Enrichment of FGF8-expressing cells from neurally induced human pluripotent stem cell cultures.

In early vertebrate development, organizer regions-groups of cells that signal to and thereby influence neighboring cells by secreted morphogens-play pivotal roles in the establishment and maintenance of cell identities within defined tissue territories. The midbrain-hindbrain organizer drives regionalization of neural tissue into midbrain and hindbrain territories with fibroblast growth factor 8 (FGF8) acting as a key morphogen. This organizer has been extensively studied in chicken, mouse, and zebrafish. Here, we demonstrate the enrichment of FGF8-expressing cells from human pluripotent stem cells (hPSCs), cultured as attached embryoid bodies using antibodies that recognize "Similar Expression to Fgf" (SEF) and Frizzled proteins. The arrangement of cells in embryoid body subsets of these cultures and the gene expression profile of the FGF8-expressing population show certain similarities to the midbrain-hindbrain organizer in animal models. In the embryonic chick brain, the enriched cell population induces formation of midbrain structures, consistent with FGF8-organizing capability.

The putative tumor suppressor gene EphA7 is a novel BMI-1 target.

Bmi1 was originally identified as a gene that contributes to the development of mouse lymphoma by inhibiting MYC-induced apoptosis through repression of Ink4a and Arf. It codes for the Polycomb group protein BMI-1 and acts primarily as a transcriptional repressor via chromatin modifications. Although it binds to a large number of genomic regions, the direct BMI-1 target genes described so far do not explain the full spectrum of BMI-1-mediated effects. Here we identify the putative tumor suppressor gene EphA7 as a novel direct BMI-1 target in neural cells and lymphocytes. EphA7 silencing has been reported in several different human tumor types including lymphomas, and our data suggest BMI1 overexpression as a novel mechanism leading to EphA7 inactivation via H3K27 trimethylation and DNA methylation.

Neurobehavioral and cytotoxic effects of vanadium during oligodendrocyte maturation: a protective role for erythropoietin.

Vanadium exposure has been known to lead to lipid peroxidation, demyelination and oligodendrocytes depletion. We investigated behaviour and glial reactions in juvenile mice after early neonatal exposure to vanadium, and examined the direct effects of vanadium in oligodendrocyte progenitor cultures from embryonic mice. Neonatal pups exposed to vanadium via lactation for 15 and 22 days all had lower body weights. Behavioural tests showed in most instances a reduction in locomotor activity and negative geotaxis. Brain analyses revealed astrocytic activation and demyelination in the vanadium exposed groups compared to the controls. In cell culture, exposure of oligodendrocytes to 300 µM sodium metavanadate significantly increased cell death. Expression of the oligodendrocyte specific proteins, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and oligodendrocyte specific protein (OSP/Claudin) were reduced upon vanadium treatment while simultaneous administration of erythropoietin (EPO; 4-12 U/ml) counteracted vanadium-toxicity. The data suggest that oligodendrocyte damage may explain the increased vulnerability of the juvenile brain to vanadium and support a potential for erythropoietin as a protective agent against vanadium-toxicity during perinatal brain development and maturation.

Retinoic acid is involved in the metamorphosis of the anal fin into an intromittent organ, the gonopodium, in the green swordtail (Xiphophorus hellerii).

In poeciliid fish the male anal fin has been transformed into a gonopodium, an intromittent organ required for internal fertilization. Elevated testosterone levels induce metamorphosis of a subset of anal fin rays to grow and form the specialized terminal structures of the gonopodium. The molecular mechanisms underlying these processes are largely unknown. Here, we investigated whether retinoic acid (RA) signaling is involved in gonopodium development in the swordtail Xiphophorus hellerii. We showed that aldh1a2, a RA synthesizing enzyme, and the RA receptors, rar-ga and rar-gb, are expressed in anal fins during metamorphosis. aldh1a2 expression is regulated by testosterone in a concentration-dependent manner and is up-regulated in both hormone-induced and naturally developing gonopodia. Androgen receptor (ar), a putative regulator of gonopodial development, is co-expressed with aldh1a2 and the RA receptors in gonopodial rays. Importantly, experimental increase of RA signaling promoted growth of the gonopodium and increased the number of new segments. Based on gene expression analyses and pharmacological manipulation of gonopodium development, we show that the RA signaling pathway is activated in response to androgen signaling and promotes fin ray growth and development during the metamorphosis of the anal fin into the gonopodium.

Effects of erythropoietin in murine-induced pluripotent cell-derived panneural progenitor cells.

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1-3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of ß-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.

Ectopic expression of neurogenin 2 alone is sufficient to induce differentiation of embryonic stem cells into mature neurons.

Recent studies show that combinations of defined key developmental transcription factors (TFs) can reprogram somatic cells to pluripotency or induce cell conversion of one somatic cell type to another. However, it is not clear if single genes can define a cell̀s identity and if the cell fate defining potential of TFs is also operative in pluripotent stem cells in vitro. Here, we show that ectopic expression of the neural TF Neurogenin2 (Ngn2) is sufficient to induce rapid and efficient differentiation of embryonic stem cells (ESCs) into mature glutamatergic neurons. Ngn2-induced neuronal differentiation did not require any additional external or internal factors and occurred even under pluripotency-promoting conditions. Differentiated cells displayed neuron-specific morphology, protein expression, and functional features, most importantly the generation of action potentials and contacts with hippocampal neurons. Gene expression analyses revealed that Ngn2-induced in vitro differentiation partially resembled neurogenesis in vivo, as it included specific activation of Ngn2 target genes and interaction partners. These findings demonstrate that a single gene is sufficient to determine cell fate decisions of uncommitted stem cells thus giving insights into the role of key developmental genes during lineage commitment. Furthermore, we present a promising tool to improve directed differentiation strategies for applications in both stem cell research and regenerative medicine.

