Labeled TH+TCF7L2 Neurons in Mouse Brain

Regions	Atlas level	Total TH	Doubles	% Doubles/total TH
Arcl	41	31.6 ± 2.5	1.2 ± 0.6	4.1 ± 2.1
Arc2	43	27.2 ± 2.2	0.7 ± 0.4	2.5 ± 1.3
IHy (A13)	41	55.7 ± 1.4	3.7 ± 0.6	6.6 ± 1.0
DMH	48	28.5 ± 2.9	0.9 ± 0.2	3.3 ± 0.8
SPA/PAG	50	10.5 ± 1.4	7.1 ± 2.1	66.0 ± 14.7
SNc	55	109.3 ± 7.4	2.4 ± 0.5	2.2 ± 0.3

Values represent estimates of mean counts of cells ± SEM (n=3). We used an immunoperoxidase reaction for tyrosine hydroxylase (TH) and ³⁵S-labeled riboprobe for TCF7L2. Arcl, anterior level of the arcuate nucleus; Arc2, posterior level of the arcuate nucleus; IHy (A13), A13 dopamingergic cells of the incerto-hypothalamic area; DMH, dorsal medial hypothalamus; SPA, subventricular zone of the hypothalamus; PAG, periaqueductal gray; SNc, compact part of the substantia nigra. The atlas level designations correspond to those described by Paxinos and Franklin (2001). No dual-labeled were observed in any other brain region containing tyrosine hydroxylase neurons.

Antibody characterization

The antisera used in the present study are all commercially available and have been tested and published by different laboratories. Their key features are summarized in Table 1.

TABLE 1.

Primary Antibodies Used

Antigen	Immunogen	Manufacturer	Dilution used
ChAT	Human placental enzyme	Millipore (Billerica, MA) Goat polyclonal Cat. #AB144P lot # 23010339	1:5,000
TH	Denatured TH from rat pheochromocytoma	Millipore Rabbit polyclonal Cat. # AB152 Lot # 0607034655	1:20,000
β-End	Synthetic peptide, from rat YGGFMTSEKSQTPLVTLFKNAIIKNVHKKGQ	Phoenix Phamaceuticals (Burlingame, CA) Rabbit polyclonal Cat. # H-022-33 lot# 00569	1:10,000
MCH	Synthetic peptide, from human, rat and mouse DFDMLRCMLGRVYRPCWQV	Phoenix Phamaceuticals Cat. # H-070-47 lot # 00835	1:10,000
ORX	Synthetic peptide, from human, bovine, rat and mouse EPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL	Phoenix Phamaceuticals Cat. # H-003-30 lot # 00794	1:10,000

The ChAT antiserum recognizes a single band of ≈68–70 kDa molecular weight on Western blots of mouse brain (manufacturer's datasheet), and has been reported for use in immunohistology by others (Whitworth et al., 2005; Heinze et al., 2007; Keeley et al., 2007). In our samples the antiserum produced a staining pattern in mouse brain identical to previous reports of ChAT expression (Armstrong et al., 1983).

The TH antiserum recognizes a single band of ≈62 kDa molecular weight on Western blots of PC12 cells (manufacturer's datasheet), and its use in immunohistochemistry has been previously published (Witkovsky et al., 2004; Whitworth et al., 2005). We found that the antiserum produced staining patterns identical to previous reports of TH morphology and distribution (Zigman et al., 2006).

The β-End antiserum shows 100% crossreactivity by competitive radioimmunoassay to β- and α-endorphin, 60% crossreactivity to γ-endorphin, and 0% to Met and Leuenkephalin (manufacturer's datasheet). The antiserum has previously been reported for use in immunohistological studies of rodent hypothalamus (Helwig et al., 2006). The specificity was previously tested by preadsorption with β-End, which eliminated staining (Helwig et al., 2006). In our studies the antiserum produced a staining pattern consistent with previous reports β-End expression (Finley et al., 1981).

The MCH antiserum shows 100% crossreactivity to MCH, and 0% to ORX-A and -B, using competitive radioimmunoassay (manufacturer's datasheet). Preadsorption of the antibody to MCH to the full-length peptide eliminated staining (Glavas et al., 2008). Staining with this antibody in our samples, as well as in previously published reports (Kerman et al., 2007; Glavas et al., 2008), is consistent with previously described MCH patterns (Bittencourt et al., 1992).

