Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Neuropathology of holoprosencephaly.

Holoprosencephaly (HPE) is a primary disorder of neural induction and patterning of the rostral neural tube resulting in noncleavage of the forebrain with failure to form two separate distinct hemispheres. The spectrum of HPE is very broad and encompasses various neuropathological phenotypes of different severity. The recent literature has demonstrated that the phenotypic variability of HPE ranges from aprosencephaly-atelencephaly, at the most severe end, to milder forms such as the "middle interhemispheric variant" of HPE at the less severe end of the spectrum. Between them, different intermediate forms demonstrate a continuum in a wide phenotypic spectrum rather than well-defined categories. Although the term "HPE" suggests a disorder affecting only the prosencephalon, other brain structures are involved, underlining the complexity of the malformation. Because of close spatiotemporal interactions and common signaling pathways contributing to the development of both brain and face, concomitant facial and ocular anomalies are associated with brain malformation. In this review, the characteristic neuropathological features of the various forms of HPE are described as well as their associated brain, face, and ocular malformations, to delineate the different phenotypes.

High levels of iron supplementation prevents neural tube defects in the Fpn1ffe mouse model.

BACKGROUND: Periconception maternal nutrition and folate in particular are important factors influencing the incidence of neural tube defects (NTDs). Many but not all NTDs are prevented by folic acid supplementation and there is a pressing need for additional strategies to prevent these birth defects. Other micronutrients such as iron are potential candidates, yet a clear role for iron deficiency in contributing to NTDs is lacking. Our previous studies with the flatiron (ffe) mouse model of Ferroportin1 (Fpn1) deficiency suggest that iron is required for neural tube closure and forebrain development raising the possibility that iron supplementation could prevent NTDs. METHODS: We determined the effect of periconception iron and/or folic acid supplementation on the penetrance of NTDs in the Fpn1ffe mouse model. Concurrently, measurements of folate and iron were made to ensure supplementation had the intended effects. RESULTS: High levels of iron supplementation significantly reduced the incidence of NTDs in Fpn1ffe mutants. Fpn1 deficiency resulted in reduced folate levels in both pregnant dams and embryos. Yet folic acid supplementation did not prevent NTDs in the Fpn1ffe model. Similarly, forebrain truncations were rescued with iron. Surprisingly, the high levels of iron supplementation used in this study caused folate deficiency in wild-type dams and embryos. CONCLUSION: Our results demonstrate that iron supplementation can prevent NTDs and forebrain truncations in the Fpn1ffe model. Surprisingly, high levels of iron supplementation and iron overload can cause folate deficiency. If iron is essential for neural tube closure, it is possible that iron deficiency might contribute to NTDs. Birth Defects Research 109:81-91, 2017. © 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc.

Conditional Knockout of Breast Carcinoma Amplified Sequence 2 (BCAS2) in Mouse Forebrain Causes Dendritic Malformation via ß-catenin.

Breast carcinoma amplified sequence 2 (BCAS2) is a core component of the hPrP19 complex that controls RNA splicing. Here, we performed an exon array assay and showed that ß-catenin is a target of BCAS2 splicing regulation. The regulation of dendrite growth and morphology by ß-catenin is well documented. Therefore, we generated conditional knockout (cKO) mice to eliminate the BCAS2 expression in the forebrain to investigate the role of BCAS2 in dendrite growth. BCAS2 cKO mice showed a microcephaly-like phenotype with a reduced volume in the dentate gyrus (DG) and low levels of learning and memory, as evaluated using Morris water maze analysis and passive avoidance, respectively. Golgi staining revealed shorter dendrites, less dendritic complexity and decreased spine density in the DG of BCAS2 cKO mice. Moreover, the cKO mice displayed a short dendrite length in newborn neurons labeled by DCX, a marker of immature neurons, and BrdU incorporation. To further examine the mechanism underlying BCAS2-mediated dendritic malformation, we overexpressed ß-catenin in BCAS2-depleted primary neurons and found that the dendritic growth was restored. In summary, BCAS2 is an upstream regulator of ß-catenin gene expression and plays a role in dendrite growth at least partly through ß-catenin.

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

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/ß-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 ß-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.

A gene trap knockout of the Tiam-1 protein results in malformation of the early embryonic brain.

Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the ß-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice.

Wnt/ß-catenin signaling is disrupted in the extra-toes (Gli3(Xt/Xt) ) mutant from early stages of forebrain development, concomitant with anterior neural plate patterning defects.

