Author: screening library

Therefore, all eukaryotic cells must alter microtubule organization between the cytoplasmic array and the spindle when entering or exiting mitosis. Many studies in recent decades have identified a number of microtubule regulators. For example, the c-tubulin complex is required for microtubule nucleation and controls the distribution and polarized growth of microtubules. Also, microtubule associated proteins include motor proteins such as kinesin family proteins and dynein, factors involved in stabilization or destabilization of microtubules and factors that align or slide microtubule bundles. The functional state of certain microtubule regulators varies during the cell cycle. In animal cells, the c-tubulin complex is Schizandrin-B phosphorylated by Aurora A kinase for nucleation of microtubules to stabilize the spindle. Aurora A also phosphorylates the conserved MAP TACC to recruit the TACC to the centrosomes. Alp7, the fission yeast TACC ortholog, translocates from the cytoplasm to the nucleus upon entry into mitosis, which is critical for spindle formation. A conserved microtubule bundling factor, Ase1/PRC1, as well as Klp9 are dephosphorylated to bind to each other in anaphase, and the interaction is required for proper spindle elongation. Although there is currently vast knowledge on this subject, how each aspect of microtubule functions and regulations are linked to each other at the molecular level remains largely unknown. More specifically,Lithospermoside although much work has been done on microtubule functions and dynamics, the molecular mechanism controlling microtubule regulation at specific cell cycle transitions remain largely unknown. For instance, it is not clear how microtubules are reorganized at mitotic entry and exit. In fission yeast, the reorganization of microtubules during the cell cycle can be summarized as follows. During interphase, a cytoplasmic microtubule array forms relatively uniformly along the cylinder formed by the yeast cell. Microtubule organizing centers in interphase are thought to localize around the nucleus. Upon entry into mitosis, the cytoplasmic array of microtubules is disassembled and the mitotic spindle is formed in the nucleus. The main MTOC during mitosis is the SPB in Schizosaccharomyces pombe.

Examination of the RPCs with high G2/M gene expression was performed with the hopes of identifying novel genes involved in the process of exiting from the cell cycle. Both hierarchical and Fisher’s exact test based clustering methods yielded many significantly associated genes, but given the nature of these genes, the majority of them probably play generic roles in cytokinesis and other mitotic processes. Visual inspection of the single cell profiles in Microsoft Excel, with an extra focus on the cells receiving the highest G2/M scores,Gomisin-G revealed several interesting candidate genes that may play a role in the ability of an RPC to exit from the cell cycle. The notch ligand, delta-like 1, whose expression had previously been shown to be well correlated with the timing of retinal neurogenesis, was found to be highly expressed in several G2/M cells. Section ISH showed Dll1 staining in subsets of cells in the ONBL at all three stages and DISH performed with P0 retinas that were labeled with -thymidine for 1 hour revealed that 1/3 of Dll1+ cells were also -thymidine+. This result is consistent with Btg2 playing a role in cell cycle exit since many more retinal neurons are being generated at P0 than at E12.5. Btg2 has been shown to enhance neural differentiation upon overexpression in PC12 cells and its Gomisin-J expression in the neural tube correlates with those cells that will generate a postmitotic neuron, further suggesting a role for this gene in the control of cell cycle exit in the retina. Both Rhbdl3 and Spred1 were found in subsets of RPCs and both were in more RPC profiles at P0 than at the earlier stages. Section ISH for both genes confirmed that their expression increased as the number of retinal neurons generated increased. Neither gene has been extensively characterized in general, nor does any information exist as to the possible functions of these genes during retinal development. Drosophila homologues of Rhbdl3 have been shown to modulate both the EGF pathway and the notch pathway and, in that manner, play specific roles in the cell fate specification of neuroblasts. Alternatively, Spred1 has been shown to be a negative regulator of the ras pathway and perturbation of Spred1 function interfered with neural differentiation in tissue culture.