The ORX antiserum shows 100% crossreactivity to ORX-A, and no crossreactivity to ORX-B by competitive radioimmunoassay (manufacturer's datasheet). The antibody recognizes bands of 15 kDa and 3.5 kDa molecular weight, representing prepro-ORX-A and the ORX-A respectively, on Western blots of rat brain (manufacturer's datasheet). Preadsorption with ORX-A peptide eliminated staining (Helwig et al., 2006; Fronczek et al., 2007). Previous work (Helwig et al., 2006) and our own shows that the antiserum stains cells in a pattern and morphology consistent with that previously reported (Nambu et al., 1999).

Production of photomicrographs

Images were obtained with a Carl Zeiss Axioskop 2 microscope and a Zeiss Stemi 2000-C dissecting microscope using both brightfield and darkfield optics. Images were captured using a Zeiss digital camera and the Axiovision 3.1 software. Adobe PhotoShop CS2 (San Jose, CA), was used to combine the images into plates, make minor adjustments to contrast and brightness, and remove any obvious dust from the dark-field images.

RESULTS

Distribution of TCF7L2 mRNA

Coronal sections of the mouse brain extending from the olfactory bulb to the caudal limits of the nucleus of the solitary tract were hybridized with the TCF7L2 antisense riboprobe. We found hybridization signal in specific nuclei throughout the brain. Control sections hybridized with the sense riboprobe or that were pretreated with RNase A did not show specific hybridization signals. Levels of TCF7L2 mRNA in brain nuclei were visualized as silver grains after hybridization with radio-labeled probes and were subjectively estimated, presented in Table 2. We observed hybridization signal in the forebrain, cerebellum, and brainstem as described in detail below. Figure 1 our findings with a series of low-power magnification images.

TABLE 2.

Relative Densities of TCF7L2 mRNA Expression in the Mouse Brain

Brain areas	TCF7L235S	Brain areas	TCF7L235S
Forebrain		Subparafascicular	+ + + +
Cerebral cortex		Retroparafascicular	+ + + +
Isocortex
Corpus callosum	+	Incertohypothalamic area	+
Hippocampal formation	+/–	Hypothalamus
Septum		Median preoptic	+/–
Lateral	+	Anterior	+ + +
Medial	+	Retrochiasmatic area	+
Septofimbrial	+	Arcuate	+
Basal forebrain		Dorsomedial	+ +
Island of Calleja	+	Lateral hypothalamic area	+ +
Olfactory tubercle	+	Posterior hypothalamic area	+
Diagonal band, vertical	+ +
Diagonal band,horizontal	+ + +	II. Cerebellum
Magnocellular preoptic	+ + +	Purkinje layer	+
Basal ganglia
Substantia innominata	+ +	III. Brain stem
Ventral pallidum	+ + +	Pretectal group	+ + + +
Globus pallidus, lateral	+	Posterior comissure	+ + + +
Thalamus		Prerubral field	+ + + +
Paraventricular	+ + + +	Superior Colliculus, optic layer	+ + +
Anterodorsal	+ + + +	Superior Colliculus, internal layers	+ + +
Anteroventral	+ + +	Substantia nigra, compact	+ +
Anteromedial	+ + +	Ventral tegmental area	+/–
Parataenial	+ + + +	Deep mesencephalic	+ + +
Centromedial	+ + + +	Periaqueductal gray: anterior	+ + +
Paracentral	+ + +	dorsal	+ +
Rhomboid	+ + + +	dorsolateral	+ +
Reuniens	+ + + +	lateral	+ + +
Xiphoid	+ + + +	Subbrachial	+ + +
Parafascicular	+ + +	Interpeduncular, intermed.	+ + + +
Intermediodorsal	+ + + +	Inferior colliculus	+ + + +
Mediodorsal	+ + +	Pontine reticular formation	+ +
Habenula, medial	+ + +	Pontine central gray	+ + + +
Habenula, lateral	+	Prepositus	+
Lateral group	+ + + +	Nucleus of solitary tract	+/–
Ventral group	+ + + +
Posterior group	+ + + +	IV. Circunventricular organs
Intergeniculate	+ +	Vascular org. lamina terminalis	+ +
Geniculate (ventral, dorsal, medial)	+ + +	Subfornical organ	+
Parafascicular	+ + +	Area postrema	+

Brain nuclei are grouped into anatomical subdivisions and organized from rostral to caudal. Qualitative estimates of TCF7L2 mRNA expression are based on hybridization signal strength. + + + + , highest density; + + + , high density; + + , moderate density; +, low density; +/–, labeling inconsistently above background.