The zinc finger transcription factor Gli3 is essential for normal development of the forebrain. Mutant mice with no functional Gli3 (extra-toes, Gli3(Xt/Xt) mutants) display a massive reduction in the size of the telencephalic lobes and absence of dorsomedial telencephalic structures, including the cortical hem, which normally expresses a number of Wnt molecules essential for patterning the hippocampus. Dorsomedial telencephalic Wnt activity, transduced through the Wnt/ß-catenin signaling pathway, is also required for hippocampal specification and dorsoventral telencephalic patterning. Wnts whose normal expression is restricted to the cortical hem are completely absent in Gli3(Xt/Xt) embryos, but some expression of those Wnts with a broader expression domain persists, raising the possibility that Wnt/ß-catenin signaling may still be active in this mutant. We examined whether the Wnt expression that persists in the Gli3(Xt/Xt) mutant neocortex activates Wnt/ß-catenin signaling, using the BAT-gal transgenic reporter. We found Wnt/ß-catenin signaling consistently decreased in the forebrains of Gli3(Xt/Xt) mutants, even prior to the formation of the cortical hem. This is accompanied by a severe reduction in expression of Wnt7b and Wnt8b at the lateral edges of the anterior neural plate that will give rise to the pallium. In addition, we found a significant increase in the expression of rostroventral markers of the anterior neural plate that will give rise to the basal forebrain. Our data reveal that Gli3 is required at the neural plate stage to regulate Wnt expression and Wnt/ß-catenin signaling in the presumptive forebrain and confirm its previously proposed role in patterning the anterior neural plate.

Cecr2 mutations causing exencephaly trigger misregulation of mesenchymal/ectodermal transcription factors.

BACKGROUND: Over 200 mouse genes are associated with neural tube defects (NTDs), including Cecr2, the bromodomain-containing subunit of the CERF chromatin remodeling complex. METHODS: Gene-trap mutation Cecr2(Gt45Bic) results in 74% exencephaly (equivalent of human anencephaly) on the BALB/c strain. Gene expression altered during cranial neural tube closure by the Cecr2 mutation was identified through microarray analysis of 11-14 somites stage Cecr2(Gt45Bic)embryos. RESULTS: Analysis of Affymetrix Mouse 430 2.0 chips detected 60 transcripts up-regulated and 54 transcripts down-regulated in the Cecr2(Gt45Bic) embryos (fold > 1.5, p < 0.05). The Cecr2 transcript was reduced only approximately 7- to 14-fold from normal levels, suggesting the Cecr2(Gt45Bic) is a hypomorphic mutation. We therefore generated a novel Cecr2 null allele (Cecr2 (tm1.1Hemc)). Resulting mutants displayed a stronger penetrance of exencephaly than Cecr2(Gt45Bic) in both BALB/c and FVB/N strains, in addition to midline facial clefts and forebrain encephalocele in the FVB/N strain. The Cecr2 transcript is reduced 260-fold in the Cecr2(tm1.1Hemc) line. Subsequent qRT-PCR using Cecr2 (tm1.1Hemc) mutant heads confirmed downregulation of transcription factors Alx1/Cart1, Dlx5, Eya1, and Six1. CONCLUSIONS: As both Alx1/Cart1 and Dlx5 mouse mutations result in exencephaly, we hypothesize that changes in expression of these mesenchymal/ectodermal transcription factors may contribute to NTDs associated with Cecr2.

The corpus callosum, the other great forebrain commissures, and the septum pellucidum: anatomy, development, and malformation.

There are three telencephalic commissures which are paleocortical (the anterior commissure), archicortical (the hippocampal commissure), and neocortical. In non-placental mammals, the neocortical commissural fibers cross the midline together with the anterior and possibly the hippocampal commissure, across the lamina reuniens (joining plate) in the upper part of the lamina terminalis. In placental mammals, a phylogenetically new feature emerged, which is the corpus callosum: it results from an interhemispheric fusion line with specialized groups of mildline glial cells channeling the commissural axons through the interhemispheric meninges toward the contralateral hemispheres. This concerns the frontal lobe mainly however: commissural fibers from the temporo-occipital neocortex still use the anterior commissure to cross, and the posterior occipito-parietal fibers use the hippocampal commissure, forming the splenium in the process. The anterior callosum and the splenium fuse secondarily to form the complete commissural plate. Given the complexity of the processes involved, commissural ageneses are many and usually associated with other diverse defects. They may be due to a failure of the white matter to develop or to the commissural neurons to form or to migrate, to a global failure of the midline crossing processes or to a selective failure of commissuration affecting specific commissural sites (anterior or hippocampal commissures, anterior callosum), or specific sets of commissural axons (paleocortical, hippocampal, neocortical commissural axons). Severe hemispheric dysplasia may prevent the axons from reaching the midline on one or both sides. Besides the intrinsically neural defects, midline meningeal factors may prevent the commissuration as well (interhemispheric cysts or lipoma). As a consequence, commissural agenesis is a malformative feature, not a malformation by itself. Good knowledge of the modern embryological data may allow for a good understanding of a specific pattern in a given individual patient, paving the way for better clinical correlation and genetic counseling.