Identification of novel genes involved in the development of the sword and gonopodium in swordtail fish.

Male swordtail fish of the genus Xiphophorus develop a sword, a colourful extension of the caudal fin, that evolved by sexual selection through female choice. Swords and gonopodia, an intromittent organ developing from the male anal fin, can be prematurely induced by exogenous testosterone, offering the opportunity to examine the identity and expression profiles of genes required during various stages of fin metamorphosis. Here, we employed suppression subtractive hybridisation to identify genes specifically up-regulated during two early stages of sword and gonopodium development. We identified 128 different sequences with significant similarity to known genes and characterized the rack1, dusp1, klf2, and tmsbeta-like genes as specifically up-regulated in developing as well as regenerating fin rays of the sword and gonopodium. We show that some of these genes follow distinct expression profiles in swords and gonopodia, suggesting differences in the genetic networks underlying the development of anal and caudal fin modifications.

Fgfr1 signalling in the development of a sexually selected trait in vertebrates, the sword of swordtail fish.

BACKGROUND: One of Darwin's chosen examples for his idea of sexual selection through female choice was the "sword", a colourful extension of the caudal fin of male swordtails of the genus Xiphophorus. Platyfish, also members of the genus Xiphophorus, are thought to have arisen from within the swordtails, but have secondarily lost the ability to develop a sword. The sustained increase of testosterone during sexual maturation initiates sword development in male swordtails. Addition of testosterone also induces sword-like fin extensions in some platyfish species, suggesting that the genetic interactions required for sword development may be dormant, rather than lost, within platyfish. Despite considerable interest in the evolution of the sword from a behavioural or evolutionary point of view, little is known about the developmental changes that resulted in the gain and secondary loss of the sword. Up-regulation of msxC had been shown to characterize the development of both swords and the gonopodium, a modified anal fin that serves as an intromittent organ, and prompted investigations of the regulatory mechanisms that control msxC and sword growth. RESULTS: By comparing both development and regeneration of caudal fins in swordtails and platyfish, we show that fgfr1 is strongly up-regulated in developing and regenerating sword and gonopodial rays. Characterization of the fin overgrowth mutant brushtail in a platyfish background confirmed that fin regeneration rates are correlated with the expression levels of fgfr1 and msxC. Moreover, brushtail re-awakens the dormant mechanisms of sword development in platyfish and activates fgfr1/msxC-signalling. Although both genes are co-expressed in scleroblasts, expression of msxC in the distal blastema may be independent of fgfr1. Known regulators of Fgf-signalling in teleost fins, fgf20a and fgf24, are transiently expressed only during regeneration and thus not likely to be required in developing swords. CONCLUSION: Our data suggest that Fgf-signalling is involved upstream of msxC in the development of the sword and gonopodium in male swordtails. Activation of a gene regulatory network that includes fgfr1 and msxC is positively correlated with fin ray growth rates and can be re-activated in platyfish to form small sword-like fin extensions. These findings point towards a disruption between the fgfr1/msxC network and its regulation by testosterone as a likely developmental cause for sword-loss in platyfish.

Divergent functions of orthodenticle, empty spiracles and buttonhead in early head patterning of the beetle Tribolium castaneum (Coleoptera).

The head gap genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd) are required for metamerization and segment specification in Drosophila. We asked whether the function of their orthologs is conserved in the red flour beetle Tribolium castaneum which in contrast to Drosophila develops its larval head in a way typical for insects. We find that depending on dsRNA injection time, two functions of Tc-orthodenticle1 (Tc-otd1) can be identified. The early regionalization function affects all segments formed during the blastoderm stage while the later head patterning function is similar to Drosophila. In contrast, both expression and function of Tc-empty spiracles (Tc-ems) are restricted to the posterior part of the ocular and the anterior part of the antennal segment and Tc-buttonhead (Tc-btd) is not required for head cuticle formation at all. We conclude that the gap gene like roles of ems and btd are not conserved while at least the head patterning function of otd appears to be similar in fly and beetle. Hence, the ancestral mode of insect head segmentation remains to be discovered. With this work, we establish Tribolium as a model system for arthropod head development that does not suffer from the Drosophila specific problems like head involution and strongly reduced head structures.

piggyBac-based insertional mutagenesis and enhancer detection as a tool for functional insect genomics.

Transposon mutagenesis provides a fundamental tool for functional genomics. Here we present a non-species-specific, combined enhancer detection and binary expression system based on the transposable element piggyBac: For the different components of this insertional mutagenesis system, we used widely applicable transposons and distinguishable broad-range transformation markers, which should enable this system to be operational in nonmodel arthropods. In a pilot screen in Drosophila melanogaster, piggyBac mutator elements on the X chromosome were mobilized in males by a Hermes-based jumpstarter element providing piggyBac transposase activity under control of the alpha1-tubulin promoter. As primary reporters in the piggyBac mutator elements, we employed the heterologous transactivators GAL4delta or tTA. To identify larval and adult enhancer detectors, strains carrying UASp-EYFP or TRE-EYFP as secondary reporter elements were used. Tissue-specific enhancer activities were readily observed in the GAL4delta/UASp-based systems, but only rarely in the tTA/TRE system. Novel autosomal insertions were recovered with an average jumping rate of 80%. Of these novel insertions, 3.8% showed homozygous lethality, which was reversible by piggyBac excision. Insertions were found in both coding and noncoding regions of characterized genes and also in noncharacterized and non-P-targeted CG-number genes. This indicates that piggyBac will greatly facilitate the intended saturation mutagenesis in Drosophila.