SOX2 plays a critical role in the pituitary, forebrain, and eye during human embryonic development

Abstract

Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus.

Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues.

Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit beta-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke's pouch, the developing anterior pituitary, and the eye.

Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-beta-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

FIG. 1

Midline sagittal MRI section of patient 1 showing hypoplasia of the anterior pituitary (ap), normal infundibulum (i), with an eutopic posterior pituitary (pp) and a thin abnormal corpus callosum (cc).

FIG. 2

Analysis of SOX2 mutations. A, SOX2 gene deletion in patient 1. Metaphase chromosomes were hybridized with BAC clone RP11–43F17 containing SOX2 (red signal; arrowhead) and a chromosome 3 centromeric clone (green signal). The normal chromosome 3 shows both red and green signals. The der(3) shows only a green signal indicating that one copy of SOX2 is deleted. B, Array-CGH profile from patient 1. The chromosome 3 ideogram (left) and enlargement of the deleted region (right) show the ratio of probes (each represented by a single dot) plotted as a function of chromosomal position; loss of copy number of a probe shifts the ratio to the left. C, Electropherogram showing the c.181C>T transition (p.Q61X) in patient 2 (arrow). BfaI digestion of PCR products showing that the heterozygous mutation occurred de novo in patient 2 (filled circle) and is not present in the unaffected parents or in a normal control individual [wild type (WT)]. M, 100-bp DNA ladder. D–F, Functional effects of the p.Q61X mutation. D, The p.Q61X mutation failed to activate the reporter showing similar levels of activation to empty expression vector compared with wild-type SOX2. E, EMSA of wild-type and p.Q61X SOX2 proteins shows that the mutant SOX2 is unable to bind DNA. F, The p.Q61X mutation results in impaired nuclear localization of the mutant protein compared with wild-type SOX2, which stains predominantly within the nucleus. DAPI, 4′,6-diamidino-2-phenylindole; IVT, in vitro translated empty expression vector.

FIG. 3

SOX2 disrupts β-catenin-TCF/LEF mediated transcriptional activation. A, Human embryonic kidney 293 cells were cotransfected with the TCF/LEF reporter construct TOPFLASH with human SOX2 expression construct alone (70 ng) or human β-catenin expression construct with variable amounts of SOX2 (1–70 ng). Increasing amounts of SOX2 led to dose-dependent repression of β-catenin-mediated activation of the reporter. B, SOX2 mutations disrupt the interaction with β-catenin. SOX2 mutant constructs were tested and compared with wild-type (WT) SOX2 using 20 ng TOPFLASH, 30 ng β-catenin, and 20 ng SOX2 expression construct. Truncating SOX2 mutations fail to repress β-catenin-mediated activation, showing levels of activation comparable to cotransfection with β-catenin alone or empty expression vector. Schematic representation of the SOX2 gene with approximate positions of the mutations is shown in the top right.

FOG. 4

SOX2 expression pattern in human embryonic and fetal tissue (mRNA: A–C, E, F, and I–L; protein: D, G, and H). Transverse (A) and sagittal (B) sections show expression of SOX2 within the developing Rathke’s pouch (Rp) and overlying neural ectoderm at CS16 and CS19, respectively. C and D, Coronal sections of F2 tissue showing SOX2 transcripts and protein continue to be expressed in the cells lining the lumen of the anterior pituitary (AP). Note the absence of expression in the infundibulum (inf) and posterior pituitary (PP). E, Transverse section showing SOX2 expression in the forebrain, including the region of the presumptive hippocampus (arrowhead), and throughout the midbrain and hindbrain in addition to the cerebellum (Ce). F–H, Transverse sections of the forebrain in human fetal tissues at approximately 8-wk development showing expression of SOX2 in the hypothalamus (Hyp). H, Magnified section corresponding to the boxed region in G. I and J, Transverse sections showing expression of SOX2 in the lens (l) vesicle and developing neural retina (nr) at CS16, where expression is restricted to the proximal inner layer, with a boundary of expression in the distal region adjacent to the lens (arrowhead). J, Expression within the lens diminishes by CS20. SOX2 is also expressed in the nasal epithelium (K) and in the tracheoesophageal tract (L), as shown in transverse section. Scale bars: 500 μm (E); 300 μm (B–D, F, G, J, and K); and 100 μm (A, H, I, and L). Di, Diencephalon; Me, mesencephalon; oc, oral cavity; Oe, developing esophagus; phy, presumptive hypothalamus; Rh, rhombencephalon; T, telencephalon; Tr, developing trachea; 3V, third ventricle.