Risk factors for non-syndromic holoprosencephaly in the National Birth Defects Prevention Study.

Holoprosencephaly (HPE) is a complex structural brain anomaly that results from incomplete cleavage of the forebrain. The prevalence of HPE at birth is low, and risk factors have been difficult to identify. Using data from a large multi-state population-based case-control study, we examined risk factors for non-syndromic HPE. Data from maternal telephone interviews were available for 74 infants with HPE and 5871 controls born between 1997 and 2004. Several characteristics and exposures were examined, including pregnancy history, medical history, maternal diet and use of nutritional supplements, medications, tobacco, alcohol, and illegal substances. We used chi(2)-tests and logistic regression (excluding women with pre-existing diabetes) to examine associations with HPE. Except for diet (year before pregnancy) and sexually transmitted infections (STIs) (throughout pregnancy), most exposures were examined for the time period from the month before to the third month of pregnancy. HPE was found to be associated with pre-existing diabetes (chi(2) = 6.0; P = 0.01), aspirin use [adjusted odds ratio (aOR) = 3.4; 95% confidence interval (CI) 1.6-6.9], lower education level (aOR = 2.5; 95%CI 1.1-5.6), and use of assisted reproductive technologies (ART) (crude OR = 4.2; 95%CI 1.3-13.7). Consistent maternal folic acid use appeared to be protective (aOR = 0.4; 95%CI 0.2-1.0), but the association was of borderline statistical significance. While some of these findings support previous observations, other potential risk factors identified warrant further study.

Replacement of related POU transcription factors leads to severe defects in mouse forebrain development.

Related transcription factors of the POU protein family show extensive overlap of expression in vivo and exhibit very similar biochemical properties in vitro. To study functional equivalence of class III POU proteins in vivo, we exchanged the Oct-6 gene by Brn-1 in the mouse. Brn-1 can fully replace Oct-6 in Schwann cells and rescue peripheral nervous system development in these mice. The same mice, however, exhibit severe defects in forebrain development arguing that Oct-6 and Brn-1 are not functionally equivalent in the central nervous system. The cause of the observed forebrain phenotype is complex, but anteriorly expanded Wnt1 expression contributes. Oct-6 normally represses Wnt1 expression in the early diencephalon and replacement by Brn-1 as a weaker inhibitor is no longer sufficient to maintain the necessary level of repression in the mouse mutant. The extent of functional equivalence between related transcription factors is thus strongly dependent on the analyzed tissue.

Disorders of prosencephalic development.

Abnormal ventral induction may result in disorders of formation, cleavage, and midline development of prosencephalic structures. Holoprosencephaly is a developmental field defect of impaired cleavage of prosencephalon. The most widely accepted classification of holoprosencephaly recognizes three major varieties: the alobar, semilobar and lobar types, according to the severity of the malformation. The brain malformations, characterized by the fusion of the cerebral hemisphere along the midline are commonly associated with facial anomalies. Corpus callosum agenesis and septo-optic dysplasia are disorders of prosencephalic midline development, and usually have less severe presentations but still, affected subjects may suffer from neurodevelopmental retardation, and/or endocrinologic and visual disorders. In this article we report an up-to-date of pathogenesis, prenatal sonographic findings, differential diagnosis and prognosis of the aforementioned anomalies.

Analysis of mouse models carrying the I26T and R160C substitutions in the transcriptional repressor HESX1 as models for septo-optic dysplasia and hypopituitarism.

A homozygous substitution of the highly conserved isoleucine at position 26 by threonine (I26T) in the transcriptional repressor HESX1 has been associated with anterior pituitary hypoplasia in a human patient, with no forebrain or eye defects. Two individuals carrying a homozygous substitution of the conserved arginine at position 160 by cysteine (R160C) manifest septo-optic dysplasia (SOD), a condition characterised by pituitary abnormalities associated with midline telencephalic structure defects and optic nerve hypoplasia. We have generated two knock-in mouse models containing either the I26T or R160C substitution in the genomic locus. Hesx1(I26T/I26T) embryos show pituitary defects comparable with Hesx1(-/-) mouse mutants, with frequent occurrence of ocular abnormalities, although the telencephalon develops normally. Hesx1(R160C/R160C) mutants display forebrain and pituitary defects that are identical to those observed in Hesx1(-/-) null mice. We also show that the expression pattern of HESX1 during early human development is very similar to that described in the mouse, suggesting that the function of HESX1 is conserved between the two species. Together, these results suggest that the I26T mutation yields a hypomorphic allele, whereas R160C produces a null allele and, consequently, a more severe phenotype in both mice and humans.

