Murine Otx1 and Drosophila otd genes share conserved genetic functions required in invertebrate and vertebrate brain development

Despite the obvious differences in anatomy between invertebrate and vertebrate brains, several genes involved in the development of both brain types belong to the same family and share similarities in expression patterns. Drosophila orthodenticle (otd) and murine Otx genes exemplify this, both in terms of expression patterns and mutant phenotypes. In contrast, sequence comparison of OTD and OTX gene products indicates that homology is restricted to the homeodomain suggesting that protein divergence outside the homeodomain might account for functional differences acquired during brain evolution. In order to gain insight into this possibility, we replaced the murine Otx1 gene with a Drosophila otd cDNA. Strikingly, epilepsy and corticogenesis defects due to the absence of Otx1 were fully rescued in homozygous otd mice. A partial rescue was also observed for the impairments of mesencephalon, eye and lachrymal gland. In contrast, defects of the inner ear were not improved suggesting a vertebrate Otx1-specific function involved in morphogenesis of this structure. Furthermore, otd, like Otx1, was able to cooperate genetically with Otx2 in brain patterning, although with reduced efficiency. These data favour an extended functional conservation between Drosophila otd and murine Otx1 genes and support the idea that conserved genetic functions required in mammalian brain development evolved in a primitive ancestor of both flies and mice.     

Otx/otd homeobox genes specify distinct sensory neuron identities in C. elegans

The mechanisms by which the diverse functional identities of neurons are generated are poorly understood. C. elegans responds to thermal and chemical stimuli using 12 types of sensory neurons. The Otx/otd homolog ttx-1 specifies the identities of the AFD thermosensory neurons. We show here that ceh-36 and ceh-37, the remaining two Otx-like genes in the C. elegans genome, specify the identities of AWC, ASE, and AWB chemosensory neurons, defining a role for this gene family in sensory neuron specification. All C. elegans Otx genes and rat Otx1 can substitute for ceh-37 and ceh-36, but only ceh-37 functionally substitutes for ttx-1. Functional substitution in the AWB neurons is mediated by activation of the same downstream target lim-4 by different Otx genes. Misexpression experiments indicate that although the specific identity adopted upon expression of an Otx gene may be constrained by the cellular context, individual Otx genes preferentially promote distinct neuronal identities.     

Functional equivalency between Otx2 and Otx1 in development of the rostral head

Mice have two Otx genes, Otx1 and Otx2. Prior to gastrulation, Otx2 is expressed in the epiblast and visceral endoderm. As the primitive streak forms, Otx2 expression is restricted to the anterior parts of all three germ layers. Otx1 expression begins at the 1 to 3 somite stage in the anterior neuroectoderm. Otx2 is also expressed in cephalic mesenchyme. Otx2 homozygous mutants fail to develop structures anterior to rhombomere 3 (r3), and Otx2 heterozygotes exhibit craniofacial defects. Otx1 homozygous mutants do not show apparent defects in early brain development. In Otx1 and Otx2 double heterozygotes, rostral neuroectoderm is induced normally, but development of the mes/diencephalic domain is impaired starting at around the 3 to 6 somite stage, suggesting cooperative interactions between the two genes in brain regionalization. To determine whether Otx1 and Otx2 genes are functionally equivalent, we generated knock-in mice in which Otx2 was replaced by Otx1. In homozygous mutants, gastrulation occurred normally, and rostral neuroectoderm was induced at 7.5 days postcoitus (7.5 dpc), but the rostral brain failed to develop. Anterior structures such as eyes and the anterior neural ridge were lost by 8.5 dpc, but the isthmus and r1 and r2 were formed. In regionalization of the rostral neuroectoderm, the cooperative interaction of Otx2 with Otx1 revealed by the phenotype of Otx2 and Otx1 double heterozygotes was substitutable by Otx1. The otocephalic phenotype indicative of Otx2 haploinsufficiency was also largely restored by knocked-in Otx1. Thus most Otx2 functions were replaceable by Otx1, but the requirement for Otx2 in the anterior neuroectoderm prior to onset of Otx1 expression was not. These data indicate that Otx2 may have evolved new functions required for establishment of anterior neuroectoderm that Otx1 cannot perform.     