For instance, it is possible that when two TFs are expressed together in the same single cell they lead to a certain cellular outcome. However, these same two factors might also be expressed separately in other single cells. A combination of more sophisticated algorithms and functional studies will be necessary to fully understand the extensive heterogeneity of TF expression in developing RPCs. Neurogenic basic helix-loop-helix transcription factors have been shown to play crucial roles in the generation of many postmitotic retinal cell types. Recently the loss of one bHLH, Math5,Oleuropein was shown to lead to deficiencies in cell cycle progression in RPCs, revealing a possible additional coordinating role for this class of TF in RPCs. Understanding the mechanism of action of these bHLH factors requires a detailed knowledge of their expression patterns. In the 42 single RPC profiles, the neurogenic bHLH genes were found in subsets of cells. For example, at least three cells at three different timepoints expressed significant levels. However, these previous conclusions were based upon upregulation of these bHLHs in the absence of Math5 and not a direct observation of their co-expression. Interestingly,Imperialine-D-glucoside single cell RTPCR in the chick retina revealed that a few cells could co-express certain bHLHs. It is possible that these reporters did not fully recapitulate the entire spectrum of expression for these genes, perhaps due to differences in the regulation of transcription or translation, as has been shown for certain homeobox TFs in Xenopus. These single cell profiles demonstrate the expression of multiple neurogenic bHLHs in single RPCs and suggest that the interplay among these TFs is perhaps not as simple as previously postulated. These data provide a potential explanation for the observed redundancy of these bHLH factors in retinal development. Furthermore, Xenopus NeuroD1 has been shown to be regulated by phosphorylation and if similar regulatory mechanisms exist in the mouse, this could provide a method for independently controlling bHLHs that are coexpressed. Additionally, these TFs are part of much larger families of factors and many other family members were found in subsets of RPCs as well.

First, the RPC genes identified using the Fisher’s exact test that were used for classifying the single cells were examined. Second, hierarchical clustering was performed on the set of single cells including the 42 RPCs and the 21 developing neurons previously characterized using Gene Cluster software. Finally, genes with potentially interesting RPC expression patterns were identified by visual inspection of the microarray data in Microsoft Excel. The types of genes identified ranged from transcription factors to secreted molecules. The presence of these transcripts in many of the RPCs continued to demonstrate the robustness of the single cell profiling method since these genes have been shown to be Angoroside-C expressed in the retina by other means. Classical birthdating experiments have shown that the different retinal cell fates are produced at different times during the course of retinal development. In addition, heterochronic mixing experiments demonstrated that RPCs could only produce the temporally appropriate cell fates when placed in an environment of a different developmental stage. Given these results, it was expected that a comparison of the single cell profiles from E12.5 RPCs to those of P0 RPCs would reveal genes that were expressed primarily in either early or late RPCs. Secreted frizzled-related protein 2,Atractylenolide-I a gene previously identified in a retina SAGE screen, was in fact only observed in early RPCs and its expression was almost completely extinguished by P0. Examining gene clusters generated around Sfrp2 either by hierarchical clustering methods or by using a Fisher’s exact test did reveal some genes with correlated expressions in RPCs, but consistently failed to yield genes with a close match for the temporal expression pattern of Sfrp2. Most of the associated genes were expressed in RPCs at timepoints beyond when Sfrp2 was detected. Comparing the gene expression profiles of E12.5 RPCs and P0 RPCs by visual inspection in Microsoft Excel, however, did reveal several candidate genes whose expression appeared mainly confined to early RPCs. There were not a large number of these genes and their expression was restricted to a small subset of the profiled RPCs, unlike Sfrp2, which was more broadly expressed in early RPCs.

Media controls consistently failed to exhibit significant cDNA smears. To further evaluate the quality of the single cell cDNA mixtures, gene specific RT-PCR was performed using three genes known to be highly expressed in the developing retina. Robust bands were detected in those preparations that displayed the most robust cDNA smears and bands were routinely not observed with the media controls. One final, more comprehensive, approach was utilized to assess the single cell cDNAs. Ten micrograms of cDNA were labeled with Cy5 and hybridized to cDNA microarrays spotted in our laboratory. These microarrays contained,Ganoderenic-acid-D 12,000 ESTs derived from the retina and many retinal expressed genes from our laboratory. Many of the transcripts spotted on these cDNA microarrays showed significant signal when hybridized with cDNA from the single cells, whereas amplifications from media controls did not show signals above background. Taken together, these data demonstrated that more than 50% of isolated single retinal cells yielded cDNA of sufficient quantity and quality for more complete gene expression profiling on Affymetrix microarrays. Ten micrograms of cDNA from each single cell to be profiled was DNase treated,Ganoderic-acid-D labeled with biotinylated ddATP using TdT, and hybridized to Affymetrix mouse 430 2.0 oligonucleotide arrays using standard Affymetrix protocols. Since previous work on these genes did not assess retinal expression, it was not clear whether these signals were due to transcriptional activity of these loci in the retina, or were false positives due to the single cell method. Examination of SAGE tags for these genes showed that expression was detected for 2 of these 5 genes, suggesting that at least in these cases, there was bona fide retinal expression. To further assess the robustness of the single cell data, the levels of housekeeping genes were examined. It was not clear which genes should be used for this test, as several studies have demonstrated that housekeeping gene expression is highly variable, as assayed by microarray, SAGE, or other profiling methods. Similar variability has been observed using preparations of retinal tissue.