Figure 1.

A series of low-power darkfield photomicrographs summarizing the distribution of TCF7L2 mRNA in the mouse brain. AD, anterodorsal thalamic nucleus; AHA; anterior hypothalamic area; AP; area postrema; Arc, arcuate nucleus; cc, corpus callosum; CG; central gray, pontine; DLG; dorsal lateral geniculate nucleus; dlPAG, dorsolateral periaqueductal gray matter; DMH, dorsomedial nucleus of hypothalamus; HDB; horizontal, diagonal band of Broca; IC, inferior colliculus; IPI, lateral interpeduncular nucleus, intermediate subdivision; lPAG, lateral periaqueductal gray matter; LV, lateral ventricle; MCPO, magnocellular preoptic nucleus; MD, mediodorsal thalamic nucleus; MG, medial geniculate nucleus; MHb, medial habenular nucleus; NTS, nucleus of solitary tract; OVLT, vascular organ of lamina terminalis; PF, parafascicular nucleus; Po, posterior nucleus of thalamus; PVT, paraventricular nucleus of thalamus; Re, reuniens thalamic nucleus; SC, superior colliculus; SFO, subfornical organ; SI, substantia innominata; SN, substantia nigra; VPL, ventral posteromedial thalamic nucleus; VPM; ventral posterolateral thalamic nucleus. Scale bars = 2 mm.

Forebrain

In the cerebral cortex we found low hybridization signal distributed throughout the layers of the neocortex, mainly in more rostral levels, including the motor and somatosensorial subdivisions. We also found a low and consistent signal in the corpus callosum, above the lateral ventricles, and close to the midline, in more anterior levels (Fig. 2). We found an inconsistent signal close to background levels in the hippocampal formation. Low signal was also found in the lateral and medial septal nucleus, in the septofimbrial nucleus, in the Island of Calleja, and in the olfactory tubercle. Moderate to high density of silver grains were found in the vertical and horizontal limbs of the diagonal band of Broca, substantia innominata, ventral pallidum, and in the magnocellular preoptic nucleus. In addition, we found a low hybridization signal throughout the globus pallidus.

Figure 2.

Distribution of TCF7L2 mRNA in the corpus callosum (cc). A: Bright-field photomicrograph showing the cytoarchitecture of a brain section containing the cc. B: Darkfield photomicrograph showing the TCF7L2 mRNA in the cc. C: Brightfield photomicrograph under high magnification showing silver grains (³⁵S-labeled riboprobe, arrows) in the cc. LV, lateral ventricle. Scale bar = 250 μm for A,B; and 100 μm for C.

In the thalamus we found strong signal in all rostro-tocaudal levels in virtually every nucleus, with the exception of the reticular nucleus (Fig. 3). We found a strong signal in all of the midline nuclei including the paraventricular, the centromedial, the dorsomedial, the rhomboid, the reunions, and the xiphoid nuclei. We also found a dense signal in the medial habenula, but a low signal in the lateral habenula. High hybridization signal was also observed in all nuclei of the anterior group including the anterodorsal, anteroventral, and anteromedial, in all nuclei of the lateral group including the laterodorsal and lateroposterior nuclei, and in all nuclei of the ventral and posterior groups including the ventral, the ventroposterolateral and the ventroposterome-dial, the dorsal and ventral geniculate, the parafascicular, the subfascicular, and the retroparafascicular nuclei. In the transition to the midbrain we found a high density of silver grains in the pretectal area, in the precommissural nucleus, in the subparafascicular area, in the prerubral field, in the nucleus of the posterior commissure, and in the more anterior levels of the periaqueductal gray matter.

Figure 3.

