Thomas Czerny

Otx2 regulates the extent, identity and fate of neuronal progenitor domains in the ventral midbrain

The specification of distinct neuronal cell-types is controlled by inducing signals whose interpretation in distinct areas along the central nervous system provides neuronal progenitors with a precise and typical expression code of transcription factors. To gain insights into this process, we investigated the role of Otx2 in the specification of identity and fate of neuronal progenitors in the ventral midbrain. To achieve this, Otx2 was inactivated by Cre recombinase under the transcriptional control of En1. Lack of Otx2 in the ventrolateral and posterior midbrain results in a dorsal expansion of Shh expression and in a dorsal and anterior rotation of the midbrain-hindbrain boundary and Fgf8 expression. Indeed, in this mutant correct positioning of the ventral site of midbrain-hindbrain boundary and Fgf8 expression are efficiently controlled by Otx1 function, thus allowing the study of the identity and fate of neuronal progenitors of the ventral midbrain in the absence of Otx2. Our results suggest that Otx2 acts in two ways: by repressing Nkx2.2 in the ventral midbrain and maintaining the Nkx6.1-expressing domain through dorsal antagonism on Shh. Failure of this control affects the identity code and fate of midbrain progenitors, which exhibit features in common with neuronal precursors of the rostral hindbrain even though the midbrain retains its regional identity and these neuronal precursors are rostral to Fgf8 expression. Dopaminergic neurons are greatly reduced in number, red nucleus precursors disappear from the ventral midbrain where a relevant number of serotonergic neurons are generated. These results indicate that Otx2 is an essential regulator of the identity, extent and fate of neuronal progenitor domains in the ventral midbrain and provide novel insights into the mechanisms by which neuronal diversity is generated in the central nervous system.

Wnt/Axin1/β-Catenin Signaling Regulates Asymmetric Nodal Activation, Elaboration, and Concordance of CNS Asymmetries

Summary Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/β-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries.

Gbx2 and Otx2 Interact with the WD40 Domain of Groucho/Tle Corepressors

ABSTRACT One of the earliest organizational decisions in the development of the vertebrate brain is the division of the neural plate into Otx2-positive anterior and Gbx2-positive posterior territories. At the junction of these two expression domains, a local signaling center is formed, known as the midbrain-hindbrain boundary (MHB). This tissue coordinates or “organizes” the development of neighboring brain structures, such as the midbrain and cerebellum. Correct positioning of the MHB is thought to depend on mutual repression involving these two homeobox genes. Using a cell culture colocalization assay and coimmunoprecipitation experiments, we show that engrailed homology region 1 (eh1)-like motifs of both transcription factors physically interact with the WD40 domain of Groucho/Tle corepressor proteins. In addition, heat shock-induced expression of wild-type and mutant Otx2 and Gbx2 in medaka embryos demonstrates that Groucho is required for the repression of Otx2 by Gbx2. On the other hand, the repressive functions of Otx2 on Gbx2 do not appear to be dependent on corepressor interaction. Interestingly, the association of Groucho with Otx2 is also required for the repression of Fgf8 in the MHB. Therefore Groucho/Tle family members appear to regulate key aspects in the MHB development of the vertebrate brain.

Improved translation efficiency of injected mRNA during early embryonic development

Injection techniques are a powerful approach to study gene function in fish and frog model systems. In particular, in vitro transcribed mRNA is broadly used for such misexpression experiments. Sequence elements flanking the coding region, such as untranslated repeats and polyadenylation sequences, are known to affect the stability and the translation efficiency of mRNA. Here we show that in early embryos, poly(A) signals strongly contribute to the activity of the injected mRNA. Of interest, they only marginally affect mRNA stability, whereas the translation efficiency is dramatically enhanced. Combination of a poly(A) tail and an SV40 late poly(A) signal leads to highly synergistic effects of the two elements for injected mRNA. Compared with established vector systems, we detected a 20‐fold improvement for mRNA derived from the novel transcription vector pMC. Developmental Dynamics 235:3370–3378, 2006.

