Transcriptional control of cone photoreceptor diversity by a thyroid hormone receptor.

Cone photoreceptor diversity allows detection of wavelength information in light, the first step in color (chromatic) vision. In most mammals, cones express opsin photopigments for sensitivity to medium/long (M, "green") or short (S, "blue") wavelengths and are differentially arrayed over the retina. Cones appear early in retinal neurogenesis but little is understood of the subsequent control of diversity of these postmitotic neurons, because cone populations are sparse and, apart from opsins, poorly defined. It is also a challenge to distinguish potentially subtle differences between cell subtypes within a lineage. Therefore, we derived a Cre driver to isolate individual M and S opsin-enriched cones, which are distributed in counter-gradients over the mouse retina. Fine resolution transcriptome analyses identified expression gradients for groups of genes. The postnatal emergence of gradients indicated divergent differentiation of cone precursors during maturation. Using genetic tagging, we demonstrated a role for thyroid hormone receptor ß2 (TRß2) in control of gradient genes, many of which are enriched for TRß2 binding sites and TRß2-regulated open chromatin. Deletion of TRß2 resulted in poorly distinguished cones regardless of retinal location. We suggest that TRß2 controls a bipotential transcriptional state to promote cone diversity and the chromatic potential of the species.

Mutually repressive interaction between Brn1/2 and Rorb contributes to the establishment of neocortical layer 2/3 and layer 4.

Although several molecules have been shown to play important roles in subtype specification of neocortical neurons, the entire mechanism involved in the specification, in particular, of upper cortical plate (UCP) neurons still remains unclear. The UCP, which is responsible for intracortical connections in the neocortex, comprises histologically, functionally, and molecularly different layer 2/3 (L2/3) and L4. Here, we report the essential interactions between two types of transcription factors, Rorb (RAR-related orphan receptor beta) and Brn1/2 (Brain-1/Brain-2), for UCP specification. We found that Brn2 expression was detected in all upper layers in the immature UCP, but was subsequently restricted to L2/3, accompanied by up-regulation of Rorb in L4, suggesting demarcation of L2/3 and L4 during cortical maturation. Rorb indeed inhibited Brn2 expression and the expression of other L2/3 characteristics, revealed by ectopic expression and knockdown studies. Moreover, this inhibition occurred through direct binding of Rorb to the Brn2 locus. Conversely, Brn1/2 also inhibited Rorb expression and the expression of several L4 characteristics. Together, these results suggest that a mutually repressive mechanism exists between Brn1/2 and Rorb expression and that the established expression of Brn1/2 and Rorb further specifies those neurons into L2/3 and L4, respectively, during UCP maturation.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain.

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

Neuronal Heterotopias Affect the Activities of Distant Brain Areas and Lead to Behavioral Deficits.

Neuronal heterotopia refers to brain malformations resulting from deficits of neuronal migration. Individuals with heterotopias show a high incidence of neurological deficits, such as epilepsy. More recently, it has come to be recognized that focal heterotopias may also show a range of psychiatric problems, including cognitive and behavioral impairments. However, because focal heterotopias are not always located in the brain areas responsible for the symptoms, the causal relationship between the symptoms and heterotopias remains elusive. In this study, we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited spatial working memory deficit and low competitive dominance behavior, which have been shown to be closely associated with the activity of the medial prefrontal cortex (mPFC) in rodents. Analysis of the mPFC activity revealed that the immediate-early gene expression was decreased and the local field potentials of the mPFC were altered in the mice with heterotopias compared with the control mice. Moreover, activation of these ectopic and overlying sister neurons using the DREADD (designer receptor exclusively activated by designer drug) system improved the working memory deficits. These findings suggest that cortical regions containing focal heterotopias can affect distant brain regions and give rise to behavioral abnormalities. Significance statement: Recent studies reported that patients with heterotopias have a variety of clinical symptoms, such as cognitive disturbance, psychiatric symptoms, and autistic behavior. However, the causal relationship between the symptoms and heterotopias remains elusive. Here we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited behavioral deficits that have been shown to be associated with the mPFC activity in rodents. The existence of heterotopias indeed altered the neural activities of the mPFC, and direct manipulation of the neural activity of the ectopic neurons and their sister neurons in the overlying cortex improved the behavioral deficit. Thus, our results indicate that focal heterotopias could affect the activities of distant brain areas and cause behavioral abnormalities.