Distribution of TCF7L2 mRNA in the thalamus. A,C,E,G: Brightfield photomicrographs showing the cytoarchitecture of four rostro-tocaudal levels of the thalamus. B,D,F,H: Darkfield photomicrographs showing the distribution of TCF7L2 mRNA in the same levels of the thalamus. AD, anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus; APT, anterior pretectal nucleus; AV, anteroventral thalamic nucleus; CM, central medial thalamic nucleus; DLG, dorsal lateral geniculate nucleus; fi, fimbria; fr, fasciculus retroflexus; LGP, lateral globus pallidus; ic, internal capsule; LD, laterodorsal thalamic nucleus; LHb, lateral habenular nucleus; LV, lateral ventricle; LP, lateral posterior thalamic nucleus; MD, medial dorsal thalamic nucleus; MHb, medial habenular nucleus; ml, medial lemniscus; mt, mammillothalamic tract; PC, paracentral thalamic nucleus; PF, parafascular nucleus; Po, posterior thalamic nucleus; PrC, precomissural nucleus; PVH, paraventricular nucleus of the hypothalamus; PVT, paraventricular nucleus of thalamus; Re, reuniens thalamic nucleus; Rh, rhomboid thalamic nucleus; Rt, reticular thalamic nucleus; sm, stria medularis; SPF, subparafascicular nucleus; VL, ventral lateral thalamic nucleus; VLG, ventral lateral geniculate nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; Xi, xiphoid nucleus, ZI, zona incerta. Scale bar = 500 μm (applies to all).

In the hypothalamus we found a more limited distribution (Fig. 4). We observed an inconsistently low signal in the median preoptic nucleus, a high signal in the anterior hypothalamic nucleus, and a low to moderate signal in the retrochiasmatic area, arcuate nucleus, ventral subdivision of the dorsomedial nucleus, lateral hypothalamic area, including the perifornical area, and in the posterior hypothalamus. Virtually no hybridization signal was observed in the paraventricular nucleus of the hypothalamus, in the ventromedial nucleus of the hypothalamus, and in the ventral and dorsal premmammillary nuclei.

Figure 4.

Distribution of TCF7L2 mRNA in the hypothalamus. A,C,E: Brightfield photomicrographs showing the cytoarchitecture of three rostro-to-caudal levels of the hypothalamus. B,D,F: Darkfield photomicrographs showing the distribution of TCF7L2 mRNA in the same levels of the hypothalamus. 3V, third ventricle; Arc, arcuate nucleus; AHA, anterior hypothalamic area; DMH, dorsomedial nucleus of the hypothalamus; f, fornix; PFx, perifornical area; RCA, retrochiasmatic area. Scale bar = 400 μm (applies to all).

In the cerebellum, hybridization signal was virtually absent with the exception of the Purkinje layer, where we found a low signal.

In the brainstem we found a high density of hybridization in the optic and internal layers of the superior colliculus, throughout the inferior colliculus, and in the deep mesencephalic nucleus (Fig. 5). We found a low and inconsistent signal in the ventral tegmental area and low to moderate levels in the compact subdivision of the substantia nigra, with higher density in its dorsolateral aspects. In the periaqueductal gray matter we found a moderate to high density of hybridization signal in the more rostral aspects, with the dorsal and dorsolateral columns showing moderate signal, and the lateral column with high signal. We also found dense hybridization in the subbrachial nucleus, in the intermediate subdivision of the interpeduncular nucleus, and in the pontine central gray. Low signal was observed scattered in the pontine reticular formation and in the prepositus nucleus. In addition, we found a low and inconsistent signal in the nucleus of the solitary tract.

Figure 5.

Distribution of TCF7L2 mRNA in the tectum of the mesencephalon. A,C: Brightfield photomicrographs showing the cytoarchitecture of two rostro-to-caudal levels of the mesencephalon. B,D: Darkfield photomicrographs showing the distribution of TCF7L2 mRNA in the same levels of the mesencephalon. Aq, aqueduct; dlPAG, dorsolateral periaqueductal gray mater; DR, dorsal raphé; lPAG, lateral periaqueductal gray matter; IC, inferior colliculus; SC, superior colliculus; vlPAG, ventrolateral periaqueductal gray matter. Scale bar = 500 μm (applies to all).

We also observed some hybridization signal in circumventricular organs (Fig. 6). We found moderate signal in the vascular organ of lamina terminalis and low signal in the subfornical organ and in the area postrema.

Figure 6.

