An astonishing journey into reproductive genetics since the 1950's

Training in genetics in Edinburgh in the 1950s led to a PhD on the developmental biology of mouse embryos with unusual chromosomal complements. Fundamental aspects of reproduction under study included ovulation induction, oocyte maturation and embryonic growth to blastocysts. It led to the introduction of embryo stem cells, preimplantation genetic diagnosis, the exact timing of human oocyte maturation in vitro and studies on fertilising human eggs in vitro to alleviate human infertility. My work was helped by studies on sperm capacitation and the physiology of fertilization in domestic and laboratory species by Thibault, Dauzier, Austin, Chang, Yanagimachi and others. I met Charles Thibault at a meeting in Cambridge U.K. where he criticised the work of Moricard, and then frequently on lecture circuits. Impressed by his grandeur but not his doubts about human IVF, Steptoe and I initiated human embryo transfers and the birth of Louise Brown. Details of her pregnancy had to be confidential to reduce the risks of abortion associated with the intrusion of numerous newsmen chasing the story. I was compelled to withold this information at a meeting in Paris in the late 1960s when I had to leave early to return to UK. This omission annoyed Thibault and led to our celebrated quarrel. I felt he failed to appreciate the complexity, the implications of this pregnancy and an astonishing future. So much was at stake, including IVF, stem cells and preimplantation diagnosis to help millions of patients. Some months later, our dispute was ended even if somewhat formally. Nevertheless it is a pleasure to recall how we shared so much in common. I still admire him as an inspiration to many colleagues and students, and a father figure in French agricultural research.     

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.     

The incredible ULKs

ABSTRACT: Macroautophagy (commonly abbreviated as autophagy) is an evolutionary conserved lysosome-directed vesicular trafficking pathway in eukaryotic cells that mediates the lysosomal degradation of intracellular components. The cytoplasmic cargo is initially enclosed by a specific double membrane vesicle, termed the autophagosome. By this means, autophagy either helps to remove damaged organelles, long-lived proteins and protein aggregates, or serves as a recycling mechanism for molecular building blocks. Autophagy was once invented by unicellular organisms to compensate the fluctuating external supply of nutrients. In higher eukaryotes, it is strongly enhanced under various stress conditions, such as nutrient and growth factor deprivation or DNA damage. The serine/threonine kinase Atg1 was the first identified autophagy-related gene (ATG) product in yeast. The corresponding nematode homolog UNC-51, however, has additional neuronal functions. Vertebrate genomes finally encode five closely related kinases, of which UNC-51-like kinase 1 (Ulk1) and Ulk2 are both involved in the regulation of autophagy and further neuron-specific vesicular trafficking processes. This review will mainly focus on the vertebrate Ulk1/2-Atg13-FIP200 protein complex, its function in autophagy initiation, its evolutionary descent from the yeast Atg1-Atg13-Atg17 complex, as well as the additional non-autophagic functions of its components. Since the rapid nutrient- and stress-dependent cellular responses are mainly mediated by serine/threonine phosphorylation, it will summarize our current knowledge about the relevant upstream signaling pathways and the altering phosphorylation status within this complex during autophagy induction.     

Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity

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Otx1 and Otx2 activities are required for the normal development of the mouse inner ear

The Otx1 and Otx2 genes are two murine orthologues of the Orthodenticle (Otd) gene in Drosophila. In the developing mouse embryo, both Otx genes are expressed in the rostral head region and in certain sense organs such as the inner ear. Previous studies have shown that mice lacking Otx1 display abnormal patterning of the brain, whereas embryos lacking Otx2 develop without heads. In this study, we examined, at different developmental stages, the inner ears of mice lacking both Otx1 and Otx2 genes. In wild-type inner ears, Otx1, but not Otx2, was expressed in the lateral canal and ampulla, as well as part of the utricle. Ventral to the mid-level of the presumptive utricle, Otx1 and Otx2 were co-expressed, in regions such as the saccule and cochlea. Paint-filled membranous labyrinths of Otx1-/- mutants showed an absence of the lateral semicircular canal, lateral ampulla, utriculosaccular duct and cochleosaccular duct, and a poorly defined hook (the proximal part) of the cochlea. Defects in the shape of the saccule and cochlea were variable in Otx1-/- mice and were much more severe in an Otx1-/-;Otx2(+/)- background. Histological and in situ hybridization experiments of both Otx1-/- and Otx1-/-;Otx2(+/)- mutants revealed that the lateral crista was absent. In addition, the maculae of the utricle and saccule were partially fused. In mutant mice in which both copies of the Otx1 gene were replaced with a human Otx2 cDNA (hOtx2(1)/ hOtx2(1)), most of the defects associated with Otx1-/- mutants were rescued. However, within the inner ear, hOtx2 expression failed to rescue the lateral canal and ampulla phenotypes, and only variable rescues were observed in regions where both Otx1 and Otx2 are normally expressed. These results suggest that both Otx genes play important and differing roles in the morphogenesis of the mouse inner ear and the development of its sensory organs.     