Caudal regression and tracheoesophageal malformation induced by adriamycin: a novel chick model of VATER association.

VATER association represents a cluster of Vertebral, Anal, Tracheo-Esophageal, Radial and Renal malformations, and caudal regression syndrome is an entity consisting of a spectrum of congenital anomalies of lower spine and hips associated with genitourinary and lower limb defects. The concurrence of various malformations may be explained by a common defect in blastogenesis, but direct evidence is yet to be accumulated. Here, by the use of autofluorescence and the teratogenic effect of adriamycin, we demonstrated that adriamycin administered to eggs of White Leghorns distributes to the caudal portion of the embryo and foregut epithelium and induces caudal regression and tracheal and pulmonary agenesis. The induction of caudal regression syndrome-like anomaly was developmental stage and dose dependent. Embryos with caudal regression demonstrated tracheoesophageal anomalies, one of the defects included in VATER association. The stages at which anomalies were produced corresponded to that of human embryos between days 22 and 26 (Carnegie stages 10-11). In view of the antitumor activity of adriamycin by intercalating to double-stranded DNA of undifferentiated cells undergoing rapid cell division, it is possible that adriamycin had preferentially attacked cells in the caudal end where pronounced proliferation takes place during this narrow period of greatest susceptibility.

Neocentromere marker chromosome of distal 3q mimicking dup(3q) syndrome phenotype.

Supernumerary marker chromosomes (SMCs) lacking alpha-satellite sequences and possessing a newly derived functional centromere are referred to as neocentromere marker chromosomes (NMCs). Although the delineation of the chromosome content of these NMCs would be helpful for genetic counseling, such fine mapping has been difficult because of the limited sizes of the involved segments. We report on a female patient with mosaic NMC involving 3q26.3-3qter, the content of which was determined using an array CGH analysis. Our results support the validity of an array CGH-based approach to investigating the origins of SMCs. Further FISH analyses revealed that the NMC is characterized by an asymmetric inv-dup structure separated by a single-copy region. The present case had many manifestations of dup(3q) syndrome, the critical interval of which is considered to be 3q26.3-q27. Common features included mental and growth retardation, hirsutism, synophrys, a broad nasal root, anteverted nares, downturned corners of the mouth, and malformed ears. The observation gives further credence to the concept that the critical region responsible for the dup(3q) phenotype to 3q26.3-q27.

Retinoid-Related Orphan Receptor ß and Transcriptional Control of Neuronal Differentiation.

The ability to generate neuronal diversity is central to the function of the nervous system. Here we discuss the key neurodevelopmental roles of retinoid-related orphan receptor ß (RORß) encoded by the Rorb (Nr1f2) gene. Recent studies have reported loss of function of the human RORB gene in cases of familial epilepsy and intellectual disability. Principal sites of expression of the Rorb gene in model species include sensory organs, the spinal cord, and brain regions that process sensory and circadian information. Genetic analyses in mice have indicated functions in circadian behavior, vision, and, at the cellular level, the differentiation of specific neuronal cell types. Studies in the retina and sensory areas of the cerebral cortex suggest that this orphan nuclear receptor acts at decisive steps in transcriptional hierarchies that determine neuronal diversity.

Association of impaired neuronal migration with cognitive deficits in extremely preterm infants.

