Structural insights into the impact of two holoprosencephaly-related mutations on human TGIF1 homeodomain.

Human protein TGIF1 is an essential regulator of cell fate with broad roles in different tissues, and has been implicated in holoprosencephaly (HPE) and many cancers. The function of TGIF1 in transcriptional regulation depends on its three-amino acid loop extension (TALE) type of homeodomain (HD). Two missense mutations that led to P192A and R219C substitutions in TGIF1-HD were previously found in HPE patients and suggested to be the causes for these cases. However, how these mutations affected TGIF1 function has not been investigated from a structural view. Here, we investigated the roles of P192 and R219 in TGIF1-HD structure packing through determining the NMR structure of TGIF1-HD. Surprisingly, P192 and R219 were found to play roles in packing α1 and α2 to α3 together with A190 and F215 through side-chain interactions. Circular dichroism (CD) showed that P192A and R219C mutants displayed structural change and less folding compared with wild-type TGIF1-HD, and 1H-15N HSQC spectrum of P192A mutant exhibited chemical shift perturbations in all three helices of TGIF1-HD. Thus, it is suggested that P192A and R219C mutations led to structure disturbances of TGIF1-HD, which subsequently reduced the DNA-binding affinity of TGIF1-HD by 23-fold and 10-fold respectively, as revealed by the isothermal titration calorimetry (ITC) experiments. Our study provides structural insights of the probable pathogenesis mechanism of two TGIF1-related HPE cases, and evidences for the roles of P192 and R219 in HD folding.

Three Tctn proteins are functionally conserved in the regulation of neural tube patterning and Gli3 processing but not ciliogenesis and Hedgehog signaling in the mouse.

Tctn1, Tctn2, and Tctn3 are membrane proteins that localize at the transition zone of primary cilia. Tctn1 and Tctn2 mutations have been reported in both humans and mice, but Tctn3 mutations have been reported only in humans. It is also not clear whether the three Tctn proteins are functionally conserved with respect to ciliogenesis and Hedgehog (Hh) signaling. In the present study, we report that loss of Tctn3 gene function in mice results in a decrease in ciliogenesis and Hh signaling. Consistent with this, Tctn3 mutant mice exhibit holoprosencephaly and randomized heart looping and lack the floor plate in the neural tube, the phenotypes similar to those of Tctn1 and Tctn2 mutants. We also show that overexpression of Tctn3, but not Tctn1 or Tctn2, can rescue ciliogenesis in Tctn3 mutant cells. Similarly, replacement of Tctn3 with Tctn1 or Tctn2 in the Tctn3 gene locus results in reduced ciliogenesis and Hh signaling, holoprosencephaly, and randomized heart looping. Surprisingly, however, the neural tube patterning and the proteolytic processing of Gli3 (a transcription regulator for Hh signaling) into a repressor, both of which are usually impaired in ciliary gene mutants, are normal. These results suggest that Tctn1, Tctn2, and Tctn3 are functionally divergent with respect to their role in ciliogenesis and Hh signaling but conserved in neural tube patterning and Gli3 processing.

Endocytic receptor LRP2/megalin-of holoprosencephaly and renal Fanconi syndrome.

Megalin (or LRP2) is an endocytic receptor that plays a central role in embryonic development and adult tissue homeostasis. Loss of this receptor in congenital or acquired diseases results in multiple organ dysfunctions, including forebrain malformation (holoprosencephaly) and renal reabsorption defects (renal Fanconi syndrome). Here, we describe current concepts of the mode of receptor action that include co-receptors and a repertoire of different ligands, and we discuss how these interactions govern functional integrity of the kidney and the brain, and cause disease when defective.

Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog.