A crucial role for primary cilia in cortical morphogenesis.

Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70-80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain.

[Serious cerebral malformations (corpus callosum aplasia, prosencephalic cyst), internal carotid canal and facial malformations due to neural crest abnormalities, associated with choleosteatoma]. / Complesse malformazioni cerebrali (aplasia del corpo calloso, cisti prosencefalica), del canale carotico interno e facciali da alterazione delle creste neurali, associate a colesteatoma (operato).

The authors report an original case of malformative spectrum, which includes cerebral (corpus callosum aplasia, prosencephalic cyst) facial, otic and basi-cranial dysplasias associated with cholesteatoma. Cephalic neural crest cells migrate to different regions in the head and neck, where they contribute to the development of mainly the first and second branchial arches and of many structures as the anterior skull base, the face, the ear and the forebrain. Data suggest that the link between these rare malformations is abnormal neural crest development, perhaps due to defective Hh signal.

CNBP regulates forebrain formation at organogenesis stage in chick embryos.

We recently demonstrated that Cellular Nucleic acid Binding Protein (CNBP)(-/-) mouse embryos exhibit forebrain truncation due to a lack of proper morphogenetic movements of the anterior visceral endoderm (AVE) during pre-gastrulation stage (Chen, W., Liang, Y., Deng, W., Shimizu, K., Ashique, A.M., Li, E., Li, Y.P., 2003. The zinc-finger protein CNBP is required for forebrain formation in the mouse, Development 130, 1367-1379). However, CNBP expression pattern in the mouse forebrain suggests that CNBP may have more direct effects during forebrain development. Our data show that CNBP is expressed in tissues of early chick embryo that are the equivalent to the mouse embryo. Using a combination of RNAi-silencing and Retrovirus-misexpression approaches, we investigated the temporal function of CNBP in the specification/development of the chick forebrain during organogenesis. The silencing of CNBP expression resulted in forebrain truncation and the absence of BF-1, Six3 and Hesx1 expression, but not Otx2 in chick embryos. Misexpression of CNBP induced the expression of BF-1, Six3 and Hesx1 in the hindbrain, but not the expression of Otx2. These results offer novel insights into the function of CNBP during organogenesis as the regulator of forebrain formation and a number of rostral head transcription factors. Moreover, CNBP and Otx2 may play roles as regulators of forebrain formation in two parallel pathways. These new insights into CNBP functions underscore the essential role of CNBP in forebrain formation during chick embryo organogenesis.

Sinoftalmía: presentación de un caso / Presentation of a synophthalmia case

Se realiza la presentación de un caso de anencefalia asociado a cíclope que ocurrió en la misión realizada recientemente en el hermano pueblo de Haití, y en la que se obtuvo un feto muerto de 1 850 g con fusión de los 2 ojos y esbozo de nariz en forma de trompa, achatada. Se hace la discusión y revisión de la literatura al respecto

Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents the expression of both activator and repressor functions of Gli.

Sonic Hedgehog (Shh) signals are transduced into nuclear ratios of Gli transcriptional activator versus repressor. The initial part of this process is accomplished by Shh acting through Patched (Ptc) to regulate Smoothened (Smo) activity. The mechanisms by which Ptc regulates Smo, and Smo activity is transduced to processing of Gli proteins remain unclear. Recently, a forward genetic approach in mice identified a role for intraflagellar transport (IFT) genes in Shh signal transduction, downstream of Patched (Ptc) and Rab23. Here, we show that the retrograde motor for IFT is required in the mouse for the phenotypic expression of both Gli activator and repressor function and for effective proteolytic processing of Gli3. Furthermore, we show that the localization of Smo to primary cilia is disrupted in mutants. These data indicate that primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli3 protein.

Idiopathic hypogonadotrophic hypogonadism associated with arachnoid cyst of the middle fossa and forebrain anomalies: presentation of an unusual case.

We report a 22-yr-old male patient with hypogonadotrophic hypogonadism (HH) associated with a giant middle fossa arachnoid cyst (AC) diagnosed by magnetic resonance imaging (MRI). He presented with pubertal and growth delay. He also had learning disabilities and anosmia. Laboratory investigation revealed pre-pubertal levels of testosterone and normal results of the combined test of anterior pituitary function, except for in GnRH acute and prolonged test. Cranial MRI showed an AC in left middle fossa with expansion to suprasellar cisterna and several abnormalities like left temporal lobe hypoplasia, left optic tract and bilateral olfactory bulb hypoplasia and left hypothalamic hypoplasia.