Many extremely preterm infants (born before 28 gestational weeks [GWs]) develop cognitive impairment in later life, although the underlying pathogenesis is not yet completely understood. Our examinations of the developing human neocortex confirmed that neuronal migration continues beyond 23 GWs, the gestational week at which extremely preterm infants have live births. We observed larger numbers of ectopic neurons in the white matter of the neocortex in human extremely preterm infants with brain injury and hypothesized that altered neuronal migration may be associated with cognitive impairment in later life. To confirm whether preterm brain injury affects neuronal migration, we produced brain damage in mouse embryos by occluding the maternal uterine arteries. The mice showed delayed neuronal migration, ectopic neurons in the white matter, altered neuronal alignment, and abnormal corticocortical axonal wiring. Similar to human extremely preterm infants with brain injury, the surviving mice exhibited cognitive deficits. Activation of the affected medial prefrontal cortices of the surviving mice improved working memory deficits, indicating that decreased neuronal activity caused the cognitive deficits. These findings suggest that altered neuronal migration altered by brain injury might contribute to the subsequent development of cognitive impairment in extremely preterm infants.

Screening for CHARGE syndrome mutations in the CHD7 gene using denaturing high-performance liquid chromatography.

Mutations in the CHD7 (chromodomain helicase DNA binding protein 7) gene cause CHARGE syndrome. At present, however, genetic testing of the CHD7 gene is not commonly applied in clinical settings because the currently available assays are technically and financially demanding, mainly because of the size of the gene. In the present study, we optimized the highly sensitive and specific mutation scanning method automated denaturing high-performance liquid chromatography (DHPLC) to analyze the entire coding region of CHD7. The coding region was amplified by 39 primer pairs, all of which have the same cycling conditions, aliquoted on a 96-well format polymerase chain reaction (PCR) plate. In this manner, all of the exons were amplified simultaneously using a single block in a thermal cycler. We then wrote a computer script to analyze each segment of the CHD7 gene by DHPLC in a serial manner using conditions that were optimized for each amplicon. The implementation of this screening method for CHD7 will help medical geneticists confirm their clinical impressions and provide accurate genetic counseling to the patients with CHARGE syndrome and their families.

Identity of neocortical layer 4 neurons is specified through correct positioning into the cortex.

Many cell-intrinsic mechanisms have been shown to regulate neuronal subtype specification in the mammalian neocortex. However, how much cell environment is crucial for subtype determination still remained unclear. Here, we show that knockdown of Protocadherin20 (Pcdh20), which is expressed in post-migratory neurons of layer 4 (L4) lineage, caused the cells to localize in L2/3. The ectopically positioned "future L4 neurons" lost their L4 characteristics but acquired L2/3 characteristics. Knockdown of a cytoskeletal protein in the future L4 neurons, which caused random disruption of positioning, also showed that those accidentally located in L4 acquired the L4 characteristics. Moreover, restoration of positioning of the Pcdh20-knockdown neurons into L4 rescued the specification failure. We further suggest that the thalamocortical axons provide a positional cue to specify L4 identity. These results suggest that the L4 identity is not completely determined at the time of birth but ensured by the surrounding environment after appropriate positioning.

Comprehensive screening of the thiopurine methyltransferase polymorphisms by denaturing high-performance liquid chromatography.

The drug-metabolizing enzyme thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathiopurine, which are used as immunosuppressants and in the treatment of acute lymphoblastic leukemia and rheumatoid arthritis. TPMT enzymatic activity is a polymorphic trait, and poor metabolizers may develop life-threatening bone marrow failure. To avoid such adverse effects, the TPMT enzymatic activity in patients' red blood cells (RBCs) is routinely measured prior to thiopurine administration in a limited number of oncology clinics. In the present study, we took advantage of a highly sensitive and specific automated denaturing high-performance liquid chromatography (dHPLC) technique that not only detects known polymorphic alleles, but also identifies previously uncharacterized sequence variants. We developed a dHPLC-based protocol to analyze the entire coding region and validated the protocol to detect all 16 previously described variant alleles. We further analyzed the entire coding region of the TPMT gene in 288 control samples collected worldwide and identified two novel amino acid substitutions Arg163Cys (487C>T) and Arg226Gln (677G>A) within exons 7 and 10, respectively. The clinical application of this comprehensive screening system for examining the entire TPMT gene would help to identify patients at risk for bone marrow failure prior to 6-mercaptopurine therapy.