Electrokinetic chromatography without electroosmotic flow

Otx1 and Otx2 genes are mouse cognates of a Drosophila head gap gene orthodenticle. The homozygous mutants have previously indicated that Otx2 is essential to development of structures anterior to rhombomere 3, probably reflecting its expression around the early primitive streak stage. Otx2 mutation also exhibits craniofacial defects by haplo-insufficiency. Affected structures correspond to the most anterior and most posterior parts of the Otx2 expression where Otx1 is not, or is only weakly, expressed at the time of brain regionalization. No apparent defects are found in early brain development by the Otx1 mutation, suggesting that the Otx1 and Otx2 functions overlap in the regions where both are expressed. To demonstrate this, the Otx1/Otx2 double heterozygous phenotype was examined in this study. Analyses with molecular markers at 9.5 days post coitus suggested the failure in development of mesencephalon and caudal diencephalon with the expansion of anterior metencephalon. Genes expressed in isthmus exhibited a characteristic lateral stripe normally, although rostrally shifted, except that Fgf8 expression was expanded dorsally. The defects were apparent at the 6-somite stage, but not at the 3-somite stage. Broad Fgf8 expression at the 3-somite stage took place normally, but it did not concentrate into a spot corresponding to future isthmus. The double heterozygous phenotype implicates a previously unsuspected mechanism for development of the mes/metencephalic territory; at the 3- to 6-somite stage Otx1 cooperates with Otx2 to establish the mes/diencephalic domain, allowing for the correct development of isthmus/ rhombomere 1.     

Otx1 and Otx2 in the development and evolution of the mammalian brain

In the last decade, a number of genes related to the induction, specification and regionalization of the brain were isolated and their functional properties currently are being dissected. Among these, Otx1 and Otx2 play a pivotal role in several processes of brain morphogenesis. Findings from several groups now confirm the importance of Otx2 in the early specification of neuroectoderm destined to become fore-midbrain, the existence of an Otx gene dosage-dependent mechanism in patterning the developing brain, and the involvement of Otx1 in corticogenesis. Some of these properties appear particularly fascinating when considered in evolutionary terms and highlight the central role of Otx genes in the establishment of the genetic program defining the complexity of a vertebrate brain. This review deals with the major aspects related to the roles played by Otx1 and Otx2 in the development and evolution of the mammalian brain.     

Fibroblast growth factor 2 increases Otx2 expression in precursor cells from mammalian telencephalon

Dissociated primary cultures from rat telencephalon at different developmental stages were used to study the effect of basic fibroblast growth factor (FGF2) on Otx2, Dlx1, and Emx1, three homeobox genes expressed in different regions of the developing mammalian forebrain. At embryonic day (E)13.5. the regional pattern of expression of Otx1, Otx2, Dlx1, Dlx2, Dlx5, and Emx1 is maintained in primary culture, suggesting that cells are already committed to a regional identity at this stage. In these cultures, Otx2 is expressed by precursor cells, whereas Dlx1 and Emx1 are predominantly expressed by postmitotic cells. We found that FGF2 increased Otx2 expression within precursor cells and the total number of Otx2-expressing cells. This effect was gene-specific, dose-dependent, and temporally regulated, with larger effects at earlier stages of development (E11.5). At E13.5, the effect of FGF2 on Otx2 expression was restricted to the basal telencephalon. Our results suggest that a restricted population of neuroblasts respond to FGF2 in a temporally regulated fashion by proliferating and increasing Otx2 expression. This interaction between FGF2 and Otx2 may be important for the regulation of neurogenesis in the forebrain.     

Bupropion and desipramine increase dopamine transporter mRNA expression in the ventral tegmental area/substantia nigra of rat brain

The homeobox gene Otx2 is a mouse cognate of the Drosophila orthodenticle gene, which is required for development of the brain, rostral to rhombomere three. We have investigated the mechanisms involved in this neural function and specifically the requirement for Otx2 in the visceral endoderm and the neuroectoderm using chimeric analysis in mice and explant recombination assay. Analyses of chimeric embryos composed of more than 90% of Otx2-/- ES cells identified an essential function for Otx2 in the visceral endoderm for induction of the forebrain and midbrain. The chimeric studies also demonstrated that an anterior neural plate can form without expressing Otx2. However, in the absence of Otx2, expression of important regulatory genes, such as Hesx1/Rpx, Six3, Pax2, Wnt1 and En, fail to be initiated or maintained in the neural plate. Using explant-recombination assay, we could further demonstrate that Otx2 is required in the neuroectodem for expression of En. Altogether, these results demonstrate that Otx2 is first required in the visceral endoderm for the induction, and subsequently in the neuroectoderm for the specification of forebrain and midbrain territories.     

Emx1 and Emx2 show different patterns of expression during proliferation and differentiation of the developing cerebral cortex in the mouse

Insights into the complex structure of the forebrain and its regulation have recently come from the analysis of the expression of genes that are likely to be involved in regionalization of this structure. We cloned four new homeo box genes, Emx1, Emx2, Otx1 and Otx2, and showed that in day 10 mouse embryos their expression domains are continuous regions of the developing brain contained within each other in the sequence Emx1 < Emx2 < Otx1 < Otx2. Recently Otx1 has been found to be specifically expressed during neurogenesis of layer 5 and 6 in the developing cerebral cortex. In order to better understand the role of Emx1 and Emx2 in the maturation of the cortex we analysed by in situ hybridization their expression patterns in the developing mouse cerebral cortex, from embryonic day 12.5 to adulthood. We found that Emx2 is expressed exclusively in proliferating cells of the ventricular zone whereas Emx1 is expressed in both proliferating and differentiated neurons, throughout the cortical layers and during all the developmental stages examined. Therefore, Emx2 gene products might control some biological parameters of the proliferation of cortical neuroblasts or of the subsequent cell migration of postmitotic neurons, leaving the cortical germinal zone. Conversely, Emx1 expression, which is confined exclusively to the dorsal telencephalon, characterizes most cortical neurons during proliferation, differentiation, migration and postnatal development and maturation.     