Distribution of TCF7L2 mRNA in circumventricular organs. A,C,E: Brightfield photomicrographs showing the cytoarchitecture of the vascular organ of lamina terminalis (OVLT, A), of the subfornical organ (SFO, C) and of the area postrema (AP, E). B,D,F: Darkfield photomicrographs showing the distribution of TCF7L2 mRNA in the OVLT (B), in the SFO (D), and in the AP (F). 3V, third ventricle; 10, dorsal motor nucleus of vagus nerve; 12 hypoglossal nucleus; cc, corpus callosum; NTS, nucleus of the solitary tract; ox, optic chiasm; SFi, septofimbrial nucleus; TS, triangular septal nucleus. Scale bar = 400 μm (applies to all).

Neurochemical specificity of TCF7L2-expressing cells

To determine the neurochemical specificity of TCF7L2 cells we combined in situ hybridization and immunohistochemistry for neurotransmitters located in the areas where we observed TCF7L2 mRNA expression. We assessed the colocalization of TCF7L2 with ChAT, TH, β-endorphin, MCH, and ORX.

ChAT

We observed that TCF7L2 mRNA is expressed in the medial septal nucleus, in the diagonal band of Broca, substantia innominata, ventral pallidum, and magnocellular preoptic nucleus, all of which are known to express ChAT (Armstrong et al., 1983). We found a small amount (≈1%–8%) of colocalization of TCF7L2 and ChAT in the medial septal nucleus, the horizontal limb of the nucleus of diagonal band, and the substantia innominata/ventral pallidum (Table 3). In addition, we found moderate colocalization of TCF7L2 and ChAT in the magnocellular preoptic nucleus, where one-third (≈31%) of the ChAT neurons coexpress TCF7L2 mRNA (Table 3; Fig. 7). We also assessed the colocalization of TCF7L2 and ChAT in other brain nuclei. We found virtually no TCF7L2 hybridization signal in the pedunculopontine tegmental nucleus, the laterodorsal tegmental nucleus, or the cranial nerve (autonomic or somatic) motor nuclei.

Figure 7.

Choline acetyl transferase immunoreactivity (ChAT-ir) and TCF7L2 mRNA distribution in the basal forebrain. A: Brightfield photomicrograph showing neurons expressing ChAT-ir (brown cytoplasm) and TCF7L2 mRNA (silver grains, ³⁵S-labeled riboprobe). B: Higher magnification of A. Note that few neurons are dual-labeled (arrows). MCPO, magnocellular preoptic nucleus; SI, substantia innominata. Scale bar = 200 μm for A; 100 μm for B.

TH

TCF7L2 mRNA was also found in brain areas that contain dopamine. We found a small amount of colocalization of TCF7L2 and TH immunoreactivity in the arcuate nucleus (in two distinct rostro-to-caudal levels), in the incertohypothalamic area (A13 group), in the dorsomedial nucleus of the hypothalamus, and in the substantia nigra, compact part (Fig. 8D–F). In these areas, only 2%–7% of TH neurons colocalized with TCF7L2 mRNA (Table 4). However, we found that a high percentage (≈66%) of TH-immunoreactive neurons in the transition of the subparafascicular area to the anterior periaqueductal gray matter also expressed TCF7L2 mRNA (Table 4; Fig. 8A–C). We also assessed the colocalization of TCF7L2 and TH in other brain stem nuclei. We found virtually no dual-labeled neurons in other catecholaminergic cell groups assessed, including the locus coeruleus, A5 and A7.

Figure 8.

Tyrosine hydroxylase immunoreactivity (TH-ir) and TCF7L2 mRNA distribution in the parafascicular nucleus (PF) and in the substantia nigra (SN). A,D: Darkfield photomicrographs showing the distribution of TCF7L2 mRNA in posterior thalamic (A) and anterior mesencephalic levels. B,E: Brightfield photomicrograph showing neurons expressing TH-ir (brown cytoplasm) and TCF7L2 mRNA (silver grains, ³⁵S-labeled riboprobe) at the same levels. C,F: Higher magnification dual-labeled neurons in the parafascicular (C) and in the substantia nigra (F). APT, anterior pretectal nucleus; Aq, aqueduct; DLG, dorsal lateral geniculate nucleus; DpMe, deep mesencephalic nucleus; fr, fasciculus retroflexus; MG, medial geniculate thalamic nucleus; PAG, periaqueductal gray matter; Po, posterior thalamic nucleus; SC, superior colliculus; SNc, compact SN; SNr, reticulate SN; VLG, ventral lateral geniculate nucleus; VTA, ventral tegmental area; ZI, zona incerta. Scale bar = 500 μm for A,B,D,E; 100 μm for C,F.

β-End, MCH, and ORX

We observed a low to moderate expression of TCF7L2 mRNA in the arcuate nucleus and in the lateral hypothalamic area. Thus, we assessed the expression of TCF7L2 mRNA in neurons related to the control of energy balance, including β-End in the arcuate nucleus, and MCH and ORX in the lateral hypothalamic area. In both regions we found very few (0.5%–2%) colocalizations with the neuropep-tides assessed.

DISCUSSION

TCF7L2 expression in the brain was initially reported in the Allen Brain Atlas (Lein et al., 2007) as part of their large-scale mapping project. In this study we extended their initial findings through a combination of radioactive in situ hybridization and dual-label immunohistochemistry techniques to provide the first comprehensive description of TCF7L2 expression in the mouse brain, as well as a detailed analysis of the neuro-chemical specificity of these neurons. We find TCF7L2 to be expressed throughout the adult mouse nervous system, and this expression can be divided into two distinct patterns. We find strong and ubiquitous expression throughout the thalamus and tectum. We also find cell-specific expression within the hypothalamus, basal forebrain, and circumventricular organs.

TCF7L2 and diabetes

It has long been known that there is a strong genetic component to the incidence of type 2 diabetes. However, identifying genes that contribute to disease development and progression has been difficult. Only in the last few years have PPARγ (Altshuler et al., 2000) and KCNJ11 (Gloyn et al., 2003) emerged as strong candidates in contributing to susceptibility to diabetes. Despite initial associations, their relevance to the general population has proven to be limited. This is in contrast to TCF7L2, whose variants are, to date, the single greatest genetic predictor for the development and progression of diabetes (Hattersley, 2007). The association of TCF7L2 with diabetes has gained even more significance because it has been replicated in numerous whole genome-wide association studies around the world (reviewed in Goodarzi and Rotter, 2007; Hattersley, 2007; Tong et al., 2009).

Before its association with diabetes, TCF7L2 was known to be a member of the WNT signaling pathway. The WNT pathway regulates numerous developmental processes, including embryogenesis, neurodevelopment, cell fate determination, maintenance of stem cells, and the development of the adipocytes, pancreas, and muscle. Of particular relevance to the pathophysiology of diabetes, WNT and TCF7L2 signaling have been shown to regulate the transcription of proglucagon, from which glucagon (GCG), and glucagon-like peptide-1 (GLP-1), and GLP-2 (Yi et al., 2005), are derived in a tissue-specific manner. In fat cells, WNT and TCF7L2 signaling inhibits adipogenesis (Ross et al., 2000), and is downregulated by insulin (Ahlzen et al., 2008).

Diabetes has traditionally been considered a peripheral disease characterized by defects in insulin secretion or insulin action, resulting in poor regulation of glucose levels. Recent work has suggested that the central nervous system (CNS) also has an important role in the development of diabetes (Obici et al., 2002, 2003; Elmquist and Marcus, 2003; Alquier and Kahn, 2004; Pocai et al., 2005; Sandoval et al., 2008). The CNS directly senses nutrients and hormones, including insulin and glucose, to produce a coordinated response targeting peripheral tissues such as the pancreas, liver, muscle, and adipose to regulate glucose and energy homeostasis. These findings have renewed interest in uncovering new signaling pathways and genes in the brain that may be important in the development of diabetes. Because of its strong expression in the brain, and its association with diabetes, TCF7L2 is an attractive candidate for further study.

Mice lacking TCF7L2 are neonatal lethal, and show development defects in the small intestine and pituitary (Korinek et al., 1998; Brinkmeier et al., 2003, 2007). In humans, however, TCF7L2 polymorphisms are linked to β-cell survival and function (Shu et al., 2008). These phenotypic discrepancies may reflect differences in human–mouse gene expression. For instance, TCF7L2 is not detectable in mouse pancreatic cells, but is expressed there at significant levels in humans (Yi et al., 2005; Cauchi et al., 2006). Our work here shows that TCF7L2 is produced in the CNS, and suggests that this expression is conserved between mice, primates, and humans (Cauchi et al., 2006; Murray et al., 2007).