Large fontanelles are a shared feature of haploinsufficiency of RUNX2 and its co-activator CBFB.

CBFB at 16q22 heterodimerizes with either RUNX2 (also known as CBFA1) or RUNX1 (CBFA2) to activate the transcription of downstream molecules. RUNX2 regulates osteoblast differentiation and chondrocyte maturation and its haploinsufficiency leads to cleidocranial dysplasia, characterized large fontanelles, hypoplasia or aplasia of the clavicles, hypoplasia of the distal phalanges, and a wide pubic symphysis. Complete loss of Runx1 or Cbfb in mice is lethal because of the absence of fetal liver hematopoiesis. Fetal rescue in Cbfb(-/-) mice by providing the Cbfb functions in the hematopoietic progenitors leads to wide fontanelle and delayed chondrocyte maturation, presumably resulting from the incomplete function of the transcriptional pathway mediated by the Cbfb-Runx2 heterodimer. The present report describes a patient with a small deletion of chromosome 16q22.1 encompassing CBFB. Skeletal abnormalities included a widely open fontanelle, multiple wormian bones along the sagittal suture, hypoplasia of the distal phalanges, and mildly shortened clavicles. G-banding analysis revealed a shortening of the 16q22.1 band. A fluorescence in situ hybridization analysis, using the BAC probe spanning the CBFB locus at 16q22.1, revealed that the CBFB probe hybridized to only one of the two homologous chromosome 16 regions. Array-comparative genomic hybridization analysis revealed that the deletion spans 1.2 megabases. In reviewing eight previously reported cases of 16q interstitial deletions involving band q22, large cranial sutures were noted in all but one case. Considering the phenotypic similarity of the 16q22 deletion case and Cbfb(-/-) mice rescued for hematopoiesis and the consistency of the phenotype among 16q22 deletion cases, we suggest that the common phenotypic feature of the 16q22 deletion, large fontanelles, can be attributed to a haploinsufficiency of CBFB.

Upper airway obstruction in neonates and infants with CHARGE syndrome.

Upper airway obstruction can be life-threatening in neonates and infants with CHARGE syndrome, many of whom undergo intratracheal intubation early in life. Although some of these patients are successfully extubated, others require tracheotomy. Deciding whether to complete tracheotomy is challenging since there are no clear criteria upon which to base this decision. We assessed 10 infants with CHARGE syndrome, 5 of whom required tracheotomy. Fiberoptic laryngoscopy showed that all of these patients shared certain features: anteroposterior flattening of the larynx; short vocal cords; anteriorly positioned, tall and hypertrophic arytenoids obscuring the glottis; uncoordinated movement of the vocal cords, epiglottis and arytenoids; salivary pooling. In addition, we observed only in those requiring tracheotomy an obstructive supraglottis that prevented visualization of the vocal cords throughout respiration. Salivary retention was much more severe in this group. These findings might be helpful for predicting the need for an early tracheotomy in situations where the vocal cords are not visible throughout the entire respiratory cycle. Given the high prevalence of malformed larynx and abnormal cranial nerve function, which are not alleviated by supraglottoplasty, we suggest that a thorough investigation of upper airway obstructive entities other than laryngomalacia be performed before embarking on supraglottoplasty in patients with CHARGE syndrome.

Embryonic expression profile of chicken CHD7, the ortholog of the causative gene for CHARGE syndrome.

