Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans

Abstract

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/β-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with β-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

Keywords: Tcf7l1; WNT pathway; hypopituitarism; pituitary; septooptic dysplasia.

Fig. S1.

Conditional deletion of Tcf3/Tcf7L1 results in forebrain defects and partially penetrant dwarfism. (A) Hesx1^Cre/+;Tcf7l1^fl/− mutant adult mouse exhibiting reduced body size compared with a control wild-type littermate. (B and C) Wild-type and Hesx1^Cre/+;Tcf7l1^fl/− mutant embryos showing exencephaly. (D and E) Growth graphs of Hesx1^Cre/+;Tcf3^fl/− mutants and control embryos. Note the severe growth failure in the mutants (triangle) relative to the control littermates (circles).

Fig. 1.

Abnormal pituitary morphogenesis in Hesx1^Cre/+;Tcf7l1^fl/− mutants. In situ hybridization (A–C) and immunohistochemistry (D–H) of transverse histological sections of the pituitary gland of control embryos (A–H) and Hesx1^Cre/+;Tcf7l1^fl/− mutants (A′–H′ and A′′–H′′) at 17.5 dpc. (A′–H′) Mildly affected embryos show pituitary hyperplasia and cleft bifurcations (arrows in D′ and E′) but mostly normal expression of differentiation markers. (A′′–H′′) Severely affected pituitaries exhibit dysmorphic pituitary tissue that is ectopically located in the oropharyngeal cavity (arrowheads), but hormone-producing cells are present. AP, anterior pituitary; BS, basisphenoid bone; Cga, glycoprotein hormone α; Gh, growth hormone; OC, oral cavity; Pomc1, pro-opiomelanocortin-α; PP, posterior pituitary. Pictures are representative of five embryos per genotype. (Scale bar: H′′, 100 μm.)

Fig. S2.

Clonogenic potential of pituitary stem cells at 18.5 dpc and 1 d postnatally (P1). Pituitaries were dissociated to single cell suspensions and cultured in stem cell-promoting media as described. Note that there are not significant differences in the proportion of clonogenic cells between genotypes.

Fig. 2.

Increased proliferation of RP progenitors but normal patterning of the developing anterior pituitary in Hesx1^Cre/+;Tcf7l1^fl/− mutants. (A–P) In situ hybridization on sagittal sections through the pituitary gland of control and Hesx1^Cre/+;Tcf7l1^fl/− mutant embryos (stage and probes are indicated; anterior to the left). (A–H) Mildly affected pituitaries show bifurcations and moderate expansion of the expression domains of Lhx3 and Prop1 at 13.5 and 15.5 dpc (arrows). (I–P) Severely affected pituitaries exhibit enlarged expression domains attributable to the abnormal morphogenesis of the developing anterior pituitary, which extends into the oropharyngeal cavity (arrowheads). (Q–R) Anti–phospho-histone H3 immunofluorescent staining on sagittal histological sections of a control embryo and a Hesx1^Cre/+;Tcf7l1^fl/− mutant. (S) Quantitative analyses showing a statistically significant increase in the mitotic index in the Hesx1^Cre/+;Tcf7l1^fl/− developing pituitaries relative to control littermates at 13.5 dpc but not at 15.5 dpc. AP, anterior pituitary; BS, basisphenoid bone; Inf, infundibulum; OC, oropharyngeal cavity. Pictures are representative of seven embryos per genotype. ***P < 0.05 (one-way ANOVA). (Scale bar: H and P, 100 μm.)

Fig. 3.