TCF7L2 in the thalamus

The strong and widespread expression of TCF7L2 in thalamic nuclei and midbrain structures suggests a potential role for TCF7L2 in the WNT signaling pathways that direct early neural development, such as differentiation of neural crest cells, patterning events, and axon guidance (Patapoutian and Reichardt, 2000; Charron and Tessier-Lavigne, 2005). In addition, TCF7L2 may be involved in WNT-associated adult processes, such as adult neurogenesis, cancer, and some neurodegenerative disorders (Malaterre et al., 2007). Peripherally, TCF7L2 has been shown to inhibit WNT signaling in a dominant-negative manner in pancreatic beta cells and preadipocytes (Ross et al., 2000; Liu and Habener, 2008).

The strongest and most striking expression of TCF7L2 was found in the thalamus. Similar to the expression pattern reported in primates (Murray et al., 2007), TCF7L2 is broadly expressed in the thalamus and respects traditional histological boundaries. While the thalamus is primarily known to be involved in the processing and relaying of sensory information, regulation of sleep, awareness, and motor activity, there is growing evidence that it may play a role in glucose sensing. For instance, there are several reports that the posterior paraventricular nucleus of the thalamus (THPVP) has been shown to respond to hypoglycemia (Teves et al., 2004; Al-Noori et al., 2008).

TCF7L2 in autonomic control sites

Moderate expression of TCF7L2 mRNA was identified in neurons localized in autonomic brain sites such as the basal forebrain, hypothalamus, and periaqueductal gray matter. We were unable to determine the neurochemical phenotype of TCF7L2-expressing cells using the commonly expressed markers ChAT, TH, β-End, MCH and ORX, suggesting that TCF7L2 is expressed in a unique and cell-specific manner.

The ability of the hypothalamus to detect and integrate peripheral hormonal signals to regulate energy homeostasis has been well studied. It is now appreciated that certain hypothalamic nuclei, such as the arcuate nucleus (ARC) and ventromedial nucleus of the hypothalamus (VMH), can also sense changes in glucose concentrations (Nogueiras et al., 2008; Sandoval et al., 2008). TCF7L2 is expressed at low levels in the ARC, but is not detectable in the VMH. However, it is expressed at moderate levels in hypothalamic nuclei such as the dorsomedial hypothalamus (DMH) and lateral hypothalamic area (LHA), which are known to be important for energy homeostasis, and which are also targets of ARC and VMH projections. Interestingly, TCF7L2 is also moderately expressed in the basal forebrain, a region that has long been the focus of sleep research, but which recently has also been shown to be responsive to hypoglycemia (Cai et al., 2001; Tkacs et al., 2007).

TCF7L2 is expressed in the three circumventricular organs (CVOs), the vascular organ of lamina terminalis (OVLT), subfornical organ (SFO), and the area postrema (AP). These are highly vascularized structures that are not sequestered by the blood– brain barrier, and thus are able to sense a variety of large molecules and peptides from the systemic circulation and relay the information to the rest of the brain (reviewed in Johnson and Gross, 1993; Fry and Ferguson, 2007). The CVOs express a wide variety of receptors that are important for osmolarity, cardiovascular, and feeding homeostasis. These sites also innervate second-order neurons that control energy balance. For instance, amylin and ghrelin receptors are distributed throughout the CVOs, while the AP can detect changes in glucose concentrations and innervates the nucleus of solitary tract (NTS), which in turn innervates the hypothalamus (reviewed in Fry and Ferguson, 2007).

Conclusion

Given the importance of the brain in regulating food intake and maintaining energy and glucose homeostasis, alterations of the activity of centrally expressed TCF7L2 may play a role in the development of diabetes. Its expression in neuronal populations that have the ability to sense circulating nutrient levels suggest that it may have a role in the development or maintenance of nutrient sensing neurons. Further work, including brain-specific and neuron-specific knockouts, will determine if central TCF7L2 is a potential regulator of energy balance and glucose homeostasis. If so, TCF7L2 will join the growing list of genes, such as cholecystokinin, insulin receptor, and glucagon-like peptide 1, which were originally identified for their roles in energy balance and metabolism in peripheral organs, but also play key roles in the CNS control of energy homeostasis.