A cis-element in the Notch1 locus is involved in the regulation of gene expression in interneuron progenitors.

Interneurons comprise approximately one third of the total cortical neurons in the mammalian cerebral cortex. Studies have revealed many details in the generation of this cell type. However, the mechanism that defines interneuron-lineage specific gene expression is not well understood. Gene regulatory elements, e.g., promoters, enhancers, and trans-acting factors, are essential for the proper control of gene expression. Here, we report that a novel evolutionarily conserved cis-element in the second intron of the Notch1 locus plays an important role in regulating gene expression in interneuron progenitors. The spatiotemporal activity of the cis-element in the developing central nervous system (CNS) was determined by both transient reporter expression in the developing chick and a transgenic mouse model. Its activity is well correlated with neurogenesis in both the chick and mouse and restricted to neural progenitor cells in the ganglionic eminence that are fated to differentiate into GABAergic interneurons of the neocortex. We further demonstrate that the cis-element activity requires the binding motif for trans-acting factors Gsh1/Barx2/Brn3. Deletion of this binding motif abolishes reporter gene expression. Together, these data provide new insights into the regulatory mechanisms of interneuron development in the vertebrate CNS.

Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.

BACKGROUND: Retinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete. RESULTS: This study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP) as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3) or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcalpha, NeuN, Pax6, Brn3a, Vimentin, etc.) allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types. CONCLUSION: The new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more complete information on retinal cell development, and they can serve as a reference for the investigations in normal retinal development and diseases.

Non-coding sequence retrieval system for comparative genomic analysis of gene regulatory elements.

BACKGROUND: Completion of the human genome sequence along with other species allows for greater understanding of the biochemical mechanisms and processes that govern healthy as well as diseased states. The large size of the genome sequences has made them difficult to study using traditional methods. There are many studies focusing on the protein coding sequences, however, not much is known about the function of non-coding regions of the genome. It has been demonstrated that parts of the non-coding region play a critical role as gene regulatory elements. Enhancers that regulate transcription processes have been found in intergenic regions. Furthermore, it is observed that regulatory elements found in non-coding regions are highly conserved across different species. However, the analysis of these regulatory elements is not as straightforward as it may first seem. The development of a centralized resource that allows for the quick and easy retrieval of non-coding sequences from multiple species and is capable of handling multi-gene queries is critical for the analysis of non-coding sequences. Here we describe the development of a web-based non-coding sequence retrieval system. RESULTS: This paper presents a Non-Coding Sequences Retrieval System (NCSRS). The NCSRS is a web-based bioinformatics tool that performs fast and convenient retrieval of non-coding and coding sequences from multiple species related to a specific gene or set of genes. This tool has compiled resources from multiple sources into one easy to use and convenient web based interface. With no software installation necessary, the user needs only internet access to use this tool. CONCLUSION: The unique features of this tool will be very helpful for those studying gene regulatory elements that exist in non-coding regions. The web based application can be accessed on the internet at: http://cell.rutgers.edu/ncsrs/.

In ovo electroporation in embryonic chick retina.

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons(1), which has been difficult with the conventional method of injection and electroporation at E1.5(2).

Epigenetic modifiers are necessary but not sufficient for reprogramming non-myelinating cells into myelin gene-expressing cells.

BACKGROUND: Modifications on specific histone residues and DNA methylation play an essential role in lineage choice and cellular reprogramming. We have previously shown that histone modifications or combinatorial codes of transcription factors (TFs) are critical for the differentiation of multipotential progenitors into myelinating oligodendrocytes. In this study we asked whether combining global manipulation of DNA methylation and histone acetylation together with the expression of oligodendrocyte-specific TFs, was sufficient to switch the identity of fibroblasts into myelin gene-expressing cells. METHODOLOGY/PRINCIPAL FINDINGS: Transfection of six oligodendrocyte-specific TFs (Olig1, Olig2, Sox10, Mash1, E47 and Nkx2.2) into NIH3T3 fibroblasts was capable of inducing expression of myelin gene promoter-driven reporters, but did not activate endogenous myelin gene expression. These results suggested the existence of a transcriptionally incompetent chromatin conformation in NIH3T3 fibroblasts. Using chromatin immunoprecipitation (ChIP) analysis, we compared the histone code on the conserved regions of myelin genes (i.e. Mbp and Mag) in differentiating oligodendrocyte progenitors and NIH3T3 fibroblasts. Chromatin at myelin gene loci was characterized by the presence of repressive histone modifications (me3K9H3 and me3K27H3) in NIH3T3 fibroblasts and active histone marks (me3K4H3 and AcH3) in oligodendrocyte lineage cells. To induce a transcriptionally competent chromatin signature, NIH3T3 fibroblasts were treated with 5-azadeoxy-citidine (5-AzaC) to decrease DNA methylation, and trichostatin A (TSA) or sirtinol, to favor histone acetylation. Treatment with 5-AzaC/TSA but not sirtinol, resulted in the detection of endogenous myelin gene transcripts in fibroblasts, although not to the levels detected in myelinating cells. Transfection of oligodendrocyte-specific TFs after 5-AzaC/TSA treatment did not further increase myelin gene expression, nor did it reprogram the transcriptional network of NIH3T3 fibroblasts into that of oligodendrocytes. CONCLUSIONS/SIGNIFICANCE: These results suggest that reprogramming of fibroblasts into myelin gene-expressing cells not only requires transcriptional activation, but also chromatin manipulations that go beyond histone acetylation and DNA methylation.