Review of the neuroanatomic landscape implicated in glucose sensing and regulation of nutrient signaling: immunophenotypic localization of diabetes gene Tcf7l2 in the developing murine brain

Abstract

Genetic variants in the transcription factor 7-like 2(Tcf7l2) gene have been found to confer a significant risk of type 2 diabetes and attenuated insulin secretion. Based on its genomic wide association Tcf7l2 is considered the single most important predictor of diabetes to date. Previous studies of Tcf7l2 mRNA localization in the adult brain suggest a putative role of Tcf7l2 in the CNS regulation of energy homeostasis. The present study further characterizes the immunophenotypic distribution of peptide expression in the brains of Tcf7l2 progeny during developmental time periods between E12.5 and P1. Tcf7l2(-/-) is lethal beyond P1. Results show that while negligible TCF7L2 expression is found in the developing brains of Tcf7l2(-/-)mice, TCF7L2 protein is relatively widespread and robustly expressed in the brain by E18.5 and exhibits specific expression within neuronal populations and regions of the brain in Tcf7l2(+/-) and Tcf7l2(+/+) progeny. Strong immunophenotypic labeling was found in the diencephalic structure of the thalamus that suggests a role of Tcf7l2 in the development and maintenance of thalamic activity. Strongly expressed TCF7L2 was localized in select hypothalamic and preoptic nuclei indicative of Tcf7l2 function within neurons controlling energy balance. Definitive neuronal staining for TCF7L2 within nuclei of the brain stem and circumventricular organs extends TCF7L2 localization within autonomic neurons and its potential integration with autonomic function. In addition robust TCF7L2 expression was found in the tectal and tegmental structures of the superior and inferior colliculi as well as transient expression in neuroepithelium of the cerebral and hippocampal cortices of E16 and E18.5. Patterns of TCF7L2 peptide localization when compared to the adult protein synthetic chemical/anatomical landscape of glucose sensing exhibit a good correlational fit between its expression and regions, nuclei, and pathways regulating energy homeostasis via integration and response to peripheral endocrine, metabolic and neuronal signaling. TCF was also found co-localized with peptides that regulate energy homeostasis including AgRP, POMC and NPY. TCF7l2, some variants of which have been shown to impair GLP-1-induced insulin secretion, was also found co-localize with GLP-1 in adult TCF wild type progeny. Impaired Tcf7l2-mediated neural regulation may contribute to the risk and/or underlying pathophysiology of type 2 diabetes that has found high expression in genomic studies of Tcf7l2 variants.

Figure 1

A-I. Medullary to midbrain coronal sections of an E18.5 Tcf7l2^+/+ mouse showing the widespread distribution of TCF7L2 peptide. A-F show the distribution of the peptide within nuclei of the hindbrain while G-I are midbrain sections illustrating the prevalence of TCF7L2 peptide within principal nuclei of the thalamus and hypothalamus. A full list of terms and their abbreviations is given in the list of abbreviations. Scale line in I = 500 microns and is the same for A-H Figure 1. J-L. The continuing series of coronal sections showing the midbrain to forebrain regions and localization of TCF7L2. K-L illustrate the peptide staining of the circumventricular organs(SFO and VOLT) as well as the stained neuroepithelium of the germinal zones lying adjacent to the walls of the lateral ventricles. Scale line in L = 500 microns and is the same for J-K.

Figure 1

A-I. Medullary to midbrain coronal sections of an E18.5 Tcf7l2^+/+ mouse showing the widespread distribution of TCF7L2 peptide. A-F show the distribution of the peptide within nuclei of the hindbrain while G-I are midbrain sections illustrating the prevalence of TCF7L2 peptide within principal nuclei of the thalamus and hypothalamus. A full list of terms and their abbreviations is given in the list of abbreviations. Scale line in I = 500 microns and is the same for A-H Figure 1. J-L. The continuing series of coronal sections showing the midbrain to forebrain regions and localization of TCF7L2. K-L illustrate the peptide staining of the circumventricular organs(SFO and VOLT) as well as the stained neuroepithelium of the germinal zones lying adjacent to the walls of the lateral ventricles. Scale line in L = 500 microns and is the same for J-K.