The Hedgehog cell-cell signaling pathway is crucial for animal development, and its misregulation is implicated in numerous birth defects and cancers. In unstimulated cells, pathway activity is inhibited by the tumor suppressor membrane protein, Patched. Hedgehog signaling is triggered by the secreted Hedgehog ligand, which binds and inhibits Patched, thus setting in motion the downstream events in signal transduction. Despite its critical importance, the mechanism by which Hedgehog antagonizes Patched has remained unknown. Here, we show that vertebrate Patched1 inhibition is caused by direct, palmitate-dependent interaction with the Sonic Hedgehog ligand. We find that a short palmitoylated N-terminal fragment of Sonic Hedgehog binds Patched1 and, strikingly, is sufficient to inhibit it and to activate signaling. The rest of Sonic Hedgehog confers high-affinity Patched1 binding and internalization through a distinct binding site, but, surprisingly, it is not absolutely required for signaling. The palmitate-dependent interaction with Patched1 is specifically impaired in a Sonic Hedgehog mutant causing human holoprosencephaly, the most frequent congenital brain malformation, explaining its drastically reduced potency. The palmitate-dependent interaction is also abolished in constitutively inhibited Patched1 point mutants causing the Gorlin cancer syndrome, suggesting that they might adopt a conformation distinct from the wild type. Our data demonstrate that Sonic Hedgehog signals via the palmitate-dependent arm of a two-pronged contact with Patched1. Furthermore, our results suggest that, during Hedgehog signaling, ligand binding inhibits Patched by trapping it in an inactive conformation, a mechanism that explains the dramatically reduced activity of oncogenic Patched1 mutants.

The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

INTRODUCTION: The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. OBJECTIVES/METHODS: We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). RESULTS: Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. CONCLUSIONS: Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.

Mutations in Hedgehog acyltransferase (Hhat) perturb Hedgehog signaling, resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects.

Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.

The relationship between sonic Hedgehog signaling, cilia, and neural tube defects.

The Hedgehog signaling pathway is essential for many aspects of normal embryonic development, including formation and patterning of the neural tube. Absence of the sonic hedgehog (shh) ligand is associated with the midline defect holoprosencephaly, whereas increased Shh signaling is associated with exencephaly and spina bifida. To complicate this apparently simple relationship, mutation of proteins required for function of cilia often leads to impaired Shh signaling and to disruption of neural tube closure. In this article, we review the literature on Shh pathway mutants and discuss the relationship between Shh signaling, cilia, and neural tube defects.

Fetal ethanol exposure activates protein kinase A and impairs Shh expression in prechordal mesendoderm cells in the pathogenesis of holoprosencephaly.

BACKGROUND: In humans, fetal ethanol exposure can cause holoprosencephaly (HPE), one of the most common birth defects that is characterized by brain, facial, and oral abnormalities. However, the pathogenesis of HPE is not clear. In the present study, we investigated the teratogenic mechanism of ethanol-induced brain and facial malformations in mice. METHODS: Pregnant C57BL/6J mice were administered ethanol on E7 and facial and brain malformations were characterized on E10.5. We examined the effect of fetal ethanol exposure on Shh expression and activation of protein kinase A (PKA) because mutations in the human Shh gene are the most frequent cause of autosomal-dominant inherited HPE and PKA is a potent endogenous antagonist of Shh signaling. RESULTS: Fetal ethanol exposure on E7 induced severe midline defects characteristic of HPE. Ethanol exposure impaired Shh expression and induced excessive apoptosis only along the anterior edge of the prechordal mesendoderm (PME). In addition, ethanol activated PKA in anterior PME cells. Pretreatment of embryos with antioxidants, such as vitamins C or E, prevented the development of ethanol-induced HPE. CONCLUSIONS: Shh expression in PME cells is involved in the pathogenesis of ethanol-induced HPE. Ethanol may impair Shh expression indirectly by activating PKA. The inhibition of excessive apoptosis in PME cells by antioxidants implies that oxidative stress may underlie the teratogenic actions of ethanol. Thus, antioxidant treatment may be a simple preventative measure that could reduce the incidence of HPE following fetal ethanol exposure.

A mechanism for mutational inactivation of the homeodomain protein TGIF in holoprosencephaly.

The homeodomain protein TGIF functions as a negative modulator for multiple classes of transcription factors. Loss of function mutations in a single copy of TGIF result in holoprosencephaly, a developmental anomaly leading to severe forebrain and craniofacial malformations. However, the mechanisms by which these mutations disrupt the functions of TGIF remain to be elucidated. Here we show that a holoprosencephaly mutation (P63R) interferes with the ability of TGIF to act as a corepressor for c-Jun and Smad2, suggesting that this holoprosencephaly mutation may lead to a general defect in the TGIF protein. In fact, we observed that the P63R mutation affects folding of the TGIF protein, resulting in the disruption of the diffuse nuclear staining pattern characteristic of wild-type (WT) TGIF and the accumulation of TGIF in nuclear aggregates. We also show that the mutant TGIF.P63R is degraded more rapidly when compared with WT TGIF and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we observed that TGIF.P63R homodimerizes with WT TGIF to sequester it into nuclear aggregates and to enhance its ubiquitin-dependent degradation. These results reveal an important mechanism for the degradation of TGIF through the ubiquitin-proteasome pathway, whose deregulation might contribute to the development of human holoprosencephaly.