The incredible shrinking organelle

The mitochondrion is probably the evolutionary remnant of a bacterial symbiont, yet contemporary mitochondria are nothing like contemporary bacteria. Evolutionary shrinkage of the mitochondrial genome is well documented, but what about wholesale shrinkage of the organelle itself?Considering its central role in energy metabolism in almost all eukaryotes, the mitochondrion is an amazingly plastic organelle, both evolutionarily and functionally. The few genes that the mitochondrial genome (mitochondrial DNA; mtDNA) encodes are clearly bacterial in origin—emanating from the α-proteobacterial lineage—supporting the widely held view that the mitochondrion is the evolutionary remnant of a bacterial symbiont (Gray et al, 2001). However, contemporary mitochondria are nothing like contemporary bacteria. For one thing, even the most gene-rich mtDNA encodes far less genetic information than the most gene-poor bacterial genome, and mitochondrial genomes are different from bacterial genomes in form, organization and mode of expression; these features vary tremendously among diverse eukaryotes. Mitochondrial genomes might be circular, linear or even highly fragmented, and they might contain highly fragmented and rearranged genes. Only within a poorly studied group of eukaryotic microbes—protists—known as jakobid flagellates does the mtDNA resemble a typical, albeit highly reduced, bacterial genome.In addition, the mitochondrial proteome is not only overwhelmingly (>90%) encoded in the nucleus, but only a small proportion (10–15%) is demonstrably α-proteobacterial in evolutionary affiliation. Thus, in the evolutionary transition from bacterial symbiont to integrated organelle, the mitochondrion has undergone an impressive degree of re-tailoring, shedding the bulk of its genetic information and taking on proteins of diverse evolutionary origins. Moreover, this re-tailoring is highly variable within different eukaryotic lineages, with an intriguing chunk of the mitochondrial proteome seeming to be organism-specific—lacking demonstrable sequence homologues other than in very close evolutionary relatives.Although the evolutionary shrinkage of the mitochondrial genome is well-documented, what is less widely appreciated is the wholesale shrinkage of the organelle itself in certain anaerobic eukaryotes. Taken to its extreme, such shrinkage involves complete loss of the mitochondrial genome, with a consequent reduction in the structural complexity and biochemical versatility of the organelle. This simplification might include elimination of the electron-transport chain (ETC) and thus lead to inability of the resulting mitochondrion-related organelle (MRO) to carry out a key function of aerobic mitochondria: ATP synthesis through coupled oxidative phosphorylation (for a full account, see Hjort et al, 2010).One such MRO, the hydrogenosome, is a hydrogen-producing organelle that was originally characterized in an anaerobic protist, Trichomonas vaginalis. The T. vaginalis hydrogenosome lacks mtDNA as well as components of the classic mitochondrial ETC, relying instead on substrate-level phosphorylation to generate ATP. Initially, the resemblance between the anaerobic biochemistry of the T. vaginalis MRO and that of anaerobic bacteria such as Clostridia raised the possibility that the hydrogenosome might have a different evolutionary origin than the classic aerobic mitochondrion. However, studies of hydrogenosomal proteins have demonstrated that the hydrogenosome is an evolutionarily derived (remnant) mitochondrion. Hydrogenosomes have been found in eukaryotes that are widely separated in phylogenetic trees, and in such trees, anaerobic, hydrogenosome-containing eukaryotes are often interspersed with close relatives that grow aerobically and contain conventional mitochondria. This punctate phylogenetic distribution suggests that the transition from mitochondrion to hydrogenosome has happened repeatedly and independently throughout eukaryotic evolution.The mitosome, an even more shrunken MRO that has not only dispensed entirely with a genome, but also has no ATP-generating capacity. This MRO was discovered in anaerobic eukaryotes that were initially thought to lack mitochondria entirely, the postulate being that they diverged away from the main line of eukaryotic evolution prior to the symbiosis that led to the mitochondrion. However, in all supposedly amitochondriate protists that have been examined, a candidate mitosome has been identified. As with hydrogenosomes, a punctate phylogenetic distribution of mitosomes is emerging.Recently, intermediate forms of ''shrinking organelle'' have been identified in the anaerobic protists Nyctotherus ovalis, Blastocystis sp. and Proteromonas lacertae (Hjort et al, 2010; Pérez-Brocal et al, 2010; de Graaf et al, 2011), relatives of brown algae and diatoms. In these cases, regions of the mtDNA that code for terminal portions of the ETC and for the mitochondrial ATP synthase have been discarded. The remaining DNA specifies genes for components of a mitochondrial translation system, as well as subunits of a proton-pumping complex I (NADH:ubiquinone oxidoreductase); a remarkable example—comparing the ciliate Nyctotherus with the stramenopiles Blastocystsis or Proteromonas—of convergent mtDNA evolution. These observations suggest that the transitional MROs of Nyctotherus, Blastocystis and Proteromonas retain a partial ETC, as well as the ability to synthesize protein, whereas other data (EST surveys) indicate that they are metabolically more complex than either hydrogenosomes or mitosomes. The discovery of these particular MROs is important because their existence argues that the transition from fully fledged aerobic mitochondrion to fully fledged anaerobic mitosome proceeds through, and might stop at, several intermediate stages: a realization that not only dramatically emphasizes the evolutionary and functional versatility of the mitochondrion, but also opens the possibility that we might yet uncover still other variations of this incredible shrinking organelle.     