Magnetic field-controlled gene expression in encapsulated cells

Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches.

Groucho corepressor proteins regulate otic vesicle outgrowth

The Groucho/Tle family of corepressor proteins is known to regulate multiple developmental pathways. Applying the dominant‐negative effect of the short member Aes, we demonstrate here a critical role of this gene family also for ear development. Misexpression of Aes in medaka embryos resulted in reduced size or loss of otic vesicles, whereas overexpression of the full‐length Groucho protein Tle4 gave the opposite phenotype. These results are in close agreement with phenotypes observed for eye formation, suggesting a similar role for Groucho/Tle proteins in the developmental pathways of both sensory organs. Furthermore, by using the heat‐inducible HSE promoter, we observed reversible branching of the embryonic axis upon Aes misexpression, indicating a transient duplication of the organizer. Groucho proteins, therefore, are critical for organizer maintenance. Developmental Dynamics 233:760–771, 2005

Duplicated members of the Groucho/Tle gene family in fish.

The highly conserved Groucho/Tle gene family has widespread functions during embryonic development and in adults. For mammalians, four full‐length Tle paralogues are known, whereas the whole spectrum of this gene family in fish species has not been analysed yet. Most detailed data exist for medaka, where 3 Tle genes have been described, Tle1, Tle3, and Tle4. We now isolated 3 additional Tle genes from the medaka genome. Sequence analysis identifies these genes as Tle2a, Tle2b, and Tle3b. Database searches of genomic sequences revealed an identical set of Tle paralogues being present in distantly related fish species, indicating duplicated Tle2 and Tle3 genes for the complete teleost lineage. Like the previously analysed medaka Tle genes, the three new genes show a broad expression pattern during embryogenesis. Nevertheless, a detailed comparison of all six Tle genes reveals critical differences in certain aspects of their expression pattern. In particular, we concentrated on the activity of Tle genes during ear development and found Tle2a and Tle2b expressed in this sensory organ. Developmental Dynamics 234:143–150, 2005.

Rapid identification of PAX2/5/8 direct downstream targets in the otic vesicle by combinatorial use of bioinformatics tools

BackgroundThe pax2/5/8 genes belonging to the PAX family of transcription factors are key developmental regulators that are involved in the patterning of various embryonic tissues. More particularly, their function in inner ear specification has been widely described. However, little is known about the direct downstream targets and, so far, no global approaches have been performed to identify these target genes in this particular tissue.ResultsHere we present an original bioinformatics pipeline composed of comparative genomics, database querying and text mining tools, which is designed to rapidly and specifically discover PAX2/5/8 direct downstream targets involved in inner ear development. We provide evidence supported by experimental validation in medaka fish that brain 2 (POU domain, class 3, transcription factor 2), claudin-7, secretory pathway component sec31-like and meteorin-like precursor are novel direct downstream targets of PAX2/5/8.ConclusionsThis study illustrates the power of extensive mining of public data repositories using bioinformatics methods to provide answers for a specific biological question. It furthermore demonstrates how the usage of such a combinatorial approach is advantageous for the biologist in terms of experimentation time and costs.

Identification of starmaker-like in medaka as a putative target gene of Pax2 in the otic vesicle.

Otoliths in bony fishes are involved in the function of the ear in the senses of balance and hearing. In a large‐scale random in situ hybridization screen of genes expressed in the medaka developing ear, we identified starmaker‐like (stm‐l) gene, a novel homologue of zebrafish starmaker and human dentine sialo‐phosphoprotein (dspp) gene. Despite the absence of sequence similarity between these genes, here we describe their similar genomic structure and expression patterns hinting for a conserved function. In medaka fry, stm‐l is expressed in various organs such as otoliths, teeth, gills, and kidney. Additionally, our results provide evidence that stm‐l is a putative downstream target gene of Pax2 transcription factor and Pax2 itself has a promoting function in otolith formation. Developmental Dynamics 238:2860–2866, 2009.

Diffusion of small molecules into medaka embryos improved by electroporation.

BackgroundDiffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules.ResultsContrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling.ConclusionsThe diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.