Figure 2

A-L show labeled cells from the boxed areas of Fig 1A-L at a higher magnification(20×). These include cells of medial longitudinal fasciculus(A), prepositus hypoglossal nucleus(B), dorsal tegmental nucleus(C), periaqueductal gray(D), microcellular tegmental nucleus(E), medial raphe nucleus(F), reticular formation of pons(G), substantia nigra compactus(H), ventromedial hypothalamic nucleus(I) arcuate nucleus(J), medial tuberal nucleus(K), and subfornical organ(l). Scale line of A,C,D-F, I, K-L = 100 microns; B,G,H,J = 50 microns.

Figure 3

A-C. Labeled cells of the lateral preoptic nucleus(A), medial preoptic nucleus(B) and septal neuroepithelium(C). 20× D. Parasagittal view of a P1 Tcf7l2^+/- head showing the distribution of TCF7L2 within the thalamus and midbrain regions. 1× E. Higher magnification of the inset from D showing TCF7L2 labeled cells clearly defining the thalamus and its adjacent areas. The fasciculus retroflexus(rf) separates the thalamic cells from the pretectal(Ptec), habenular(MHb) and commissural cells above(PrC) and the zona incerta(ZI) below. 10× F. Parasagittal view showing the stained hindbrain nuclei including the reticular formation of the pons(RFp), medial vestibular(MVe), prepositus hypoglossal nucleus(Pr), Solitary tract nucleus(Sol), reticular formation medulla(RFm) and the gigantocellular nucleus(Gi) as well as the midbrain thalamus(TH), and ventromedial hypothalamic nucleus(VMH). 5× G. A higher magnification of the inset region F showing in detail the staining of neurons within the gigantocellular reticular nucleus. Scale line of in A-C = 50 microns; D = 1mm; E = 100 microns and F = 200 microns.

Figure 4

A parasagittal view of labeled cells of the hypothalamic region including the ventromedial(VMH), retrochiasmatic(RC), dorsomedial(DM), posterior(PH) and periventricular(Pe) hypothalamic nuclei. 10× B-C. Magnified views of the inset region of A showing detailed staining of the labeled cells of the retrochiasmatic nucleus using red(B) and blue(C) counterstaining to highlight the staining of these cells lying adjacent to the optic tract(OT) which have not been reported in earlier works. 20× D. Parasagittal section showing the stained cells of the thalamus with relation to the cells of the lateral hypothalamic(LH) and lateral preoptic nuclei(LPO). 5× E. Parasagittal section showing the stained cells of the hindbrain including the gigantocellular(Gi), medullary reticular(RM), pontine reticular(RP) and the pedunculopontine(PP) nuclei and those of the midbrain including the arcuate(Arc) and ventromedial(VMH) hypothalamic nuclei. Other stained cells shown include the periaqueductal gray(PAG), hypothalamic neuroepithelium(Hn) and cells within the preoptic area(POA). Scale line in A = 100 microns; B-C = 50 microns; D-E = 300 microns.

Figure 5

A-C. Stained cells within the arcuate hypothalamic nucleus. A. NPY-stained cells showing immunoreactive fibers extending beyond the nucleus into the region adjacent to the arcuate nucleus. B. TCF7L2 stained nuclei of arcuate neurons. C. Agouti-related protein immunoreactive cells within the arcuate nucleus.

Figure 6

A. Parasagittal section of an E18.5 Tcf7l2^+/- showing the distribution of stained neuroepithelium in the neocortical(N) and hippocampal(Hi) cortices. Immunoreactive neuroepithelial cells were also found in the germinal layers of the cerebellum(Cb) and those lining the cerebral aqueduct(CAq). These also extended into regions of stained cells found in the dorsal raphe (DR, dorsal tegmental(DTg) and posterior dorsal tegmental(PdT) nuclei. 5× B-D. Magnified insets of A including details of stained cells within the ventricular(V), subventricular(SV) and granular zones of the olfactory bulb(B), olfactory lobe(C), the retrosplenial lobe(D) and the external granular(Eg), molecular layer(Ml) and Purkinje cells(Pc) of the cerebellum(E). 20× F-I. Serially stained parasagittal sections of an E16 Tcf7l2^+/- showing sequential staining for nestin(F), oligo 2(G), neuronal nuclei(H) and H3(I) highlighting the similarity in staining patterns for TCF7L2(A). 5× J-L. Magnified insets of G-I, showing detailed staining for H3(J), neuronal nuclei (K) and oligo 2(L). Compared to Fig A, TCF7L2 stained those mitotic figures of neuroepithelium in F and I, oligodendocytic maturation(G and L) and those cells lacking stained expression in post-mitotic neurons in H and K. Scale line in A = 500 microns; B-E = 100 microns; F-I = .1cm; J-L = 100 microns.