The Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila sensory organ precursors cells.

Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells, the identity of which relies on activation of Notch at cytokinesis. Although Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We have previously reported that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that their concomitant loss results in a penetrant Notch gain-of-function phenotype, indicating that they control parallel pathways. Although unequal partitioning of cell fate determinants and cell polarity were unaffected, we observed increased amounts of signalling-competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface, at the expense of the basal pool of Notch. We propose that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place.

Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo.

In Drosophila, the sensory organ precursor (SOP or pI cell) divides asymmetrically to give birth to daughter cells, the fates of which are governed by the differential activation of the Notch pathway. Proteolytic activation of Notch induced by ligand is based on the correct polarized sorting and localization of the Notch ligand Delta, the Notch receptor and its trafficking partner Sanpodo (Spdo). Here, we have identified Stratum (Strat), a presumptive guanine nucleotide exchange factor for Rab GTPases, as a regulator of Notch activation. Loss of Strat causes cell fate transformations associated with an accumulation of Notch, Delta and Spdo in the trans-Golgi network (TGN), and an apical accumulation of Spdo. The strat mutant phenotype is rescued by the catalytically active as well as the wild-type form of Rab8, suggesting a chaperone function for Strat rather than that of exchange factor. Strat is required to localize Rab8 at the TGN, and rab8 phenocopies strat We propose that Strat and Rab8 act at the exit of the Golgi apparatus to regulate the sorting and the polarized distribution of Notch, Delta and Spdo.

Novel genes upregulated when NOTCH signalling is disrupted during hypothalamic development.

BACKGROUND: The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that the Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, the role of Notch during hypothalamus formation along with its downstream effectors remains poorly defined. RESULTS: Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. CONCLUSIONS: These data give essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network.

NODAL and SHH dose-dependent double inhibition promotes an HPE-like phenotype in chick embryos.

Holoprosencephaly (HPE) is a common congenital defect that results from failed or incomplete forebrain cleavage. HPE is characterized by a wide clinical spectrum, with inter- and intrafamilial variability. This heterogeneity is not well understood and it has been suggested that HPE involves a combination of multiple gene mutations. In this model, several mutated alleles or modifying factors are presumed to act in synergy to cause and determine the severity of HPE. This could explain the various clinical phenotypes. Screening for HPE-associated genes in humans suggests the involvement of NODAL or SHH signaling, or both. To test this multigenic hypothesis, we investigated the effects of chemical inhibition of these two main HPE signaling pathways in a chick embryo model. SB-505124, a selective inhibitor of transforming growth factor-B type I receptors was used to inhibit the NODAL pathway. Cyclopamine was used to inhibit the SHH pathway. We report that both inhibitors caused HPE-like defects that were dependent on the drug concentration and on the developmental stage at the time of treatment. We also investigated double inhibition of NODAL and SHH pathways from the onset of gastrulation by using subthreshold inhibitor concentrations. The inhibitors of the NODAL and SHH pathways, even at low concentration, acted synergistically to promote an HPE-like phenotype. These findings support the view that genetic heterogeneity is important in the etiology of HPE and may contribute to the phenotypic variability.

New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases.

BACKGROUND: Holoprosencephaly (HPE) is the most common forebrain defect in humans. It results from incomplete midline cleavage of the prosencephalon. METHODS: A large European series of 645 HPE probands (and 699 relatives), consisting of 51% fetuses and 49% liveborn children, is reported. RESULTS: Mutations in the four main genes involved in HPE (SHH, ZIC2, SIX3, TGIF) were identified in 25% of cases. The SHH, SIX3, and TGIF mutations were inherited in more than 70% of these cases, whereas 70% of the mutations in ZIC2 occurred de novo. Moreover, rearrangements were detected in 22% of the 260 patients screened by array comparative genomic hybridisation. 15 probands had two mutations providing additional support for the 'multiple-hit process' in HPE. There was a positive correlation between the severity of the brain malformation and facial features for SHH, SIX3, and TGIF, but no such correlation was found for ZIC2 mutations. The most severe HPE types were associated with SIX3 and ZIC2 mutations, whereas microforms were associated with SHH mutations. The study focused on the associated brain malformations, including neuronal migration defects, which predominated in individuals with ZIC2 mutations, and neural tube defects, which were frequently associated with ZIC2 (rachischisis) and TGIF mutations. Extracraniofacial features were observed in 27% of the individuals in this series (up to 40% of those with ZIC2 mutations) and a significant correlation was found between renal/urinary defects and mutations of SHH and ZIC2. CONCLUSIONS: An algorithm is proposed based on these new phenotype-genotype correlations, to facilitate molecular analysis and genetic counselling for HPE.