BACKGROUND: CHARGE syndrome represents a constellation of malformations: C, coloboma of the iris or retina; H, heart defects; A, atresia of the choanae; R, retardation of growth and/or development; G, genital anomalies; and E, ear abnormalities. Recently, the Chromodomain helicase DNA-binding protein-7 (CHD7) at chromosome 8q12.1 was identified as a causative gene for CHARGE syndrome. Because CHD7 was identified as a causative gene using a positional cloning approach, the role of CHD7 in early embryogenesis needs to be further investigated. METHODS: Fertilized chick eggs were incubated to Hamburger and Hamilton stages 4-20 and were studied using whole mount in situ hybridization. Chicken EST clones corresponding to the chicken CHD7 (cChd7) sequence were identified using the chicken EST sequence database. From the EST clones, a digoxigenin-labeled RNA probe was synthesized and hybridized in situ to the embryonic specimens. RESULTS: The expression of cChd7 was pan-neuronal at stages 8-20. It was expressed throughout the rostral neural ectoderm and along the rostrocaudal axis but was absent from the more lateral, non-neuronal ectoderm. Adjacent to the neural tube, cChd7 transcripts were detected at the optic and otic placodes. At stage 20, cChd7 expression was observed in the branchial arches and olfactory placodes in addition to brain and optic and otic placodes. CONCLUSIONS: cChd7 was expressed in the neural epithelium, the otic placodes, the optic placodes, the branchial arches, and the olfactory placodes, which are the primordial tissues that give rise to organs affected in human CHARGE syndrome patients.

An Alu retrotransposition-mediated deletion of CHD7 in a patient with CHARGE syndrome.

CHD7 mutations account for about 60-65% among more than 200 CHARGE syndrome cases. When rare whole gene deletion cases associated with chromosomal abnormalities are excluded, all mutations of CHD7 reported to date have been point mutations and small deletions and insertions, rather than exonic deletions. To test whether exonic deletions represent a common pathogenic mechanism, we assessed exon copy number by using a recently developed method, the multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons were amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography, and quantitated by fluorescence detection using a post-column intercalation dye under the premise that the relative peak intensities for each target directly reflect exon copy number. By using MP/LC, we identified one CHARGE syndrome patient who had a de novo deletion encompassing exons 8-12 among 13 classic CHARGE patients in whom screening by denaturing high-performance liquid chromatography (DHPLC) failed to identify point mutations and small insertions/deletions in CHD7. This is the first CHARGE patient who was documented to have exonic deletion of CHD7. The deletion closely recapitulated the Alu-mediated inactivation of the human CMP-N-acetylneuraminic acid hydroxylase gene (CMP-Neu5Ac hydroxylase), which is regarded as a novel molecular mechanism in the evolution from non-human primates to humans. As demonstrated in this study, MP/LC is a promising method for characterizing exonic deletions, which are largely left unexamined in most routine mutation analysis.

Identification of a prosencephalic-specific enhancer of SALL1: comparative genomic approach using the chick embryo.

Comparative genomics is a promising approach for identifying regulatory elements governing the unique spatio-temporal expression patterns of morphogenetic genes. Conserved noncoding genomic sequences are candidate regulatory elements. Here we performed a survey for conserved noncoding elements (CNE) nested within the SALL1 gene; mutations in this gene result in the Townes-Brocks syndrome. A comparison of the genomic sequence between humans and chicken revealed five CNE. Genomic fragments corresponding to each CNE were inserted into reporter cassettes consisting of eGFP cDNA and a minimal promoter. These constructs were electroporated into chick embryos during gastrula, neurula, and pharyngula stages. Among the five CNE that were examined, one 443 bp CNE exhibited tissue-specific enhancer activity. At the neurula stage, the eGFP signal was visualized in the prosencephalon. At the pharyngula stage, the eGFP signal was confined within the anterior neural ridge, which represents one of the morphogenetic centers regulating the patterning of the anterior neural plate. This report identifies, for the first time, an enhancer element of SALL1.

Phenotypic spectrum of CHARGE syndrome with CHD7 mutations.

CHD7 gene mutations were identified in 17 (71%) of 24 children clinically diagnosed to have CHARGE syndrome (C, coloboma of the iris or retina; H, heart defects; A, atresia of the choanae; R, retardation of growth and/or development; G, genital anomalies; and E, ear abnormalities). Colobomata, hearing loss, laryngomalacia, and vestibulo-cochlear defect were prevalent. Molecular testing for CHD7 enables an accurate diagnosis and provides health anticipatory guidance and genetic counseling to families with CHARGE syndrome.