Dysregulation of Fgf8, Fgf10, and Bmp4 expression in the hypothalamus of Hesx1^Cre/+;Tcf7l1^fl/− mutants. In situ hybridization on sagittal histological sections revealing the expression of Fgf10 (A–D), Fgf8 (E–H), and Bmp4 (I–L) in the prospective hypothalamus and Lhx3 (M–P) in RP (anterior to the left). (A–H) Fgf10 and Fgf8 expression domains are anteriorized along the neural epithelium of the ventral diencephalon (i.e., prospective hypothalamus) overlaying the oral ectoderm of RP in the Hesx1^Cre/+;Tcf7l1^fl/− mutants (compare arrowheads in C and D and G and H, respectively). C and D and G and H represent enlarged images of the dotted squared areas in A and B and E and F, respectively. (I–L) The expression domain of Bmp4 is rostrally expanded in Hesx1^Cre/+;Tcf7l1^fl/− prospective hypothalamus compared with control embryos (compare arrowheads K and L). K and L represent enlarged images of the squared dotted areas in I and J. (M–P) The expression domain of Lhx3 is rostrally extended in Hesx1^Cre/+;Tcf3^fl/− compared with the control embryo (arrows in O and P), indicating an expansion of the RP epithelium. O and P are enlarged images of the dotted square areas in M and N respectively. VD, ventral diencephalon. Pictures are representative of five embryos per genotype. (Scale bar: P, 100 μm.)

Fig. 4.

SHH and its regulators Tbx2 and Tbx3 are misexpressed in the developing hypothalamus of Hesx1^Cre/+;Tcf7l1^fl/− mutants. (A–H) In situ hybridization on sagittal sections of 10.5-dpc embryos (anterior to the left) reveals the anterior shift of the Tbx3 expression domain, and to a lesser extent Tbx2, within the developing hypothalamus of the Hesx1^Cre/+;Tcf7l1^fl/− mutants compared with the control embryos. (I–L) Immunostaining showing a reduction of SHH expression in the caudal region of the preoptic area in a Hesx1^Cre/+;Tcf7l1^fl/− mutant relative to a control. Note that the reduction of SHH expression in the caudal region of the preoptic area appears to overlap with the anterior extension of Tbx3 expression. VD, ventral diencephalon. Pictures are representative of three embryos per genotype. (Scale bar: P, 100 μm.)

Fig. S3.

Immunostaining against Caspase 3 and TCF7L1. Histological sagittal sections of Hesx1^Cre/+;Tcf7l1^fl/− mutant and control embryos at 10.5 dpc (anterior to the left). (A and B) Caspase^+ve cells are detected mostly in the ventral region of RP (arrowhead) in both genotypes. (C and D) TCF7L1^+ve cells (green) are detected throughout the hypothalamus and RP regions in the wild-type embryo but are absent in the Hesx1^Cre/+;Tcf7l1^fl/− mutant. (Scale bar: 100 μm.)

Fig. 5.

Expression of a mutant form of TCF7L1 lacking the β-catenin-interacting domain (Tcf7l1^ΔN/ΔN) is sufficient to sustain normal hypothalamic-pituitary axis development. (A–H) In situ hybridization on sagittal sections showing normal expression domains of Fgf10 (A–D) and Lhx3 (E-H) in Tcf7l1^ΔN/ΔN mutants and control wild-type littermates at 9.5 dpc (anterior to the left). C, D, G, and H are enlarged images of the dotted squared areas. The apparent difference in size in the Tcf7l1^ΔN/ΔN mutants relative to the controls is caused by an overall developmental delay in the mutants. (I–R) In situ hybridization on transverse histological sections through the pituitary gland of Tcf7l1^ΔN/ΔN mutants and control embryos at 18.5 dpc reveal no gross differences in the levels of expression of several differentiation markers. Note that pituitary morphogenesis is also normal in Tcf7l1^ΔN/ΔN mutants relative to controls. AL, anterior lobe; BS, basisphenoid bone; PL, posterior lobe; VD, ventral diencephalon. Pictures are representative of five embryos per genotype. (Scale bars: H and R, 100 μm.)

Fig. 6.