NOTCH, a new signaling pathway implicated in holoprosencephaly.

Genetics of Holoprosencephaly (HPE), a congenital malformation of the developing human forebrain, is due to multiple genetic defects. Most genes that have been implicated in HPE belong to the sonic hedgehog signaling pathway. Here we describe a new candidate gene isolated from array comparative genomic hybridization redundant 6qter deletions, DELTA Like 1 (DLL1), which is a ligand of NOTCH. We show that DLL1 is co-expressed in the developing chick forebrain with Fgf8. By treating chick embryos with a pharmacological inhibitor, we demonstrate that DLL1 interacts with FGF signaling pathway. Moreover, a mutation analysis of DLL1 in HPE patients revealed a three-nucleotide deletion. These various findings implicate DLL1 in early patterning of the forebrain and identify NOTCH as a new signaling pathway involved in HPE.

Holoprosencephaly: An update on cytogenetic abnormalities.

Holoprosencephaly (HPE), the most common developmental defect of the forebrain and midface, is caused by a failure of midline cleavage early in gestation. Isolated HPE, which is highly genetically heterogeneous, can be due to major chromosomal abnormalities. Initially, karyotype approach led to the identification of several recurrent chromosomal anomalies predicting different HPE loci. Subsequently, several genes were isolated from these critical HPE regions, but point mutations and deletions in these genes were found only in 25% of the genetic cases. In order to identify other HPE genes, a more accurate investigation of the genome in HPE patients was necessary. To date, high-resolution cytogenetic techniques such as subtelomeric multiplex ligation-dependent probe amplification (MLPA) and microarray-based comparative genomic hybridization (array CGH) have enhanced chromosomal aberration analysis. In this article, we have updated the cytogenetic anomalies associated with HPE in a map listing all the subtelomeric and interstitial deletions that have been characterized either by karyotype, MLPA, or array CGH. The accumulation of recurrent genomic imbalances will lead to the further delineation of minimal critical HPE loci, which is the first step to the identification of new HPE genes.

Array-CGH analysis indicates a high prevalence of genomic rearrangements in holoprosencephaly: an updated map of candidate loci.

Holoprosencephaly (HPE) is the most frequent malformation of the brain. To date, 12 different HPE loci and 8 HPE genes have been identified from recurrent chromosomal rearrangements or from the sequencing of genes from Nodal and SHH pathways. Our cohort of HPE patients presents a high genetic heterogeneity. Point mutations were found in SHH, ZIC2, SIX3, and TGIF genes in about 20% of cases (with 10% in SHH). Deletions in these same genes were found in 7.5% of the patients and 4.4% presented with other subtelomeric gain or losses. Consequently, the molecular basis of HPE remains unknown in 70% of our cohorts. To detect new HPE candidate genes, we used array-CGH to refine the previous karyotype based HPE loci map. We analyzed 111 HPE patients with high-performance Agilent oligonucleotidic arrays and found that 28 presented anomalies involving known or new potential HPE loci located on different chromosomes but with poor redundancy. This study showed an impressive rate of 19 patients among 111 with de novo chromosomal anomalies giving evidence that microrearrangements could be a major molecular mechanism in HPE. Additionally, this study opens new insights on HPE candidate genes identification giving an updated HPE candidate loci map.

MLPA screening reveals novel subtelomeric rearrangements in holoprosencephaly.

Holoprosencephaly (HPE) is the most common developmental brain anomaly in human, associated with a wide spectrum of presentations. The etiology is heterogeneous, due to environmental and genetic factors. Out of 12 cytogenetic candidate loci previously reported, eight were subtelomeric, including the loci in which two of the four major HPE genes were identified (SHH and TGIF). Recently, we reported that these two genes could be mutated or microdeleted. Therefore, we hypothesized that subtelomeres screening in HPE patients could refine the known subtelomeric candidate loci and identify novel ones. In this study, 181 samples, 72 fetuses and 109 live-born infants, with HPE and a normal karyotype, and 10 patients deleted for SHH or TGIF (3.5 Mb from telomeres) were screened for subtelomeric rearrangements using the multiplex ligation probe-dependent amplification (MLPA) method with two kits. Quantitative PCR was performed when discrepancies were observed between these two kits. We found that known SHH and TGIF microdeletions on 7q and 18p, encompassed their subtelomeric region (3.5 Mb) and were often associated with cryptic gains. Out of the 181 samples, we detected rearrangements in known candidate HPE loci (1q, 20p, and 21q) as well as in other novel subtelomeric locations (1p, 5q, 8p, 17q, 18q, 22q, and Xq) and in the subcentromeric 15q. We also found associations between cryptic subtelomeric gain and loss that may be inherited from a parental balanced translocation, which is helpful for genetic counseling. These findings reinforce the multihit origin for HPE and contribute to the explanation of the wide phenotypic spectrum described in this developmental disorder.

Phenotypic variability of a 4q34-->qter inherited deletion: MRKH syndrome in the daughter, cardiac defect and Fallopian tube cancer in the mother.

Terminal deletions of the long arm of chromosome 4 are associated with a recognizable phenotype consisting of dysmorphic facial features, cleft palate, upper and lower limb malformations, cardiac defects and growth and mental retardation. Here we report on two female patients, a mother and her daughter, carrying the same 4q34-->qter deletion but presenting with a different phenotype. The mother's presentation is consistent with previous findings in patients with terminal deletions of the long arm of chromosome 4. However, she presented at the age of 54years with bilateral serous carcinoma of the Fallopian tubes, a rare gynaecologic cancer that might be attributed to the haploinsufficiency of the tumor suppressor gene FAT. The daughter presented isolated congenital aplasia of the uterus and vagina, the prime feature of the MRKH syndrome. This has not been described before in association with a 46,XX,del(4)(q34qter).

Molecular evaluation of foetuses with holoprosencephaly shows high incidence of microdeletions in the HPE genes.

Holoprosencephaly (HPE), the most common structural malformation of the forebrain in humans, can be detected early during pregnancy using prenatal ultrasonography . Among foetuses with a normal karyotype, 14% have mutations in the four main HPE genes (SHH, ZIC2, SIX3 and TGIF). Genomic rearrangements have now been implicated in many genetic diseases, so we hypothesized that microdeletions in the major HPE genes may also be common in HPE foetuses with severe phenotype or other associated malformations. We screened the DNA obtained from 94 HPE foetuses with a normal karyotype for the presence of microdeletions involving the four major HPE genes (SHH, ZIC2, SIX3 and TGIF). Thirteen of the foetuses had a point mutation in one of the 4 genes and 81 had no known mutations. Quantitative multiplex PCR of short fluorescent fragments (QMPSF) analysis was used for rapid determination of HPE genes copy numbers and the identified microdeletions were confirmed by real time quantitative PCR, or fluorescent in situ hybridization (FISH) (if a cell line was available). Microdeletions were detected in 8 of 94 foetuses (8.5%) (2 in SHH, 2 in SIX3, 3 in ZIC2 and 1 in TGIF genes), and only among the 81 foetuses with a normal karyotype and no point mutations. These data suggest that microdeletions in the four main HPE genes are a common cause of prenatal HPE, as well as point mutations, and increase the total diagnosis rate close to approximately 22.3% of foetuses with normal karyotype. Detection can be achieved by the QMPSF testing method that proved to be efficient for testing several genes in a single assay.

Transcriptome variations in human CaCo-2 cells: a model for enterocyte differentiation and its link to iron absorption.

Complete clinical expression of the HFE1 hemochromatosis is very likely modulated by genes linked to duodenal iron absorption, whose level is conditioned by unknown processes taking place during enterocyte differentiation. We carried out a transcriptomic study on CaCo-2 cells used as a model of enterocyte differentiation in vitro. Of the 720 genes on the microarrays, 80, 50, and 56 were significantly down-regulated up-regulated, and invariant during differentiation. With regard to iron metabolism, we showed that HEPH, SLC11A2, SLC11A3, and TF are significantly up-regulated, while ATP7B and SLC39A1 (and SFT) are down-regulated and ACO1, dCYTb, FECH, and FTH1 show constant expression. Ontological annotations highlight the decrease in the expression of cell cycle and DNA metabolism associated genes as well as transcription, protein metabolism, signal transduction, and nucleocytoplasmic transport associated genes, whereas there are increases in the expression of genes linked to cell adhesion, lipid and xenobiotic metabolism, iron transport and homeostasis, and immune response.