Molecular cloning and characterization of Drosophila genes encoding small GTPases of the rab and rho families

We have isolated eight genes from Drosophila, small GTPases. They can be classified into three rab family genes (Drab2, Drab5, Drab11) and five rho family genes (Drac1a, Drac1b, Drac3, Dcdc42, DrhoA). While Drac3 is a novel type of rac gene, others are homologues of known mammalian genes for small GTPases. Northern blot analyses showed that all the genes are expressed throughout all developmental stages from embryo to adult. In situ hybridization to embryos revealed that Drab2, Drac1b, and Drac3 are highly expressed in the nervous system, in the trunk mesoderm, and in the cephalic mesoderm, respectively. Since hemocytes are derived from the cephalic mesoderm, we carried out double stainings using a hemocyte marker anti-peroxidasin antibody and Drac3 in situ hybridization. We found that Drac3 is expressed in hemocyte precursor cells. In the Drac3 deficiency embryos, the hemocyte precursor cells start to differentiate normally, but never develop into mature hemocytes, indicating that Drac3 is essential for their maturation. The DrhoA and Dcdc42 genes complemented S. cerevisiae rho1 and cdc42 mutations in the same manner as human rhoA and CDC42, respectively. These results suggest functional similarity between Drosophila and mammalian small GTPase genes.     

Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite Archegozetes longisetosus, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost. engrailed (en) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the Drosophila genes Antennapedia (Antp), Sex combs reduced (Scr), Deformed (Dfd), proboscipedia (pb), and orthodenticle (otd) confirm direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost. We make further inferences concerning the evolution of heads and Hox genes in arthropods.     

Persistent transgene expression and normal differentiation of immortalized human keratinocytes in vivo

The homeobox gene otx2 is a key regulator for specifying the rostral part of the vertebrate head. In Xenopus, otx2 directly controls the differentiation of the cement gland, the anterior-most organ formed in the tadpole. Since embryos of a direct developing frog, Eleutherodactylus coqui, lack a cement gland, we are interested in whether altered expression of the otx2 gene is involved in this evolutionary change. We have cloned the E. coqui homologue of otx2, Ecotx2. The homeodomain of the Ecotx2 protein is identical to the mouse and zebrafish Otx2 proteins and differs by a single amino acid from the Xenopus Otx2 protein. Study of the spatiotemporal expression pattern shows that Ecotx2 RNA is progressively restricted to the anterior region of the embryo during gastrulation and becomes further restricted to the future forebrain and midbrain during neural development. In Xenopus, in addition to the conserved expression in the anterior neuroectoderm, the expression in ectoderm expands more anteriorly to the cement gland primordium. This anterior expansion of otx2 expression is not found in E. coqui, correlating with the loss of a cement gland. When misexpressed in Xenopus laevis ectoderm, Ecotx2 can activate expression of the cement-gland-specific genes XCG and XAG1, indicating that the function of activating the pathway of cement gland formation is retained by the Ecotx2 protein. Our results indicate that there are modifications in the pathway of cement gland formation, upstream of otx2 expression, in the development of E. coqui.     

Cannabis for migraine treatment: the once and future prescription? An historical and scientific review

Over the past ten years, the discovery and functional characterisation of murine Hox genes has led to a better understanding of some of the molecular mechanisms underlying limb development. It has also shed some light on the potential genetic events which have accompanied the fin-to-limb transition, an evolutionary step of critical importance which opened the way to the evolution of higher vertebrates. This convergence between developmental biology and the sciences of evolution is one of the synergistic interface that has been established recently thanks to the use of genetic engineering and transgenic animals. The increasing number of human genetic syndromes which are derived from mutations in developmental control genes remind us that many human genetic diseases are nothing else but alterations in our developmental programme. Here, we illustrate these various issues by discussing the function of Hox genes during limb development.     

Murine tumor suppressor models

Tumor suppressor genes have been shown to be necessary for proper maintenance of cell growth control. Inactivation of these genes in the germline of humans is linked to inherited cancer predisposition. Moreover, sporadically arising human tumors often have somatic mutations in tumor suppressor genes. During the past few years, advances in molecular and cellular biology have led to the creation of animal models that have germline mutations of various tumor suppressor genes. Such mice potentially represent important animal models for familial cancer predisposition syndromes, and the study of the tumorigenesis process has been greatly assisted by their development. Such models have also demonstrated the importance of tumor suppressor function in embryonic development. In this review, we describe mice with inactivated germline tumor suppressor genes that are genetically analogous to 10 different inherited cancer syndromes in humans. We describe the variable usefulness of the mutant mice as models for human disease.