Otx1l, Otx2 and Irx1b establish and position the ZLI in the diencephalon

The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region - the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.     

Genetic modifiers of otocephalic phenotypes in Otx2 heterozygous mutant mice

Mice heterozygous for the Otx2 mutation display a craniofacial malformation, known as otocephaly or agnathia-holoprosencephaly complex. The severity of the phenotype is dependent on the genetic background of a C57BL/6 (B6) strain; most of the offspring of Otx2 knock-out chimeras, which are equivalent to the F(1) of CBA and B6 strains, backcrossed with B6 females display reduction or loss of mandible, whereas those backcrossed with CBA females do not show noticeable phenotype at birth. The availability of phenotypically disparate strains renders identification of Otx2 modifier loci possible. In this study, a backcross of chimera with B6 was generated and genome-wide scans were conducted with polymorphic markers for non-mendelian distribution of alleles in Otx2 heterozygous mutant mice displaying abnormalities in the lower jaw. We identified one significant locus, Otmf18, between D18Mit68 and D18Mit120 on chromosomes 18, linked to the mandibular phenotype (LOD score 3.33). A similar replication experiment using a second backcross (N3) mouse demonstrated the presence of another significant locus, Otmf2 between D2Mit164 and D2Mit282 on chromosome 2, linked to the mandibular phenotype (LOD score 3.93). These two modifiers account for the distribution of the craniofacial malformations by the genetic effect between B6 and CBA strains. Moreover, Otmf2 contain a candidate gene for several diseases in mice and humans. These genetic studies involving an otocephalic mouse model appear to provide new insights into mechanistic pathways of craniofacial development. Furthermore, these experiments offer a powerful approach with respect to identification and characterization of candidate genes that may contribute to human agnathia-holoprosencephaly complex diseases.     

Otx2 and HNF3beta genetically interact in anterior patterning

Patterning the developing nervous system in the mouse has been proposed to depend on two separate sources of signals, the anterior visceral endoderm (AVE) and the node or organizer. Mutation of the winged-helix gene HNF3beta leads to loss of the node and its derivatives, while mutation of the homeobox gene Otx2 results in loss of head structures, apparently at least partially because of defects in the AVE. To investigate the potential genetic interactions between the two signaling centers, we crossed Otx2+/- and HNF3beta+/- mice and found that very few Otx2+/-;HNF3beta+/- double heterozygous mutants survived to weaning. Normal Mendelian ratios of genotypes were observed during gestation, but more than half the double heterozygotes displayed a severe anterior patterning phenotype that would be incompatible with postnatal survival. The phenotype was characterized by varying degrees of holoprosencephaly, cyclopia with proboscis-like structures, and anterior forebrain truncations. Regional marker analysis revealed that ventral forebrain structures of Otx2+/-;HNF3beta+/- mutant embryos were most severely affected. Shh expression was completely absent in the anterior region of Otx2+/-;HNF3beta+/- embryos, suggesting that Otx2 and HNF3beta genetically interact, directly or indirectly, to regulate Shh expression in the anterior midline. In addition, the forebrain truncations suggest an involvement of both genes in anterior patterning, through their overlapping expression domains in either the AVE and/or the prechordal mesoderm.     

MiR-206 regulates neural cells proliferation and apoptosis via Otx2

MiR-206 was involved in a series of cellular activities, such as the growth and development of skeletal muscle and the tumorigenesis. MiR-206 was characterized previously as a differentially expressed gene in sodium arsenite (SA)-induced neural tube defects (NTDs) in chick embryos via miRNA microarray analysis. However, the role of miR-206 in the pathological process of nerve cells remained elusive. In this study we found differential expression of miR-206 in SA-treated chick embryos by Northern blot analysis. Ectopic expression of miR-206 inhibited cell proliferation, and promoted cell apoptosis in U343 and SK-N-SH cell by using MTT, Edu Apollo assay and Flow cytometry analysis. Further investigation revealed that miR-206 can interact with 3'-untranslated region (UTR) of Otx2. MiR-206 mimics down-regulated the endogeneous Otx2 expression, whereas the miR-206 inhibitor obviously up-regulated the expression of Otx2. These findings indicate that overexpression of miR-206 promotes cell apoptosis and low expression of miR-206 inhibits cell apoptosis. Otx2 may play an important role in the process of miR-206-mediated cell apoptosis.