TCF7L1 is expressed in the developing hypothalamic-pituitary axis, central nervous system and eyes during human embryogenesis. In situ hybridization against hTCF7L1 on coronal (A–C and G–I) or sagittal (D–F) histological sections at Carnegie stage (CS) 13, 18, and 20. (A–C) Note the expression of hTCF7L1 in the ventral diencephalon neuroepithelium (i.e., prospective hypothalamus) (arrowheads in B), RP progenitors (arrows in B), and developing optic cups (arrows in C) at CS13. (D–F) At CS18, expression of hTCF7L1 is observed throughout the neuroepithelium of the hind-, mid-, and forebrain, including the hypothalamus (arrowheads in E) and telencephalon (arrows in F). Expression is also detected in the developing anterior pituitary gland (arrows in E). (G–I) At CS20, expression of hTCF7L1 is detected in the anterior and posterior lobes of the pituitary gland (H), neural retina (arrows in I), eyelid (arrow in G), olfactory epithelium of the nasal cavity, and trigeminal ganglia. B, C, E, F, H, and I are enlarged images of the dotted squared areas. AP, anterior pituitary; BS, basisphenoid bone; hyp, hypothalamus; NC, nasal cavity; NR, neural retina; OC, optic cup; ON, optic nerve; PP, posterior pituitary; VD, ventral diencephalon. (Scale bars: A, D, and G, 500 μm; B, C, E, F, H and I, 100 μm.)

Fig. 7.

Identification of hTCF7L1 heterozygous missense variants in patients with SOD. (A) Electropherogram showing two heterozygous missense variants in hTCF7L1: p.R92P and p.R400Q. (B) Both DNA variants result in the substitution of highly conserved amino acids. R92 is not conserved in Xenopus and zebrafish. (C) Transient luciferase assays on HEK293 cells cotransfected with either TOPflash (left graph) or hLEF1-promoter-luc (right graph) reporters and constructs expressing wild-type, p.R92P, or p.R400Q hTCF7L1 proteins. Note the significant reduction in repressing activity of the p.R92P and p.R400Q hTCF7L1 variants relative to wild-type TCF7L1 using both reporters. **P < 0.05 (one-way ANOVA). (D) Western blot analysis of transfected HEK293 cells used in C with a specific anti-TCF7L1 antibody detects the expression of the wild-type and p.R92P or p.R400Q hTCF7L1 proteins at similar levels. GAPDH was also detected in the same membrane as loading control.

Fig. S4.

The hTCF7L1 p.R92P mutant protein is hyperphosphorylated by HIPK2. Western blot analysis using antibody to detect phosphorylated TCF7L1 reveals an increase in HIPK2-mediated phosphorylation of hTCF7L1 (p.R92P) mutant protein compared with wild-type TCF7L1.

Fig. S5.

Absence of dominant-negative effects of the hTCF7L1 p.R92P and p.R400Q variants. Transient luciferase assays on HEK293 cells cotransfected with TopFlash reporter and constructs expressing wild-type hTCF7L1 and either p.R92P or p.R400Q hTCF7L1 proteins. Note the significant reduction in repressing activity of the p.R92P and p.R400Q hTCF7L1 variants relative to wild-type TCF7L1 and the absence of effect when combined with wild-type hTCF7L1. **P < 0.05 (one-way ANOVA).

Fig. 8.

Incomplete rescue of the eyeless phenotype of double tcf7l1a/b zebrafish mutants with the human hTCF7L1 p.R92P and p.R400Q variants. Lateral view of 32-h postfertilization zebrafish embryos (anterior to left). Wild-type embryos injected with 100 pg of hTCF7L1 (C), p.R92P (E), and p.R400Q (H) mRNA show no phenotype compared with wild-type untreated embryos (A). tcf7l1a^−/−;tcf3l1b^+/− double mutants fail to develop eyes (B). Injection of tcf7l1a^−/−;tcf3l1b^+/− double mutants with 100 pg of wild-type hTCFL1 mRNA rescues the eyeless phenotype (D). Injection of tcf7l1a^−/−;tcf3l1b^+/− double mutants with 100 pg of hTCF7L1 p.R92P (F and G) or p.R400Q (I and J) results in incomplete rescue of the eyeless phenotype. (K) Percentage of embryos with normal eyes is shown in blue, and percentage of embryos with very small or no eyes is shown in red. p.R92P: 64.5% SD 12.41, P < 0.009; p.R400Q: 49.65% SD 5.93, P < 0.0006 (unpaired t test).