Sonic hedgehog expression in Gli3 depressed mouse embryo, Pdn/Pdn.

The phenotype of the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is similar to Greig cephalopolysyndactyly syndrome (GCPS), which is induced by mutation of GLI3. Suppression of Gli3 gene expression has been observed in Pdn/Pdn. Thus, the gene responsible for Pdn/Pdn has been considered to be Gli3. Recently, the mutation point was demarcated, that is, a transposon was inserted into intron 3 of the Gli3 gene in the Pdn mouse. Forward and reverse primers were constructed in intron 3 near the insertion point. A forward primer in the long terminal repeat region of the transposon was also constructed. Now we can discriminate +/+, Pdn/+, Pdn/Pdn embryos from the PCR products. After genotyping of the Pdn embryos, Gli3 and other correlated gene expressions, such as sonic hedgehog (Shh), Bmp-2, Bmp-4, ptc-1, were analyzed by real-time PCR method. Gli3 gene expression in Pdn/Pdn was suppressed to 20-30% of +/+, and that in Pdn/+ was about 60% of +/+ through all the embryonic and neonatal periods examined. As Shh has been considered to be an antagonist of Gli3, Shh expression was analyzed, and a difference among genotypes was observed only on day 9 of gestation. We could not detect any alterations among genotypes in other gene expressions examined. Gli3 and Shh gene expression were also analyzed on day 9 by whole-mount in situ hybridization in the +/+ and Pdn/Pdn embryos. Neuroectoderm was positive by Gli3 probe in +/+ but not in Pdn/Pdn. Notochord, floor plate and prechordal mesoderm were positive by Shh probe both in +/+ and Pdn/Pdn embryos, but ectopic and/or over-expression of Shh were not observed in Pdn/Pdn embryos.

TGF-beta signaling in human skeletal and patterning disorders.

Members of the transforming growth factor beta (TGF-beta) family of multifunctional peptides are involved in almost every aspect of development. Model systems, ranging from genetically tractable invertebrates to genetically engineered mice, have been used to determine the mechanisms of TGF-beta signaling in normal development and in pathological situations. Furthermore, mutations in genes for the ligands, receptors, extracellular modulators, and intracellular signaling molecules have been associated with several human disorders. The most common are those associated with the development and maintenance of the skeletal system and axial patterning. This review focuses on the mechanisms of TGF-beta signaling with special emphasis on the molecules involved in human disorders of patterning and skeletal development.

Extramedullary hematopoiesis within a frontoethmoidal encephalocele in a newborn with holoprosencephaly.

We present the first report of extramedullary hematopoiesis (EMH) in an encephalocele. The patient was a new-born with semilobar holoprosencephaly, a frontoethmoidal encephalocele, and a large subdural hematoma. The encephalocele appeared as a hemorrhagic mass, protruding from the forehead to cover the right eye, without involvement of the sinuses or nasopharynx. Computerized tomography and magnetic resonance imaging studies ruled out other forms of holoprosencephaly and confirmed the continuity of the brain with the extruded mass. Immunohistochemistry confirmed the presence of an atrophic epithelium covering the mass. Histologic examination of the encephalocele revealed EMH both within and adjacent to malformed cerebral cortex, with a tendency for the hematopoietic cells to line up in columns within malformed cerebral cortex. We propose that a single event during the fourth week of gestation could both interrupt closure of the neural tube, giving rise to the encephalocele, and impair migration of the neural crest, leading to holoprosencephaly secondary to failure of neural crest derivatives to induce basomedial telencephalic differentiation. EMH may have been induced from hematopoietic stem cells in the richly vascular meningeal component of the encephalocele, in response to anemia and hypoxia.

The interaction of the carboxyl terminus-binding protein with the Smad corepressor TGIF is disrupted by a holoprosencephaly mutation in TGIF.

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. We demonstrate that TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and we show that TGIF is able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF.