Search

Chick/quail transplantation experiments were performed to analyse possible factors involved in the regionalisation of the midbrain-hindbrain domain. The caudal prosomeres, expressing Otx2, were transplanted at stage HH10 into rostrocaudal levels of the midbrain-hindbrain domain, either straddling the intra-metencephalic constriction (type 1 grafts), or at rostral and medial levels of pro-rhombomere A1 (type 2 and 3 grafts, respectively); thus, in all situations, one border of the graft was in contact with the host Gbx2- and Fgf8-expressing domains. The area containing the graft, recognised by QCPN immunohistochemistry, was first analysed 48 hours after transplantation for Otx2, Gbx2, En2 and Fgf8. Although in all three situations, a large part of the graft maintained Otx2 expression, another part became Otx2 negative and was induced to express Gbx2 and Fgf8. These inductive events occurred exclusively at the interface between the Otx2-positive transplanted domain and the ipsilateral host Gbx2-positive rhombomere 1, creating a new Otx2-Gbx2 boundary within the grafted territory. In type 1 and 2 grafts, the induced Fgf8 domain is in continuity with the host Fgf8 isthmic domain, whereas for type 3 grafts, these two domains are separate. High levels of En2 expression were also induced in the area expressing Gbx2 and Fgf8, and Wnt1 and Pax2 expressions, analysed in type 3 grafts, were induced at the intragraft Otx2-Gbx2 new boundary. Moreover, at later embryonic stages, the graft developed meso-isthmo-cerebellar structures. Thus, gene expressions induced in the grafted prosencephalon not only mimicked the pattern observed in the normal midbrain-hindbrain domain, but is followed by midbrain-hindbrain cytodifferentiation, indicating that not only Fgf8 but also confrontation of Otx2 and Gbx2 may play an essential role during midbrian-hindbrain regionalisation.

Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, show a limited amino acid sequence divergence. Their embryonic expression patterns overlap in spatial and temporal profiles with two major exceptions: until 8 days post coitum (d.p.c.) only Otx2 is expressed in gastrulating embryos, and from 11 d.p.c. onwards only Otx1 is transcribed within the dorsal telencephalon. Otx1 null mice exhibit spontaneous epileptic seizures and multiple abnormalities affecting primarily the dorsal telencephalic cortex and components of the acoustic and visual sense organs. Otx2 null mice show heavy gastrulation abnormalities and lack the rostral neuroectoderm corresponding to the forebrain, midbrain and rostral hindbrain. In order to define whether these contrasting phenotypes reflect differences in expression pattern or coding sequence of Otx1 and Otx2 genes, we replaced Otx1 with a human Otx2 (hOtx2) full- coding cDNA. Interestingly, homozygous mutant mice (hOtx21/hOtx21) fully rescued epilepsy and corticogenesis abnormalities and showed a significant improvement of mesencephalon, cerebellum, eye and lachrymal gland defects. In contrast, the lateral semicircular canal of the inner ear was never recovered, strongly supporting an Otx1-specific requirement for the specification of this structure. These data indicate an extended functional homology between OTX1 and OTX2 proteins and provide evidence that, with the exception of the inner ear, in Otx1 and Otx2 null mice contrasting phenotypes stem from differences in expression patterns rather than in amino acid sequences.

We have isolated a novel Xenopushomeobox gene, Xrxl, belonging to the paired-like class of homeobox genes. Xrxlis expressed in the anterior neural plate, and subsequently in the neural structures of the developing eye (neural retina and pigmented epithelium), and in other forebrain structures deriving from the anterior neural plate: in the pineal gland, throughout its development, in the diencephalon floor and in the hypophysis. its rostral limit of expression corresponds to the chiasmatic ridge, which some authors consider as the anteriormost limit of the neural tube: thus, Xrxlmay represent one of the most anteriorly expressed homeobox genes reported to date. Moreover, its expression in organs implicated in the establishment of circadian rhythms, may suggest for Xrxla role in the genetic control of this function. Finally, analysis of Xrxlexpression in embryos subjected to various treatments, or microinjected with different dorsalizing agents (noggin, Xwnt-8), suggests that vertical inductive signals leading to head morphogenesis are required to activate Xrxl.

The murine cripto gene encodes a 171-aminoacid epidermal growth factor-related protein, with 93% similarity to its human counterpart in the ‘EGF-like’ domain. The murine cripto mRNA contains two B1 repeats in its 3 non-coding region and a 163-nucleotide homology to the human mRNA. The mouse cripto gene is expressed at low level in specific organs of the adult animal such as spleen, heart, lung and brain. In situ hybridization analysis during murine embryogenesis (day 6.2 to day 10.5) reveals a very restricted expression pattern. cripto transcripts are first detected in a few epiblastic cells at day 6.5. During gastrulation, the transcripts are expressed in the forming mesoderm and later during development cripto gene expression is restricted to the truncus arteriosus of the developing heart. This expression pattern suggests a role for cripto gene in the determination of the epiblastic cells that subsequently give rise to the mesoderm.

We have replaced part of the mouse homeogene Otx2 coding region with the E. coli lacZ coding sequence, thus creating a null allele of Otx2. By 9.5 dpc, homozygous mutant embryos are characterized by the absence of forebrain and midbrain regions. From the early to mid-streak stages, endomesodermal cells expressing lacZ fail to be properly localized anteriorly. In the ectodermal layer, lacZ transcription is progressively extinguished, being barely detectable by the late streak stage. These data suggest that Otx2 expression in endomesoderm and ectoderm is required for anterior neuroectoderm specifi-cation. In gastrulating heterozygous embryos, a post-tran-scriptional repression acts on lacZ transcripts in the ectoderm, but not in the external layer, suggesting that different post-transcriptional mechanisms control Otx2 expression in both layers.

Morphogenesis, growth and differentiation of tissues and organs require cell interactions mediated by signal molecules, their receptors and transcriptional control systems. c-fos-induced growth factor (figf) is a new secreted member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family with mitogenic activity on fibroblasts. Here we studied figfexpression during murine embryonic development. figfexpression was detected with a dynamic pattern in several body structures and organs such as limb buds, acoustic ganglion, teeth, heart, anterior pituitary as well as lung and kidney mesenchyme, liver, derma, and periosteum of the vertebral column.

We have determined the chromosomal localization of four human homeobox-containing genes, EMX1, EMX2, OTX1, and OTX2, related to Drosophilagenes expressed in the developing head of the fly. Murine homologs of these genes are expressed in specific nested domains in the developing rostral brain of midgestation embryos. DNAs from a panel of 19 rodent-human hybrids, each carrying one or a few human chromosomes such that most human chromosome regions were represented, were tested for the presence of the four gene loci by filter hybridization to radiolabeled probes. Regional chromosomal localization was determined by similarly testing DNAs from hybrid mapping panels for each of the candidate chromosomes. Finally, fluorescence in situhybridization of cosmid clones for these loci refined the locations, two of which were in the vicinity of previously mapped orphan homeobox genes and two of which were near each other. OTX2, the earliest and most widely expressed gene, maps to chromosome region 14q21-q22; the OTX1locus maps to 2p13; EMX2maps to 10q26.1; and EMX1, the most narrowly and lately expressed, maps to 2p14-p13. Thus, these homeobox-containing genes involved in brain development are not linked to any of the four HOXclusters on 7p15-p14, 17q21-q22, 12q12-q13, and 2q31. However, the OTX1and EMX1loci may be closely linked on or near 2p13, prompting speculation that a clustered gene structure could have functional significance, as is presumably the case for the HOXclusters.

Understanding the genetic mechanisms that control brain patterning in vertebrates represents a major challenge for developmental neurobiology. The cloning of genes likely to be involved in the organization of the brain and an analysis of their roles have revealed insights into the molecular pathways leading to neural induction, tissue specification, and regionalization of the brain. Among these genes, bothOtx1andOtx2,two murine homologs of theDrosophila orthodenticle(otd) gene, contribute to several steps in brain morphogenesis. Recent findings have demonstrated thatOtx2plays a major role in gastrulation and in the early specification of the anterior neural plate whileOtx1is mainly involved in corticogenesis, andOtx1andOtx2genes cooperate in such a way that a minimal level of OTX proteins are required for proper regionalization and subsequent patterning of the developing brain. Finally, experiments have shown functional equivalence betweenDrosophila otdand vertebrateOtxgenes, suggesting a surprising conservation of function required in brain development throughout evolution.

Human first trimester extraembryonic tissues of normal and androgenetic origin (molar pregnancies) were investigated for the expression of 6 homeobox genes from the chromosome 12-encoded Hox-3 cluster by non-autoradiographic in situ hybridization with biotinylated RNA probes. By comparative in situ hybridization involving the use of exon- or region-specific RNA probes, analysis included the cellular distribution of alternative Hox-3 transcripts in chorionic villous tissues. A bias in extraembryonic distribution was seen between transcripts of the three most upstream Hox-3 genes (Hox-3.7, -3.6, and -3.1) versus transcripts of the 3 most downstream genes (Hox-3.3, 3.4, and 3.5). Only genes from the latter group are transcribed in human extra-embryonic tissues including extraembryonic tissues of androgenetic origin. Moreover, comparative in situ hybridization showed that distinct alternative transcripts of Hox-3.3, Hox 3.4 and Hox-3.5 are exclusively found in trophoblast cells while others are present in chorionic villous stromal cells as well. These data demonstrate the existence of tissue- and cell-specific use of transcriptional (alternative gene promoters) or post-transcriptional (alternative splicing) regulation of homeobox genes in extraembryonic tissues.

Sixteen HLA class I clones have been isolated from a SV40-transformed human fibroblast line (GM637) cDNA library. The clones, characterized by hybridization to ABC locus-specific probes and sequence analysis, correspond to transcripts from four different class I genes: A2, A10, Cw4, and Cw6 (or Cw7), as implied by cell typing. Only the A2 sequence was known. The nucleotide and deduced amino acid sequence of the new alleles are reported here, and their structural features are discussed. Two independent cDNAs of A2 specificity display an unusual polyadenylation site located 100 by upstream from the canonical one. Moreover, two cDNAs pertaining to the same C allele display two alternative mechanisms of splicing, which cause either presence or absence in mature transcripts of the transmembrane exon 5 sequence. Transcripts missing this region are predicted to synthesize a nonmembrane-bound, secreted antigen. A soluble protein, specifically reacting with class I-specific HLA antibodies, is found in the supernatant of the GM637 cells. The significance of HLA class I transcripts generated by differential processing is discussed.

Understanding the genetic mechanisms that control patterning of the vertebrate brain represents a major challenge for developmental neurobiology. Previous data suggest that Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, might both contribute to brain morphogenesis. To gain insight into this possibility, the level of OTX proteins was modified by altering in vivo the Otx gene dosage. Here we report that Otx genes may cooperate in brain morphogenesis and that a minimal level of OTX proteins, corresponding either to one copy each of Otx1 and Otx2, or to only two copies of Otx2, is required for proper regionalization and subsequent patterning of the developing brain. Thus, as revealed by anatomical and molecular analyses, only Otx1−/−; Otx2+/− embryos lacked mesencephalon, pretectal area, dorsal thalamus and showed an heavy reduction of the Ammon’s horn, while the metencephalon was dramatically enlarged occupying the mesencencephalic area. In 8.5 days post coitum (d.p.c.) Otx1−/−; Otx2+/− embryos, the expression patterns of mesencephalic-metencephalic (mes-met) markers such as En-1 and Wnt-1 confirmed the early presence of the area fated to give rise to mesencephalon and metencephalon while Fgf-8 transcripts were improperly localized in a broader domain. Thus, in Otx1−/−; Otx2+/− embryos, Fgf-8 misexpression is likely to be the consequence of a reduced level of specification between mes-met primitive neuroepithelia that triggers the following repatterning involving the transformation of mesencephalon into metencephalon, the establishment of an isthmic-like structure in the caudal diencephalon and, by 12.5 d.p.c., the telencephalic expression of Wnt-1 and En-2. Taken together these findings support the existence of a molecular mechanism depending on a precise threshold of OTX proteins that is required to specify early regional diversity between adjacent mes-met territories and, in turn, to allow the correct positioning of the isthmic organizer.

Genetic and molecular approaches have enabled the identification of regulatory genes critically involved in determining cell types in the pituitary gland and/or in the hypothalamus. Here we report that Otx1, a homeobox-containing gene of the Otx gene family, is postnatally transcribed and translated in the pituitary gland. Cell culture experiments indicate that Otx1 may activate transcription of the growth hormone (GH), follicle-stimulating hormone (βFSH), luteinizing hormone (βLH) and α-glycoprotein subunit (αGSU) genes. Analysis of Otx1 null mice indicates that, at the prepubescent stage, they exhibit transient dwarfism and hypogonadism due to low levels of pituitary GH, FSH and LH hormones which, in turn, dramatically affect downstream molecular and organ targets. Nevertheless, Otx1−/− mice gradually recover from most of these abnormalities, showing normal levels of pituitary hormones with restored growth and gonadal function at 4 months of age. Expression patterns of related hypothalamic and pituitary cell type restricted genes, growth hormone releasing hormone (GRH), gonadotropin releasing hormone (GnRH) and their pituitary receptors (GRHR and GnRHR) suggest that, in Otx1−/− mice, hypothalamic and pituitary cells of the somatotropic and gonadotropic lineages appear unaltered and that the ability to synthesize GH, FSH and LH, rather than the number of cells producing these hormones, is affected. Our data indicate that Otx1 is a new pituitary transcription factor involved at the prepubescent stage in the control of GH, FSH and LH hormone levels and suggest that a complex regulatory mechanism might exist to control the physiological need for pituitary hormones at specific postnatal stages. Key words: Otx1, Cell specificity, Pituitary hormone, Dwarfism, Hypogonadism, Spermiogenesis, Mouse

OTX1 and OTX2 are transcription factors containing a bicoid-likehomeodomain and play crucial roles in brain development and in few other body districts as in the case of OTX1, which is required in hematopoiesis for development of the erythroid compartment. Previous studies indicated that in human OTX2 is functionally involved in the generation of anaplastic medulloblastomas; OTX1 expression strongly correlates with nodular/desmoplastic medulloblastomas; and none of them is expressed in other brain tumors. Here we have investigated whether OTX1 and/or OTX2 may potentially be involved in tumors of nonneural origin by focusing on human B-cell non-Hodgkin's lymphomas (NHLs). We analyzed 184 cases (fig 1)encompassing different NHL subtypes according to the World Health Organization classification and discovered that, while OTX2 expression was never detected, OTX1 was selectively activated in virtually all cases of Diffuse Large B Cell Lymphoma, Burkitt Lymphoma and a relevant fraction of Follicular Cell Lymphomas. In DLBCL lymph nodes and tissue infiltrations, OTX1 protein was detected in the nucleus of PAX5+-CD20+cancer cells. Conversely, in nonmalignant lymph nodes and tonsils, OTX1 was expressed in a numerically very small and non-proliferating cell population restricted to the germinal center. These OTX1+cells were CD138+-IgG+, about 50% of them also co-expressed both CD10 and CD20 and none of them exhibited PAX5 or BCL6 expression, thus suggesting that OTX1 expression identifies a subset of GC-restricted plasmacell-like cells (fig 2). Interestingly, in contrast with lymphomas, in these cells the OTX1 protein was prevalently localized to the cytoplasm. Collectively, our data suggest that activation of OTX1 expression and its nuclear localization represent together a newly described molecular event occurring in specific NHL subtypes of GC and early post-GC origin (FCL, DLBCL, BL), and lead us to hypothesize that it may be potentially involved (directly or indirectly) in the initiation and/or maintenance of these tumors.

Background: Homeobox genes of the orthodenticle(otd)/Otxfamily have conserved roles in the embryogenesis of head and brain. Gene replacement experiments show that the Drosophila otdgene and orthologous mammalian Otxgenes are functionally equivalent, in that overexpression of either gene in null mutants of Drosophilaor mouse can restore defects in cephalic and brain development. This suggests that otdand Otxgenes control a comparable subset of downstream target genes in either organism. Here we use quantitative transcript imaging to analyze this equivalence of otdand Otxgene action at a genomic level. Results: Oligonucleotide arrays representing 13,400 annotated Drosophilagenes were used to study differential gene expression in flies in which either the Drosophila otdgene or the human Otx2gene was overexpressed. Two hundred and eighty-seven identified transcripts showed highly significant changes in expression levels in response to otdoverexpression, and 682 identified transcripts showed highly significant changes in expression levels in response to Otx2overexpression. Among these, 93 showed differential expression changes following overexpression of either otdor Otx2, and for 90 of these, comparable changes were observed under both experimental conditions. We postulate that these transcripts are common downstream targets of the fly otdgene and the human Otx2gene in Drosophila. Conclusion: Our experiments indicate that approximately one third of the otd-regulated transcripts also respond to overexpression of the human Otx2gene in Drosophila. These common otd/Otx2downstream genes are likely to represent the molecular basis of the functional equivalence of otdand Otx2gene action in Drosophila.