Linking specification to differentiation:From proneural genes to the regulation of ciliogenesis

Much of developmental biology is concerned with the processes by which cells become committed to particular fates in a regulated fashion, whereas cell biology addresses, among other things, the variety of differentiated forms and functions that cells can acquire. One open question is how the regulators of the former process lead to attainment of the latter. “High-level” regulators of cell fate specification include the proneural factors, which drive cells to commit as precursors in the sensory nervous system. Recent research has concentrated on the gene expression events downstream of proneural factor function. Here we summarize this research and describe our own research that has provided clear links between a proneural factor, atonal and the cell biological program of ciliogenesis, which is a central aspect of sensory neuron differentiation

Carboxyl-modified single-wall carbon nanotubes improve bone tissue formation in vitro and repair in an in vivo rat model.

The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH-SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH-SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH-SWCNTs' use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 μg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies

The function and regulation of the bHLH gene, cato, in Drosophila neurogenesis

Abstract Background bHLH transcription factors play many roles in neural development. cousin of atonal (cato) encodes one such factor that is expressed widely in the developing sensory nervous system of Drosophila. However, nothing definitive was known of its function owing to the lack of specific mutations. Results We characterised the expression pattern of cato in detail using newly raised antibodies and GFP reporter gene constructs. Expression is predominantly in sensory lineages that depend on the atonal and amos proneural genes. In lineages that depend on the scute proneural gene, cato is expressed later and seems to be particularly associated with the type II neurons. Consistent with this, we find evidence that cato is a direct target gene of Atonal and Amos, but not of Scute. We generated two specific mutations of cato. Mutant embryos show several defects in chordotonal sensory lineages, most notably the duplication of the sensory neuron, which appears to be caused by an extra cell division. In addition, we show that cato is required to form the single chordotonal organ that persists in atonal mutant embryos. Conclusions We conclude that although widely expressed in the developing PNS, cato is expressed and regulated very differently in different sensory lineages. Mutant phenotypes correlate with cato's major expression in the chordotonal sensory lineage. In these cells, we propose that it plays roles in sense organ precursor maintenance and/or identity, and in controlling the number of cell divisions in the neuronal branch of the lineage arising from these precursors.</p

An approximation algorithm for counting contingency tables

We present a randomized approximation algorithm for counting contingency tables , m × n non-negative integer matrices with given row sums R = ( r 1 ,…, r m ) and column sums C = ( c 1 ,…, c n ). We define smooth margins ( R , C ) in terms of the typical table and prove that for such margins the algorithm has quasi-polynomial N O (ln N ) complexity, where N = r 1 + … + r m = c 1 + … + c n . Various classes of margins are smooth, e.g., when m = O ( n ), n = O ( m ) and the ratios between the largest and the smallest row sums as well as between the largest and the smallest column sums are strictly smaller than the golden ratio (1 + ${sqrt{5}}$ )/2 ≈ 1.618. The algorithm builds on Monte Carlo integration and sampling algorithms for log-concave densities, the matrix scaling algorithm, the permanent approximation algorithm, and an integral representation for the number of contingency tables. © 2010 Wiley Periodicals, Inc. Random Struct. Alg., 2010Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77454/1/20301_ftp.pd

On the asymptotic and practical complexity of solving bivariate systems over the reals

This paper is concerned with exact real solving of well-constrained, bivariate polynomial systems. The main problem is to isolate all common real roots in rational rectangles, and to determine their intersection multiplicities. We present three algorithms and analyze their asymptotic bit complexity, obtaining a bound of \sOB(N^{14}) for the purely projection-based method, and \sOB(N^{12}) for two subresultant-based methods: this notation ignores polylogarithmic factors, where $N$ bounds the degree and the bitsize of the polynomials. The previous record bound was \sOB(N^{14}). Our main tool is signed subresultant sequences. We exploit recent advances on the complexity of univariate root isolation, and extend them to sign evaluation of bivariate polynomials over two algebraic numbers, and real root counting for polynomials over an extension field. Our algorithms apply to the problem of simultaneous inequalities; they also compute the topology of real plane algebraic curves in \sOB(N^{12}), whereas the previous bound was \sOB(N^{14}). All algorithms have been implemented in MAPLE, in conjunction with numeric filtering. We compare them against FGB/RS, system solvers from SYNAPS, and MAPLE libraries INSULATE and TOP, which compute curve topology. Our software is among the most robust, and its runtimes are comparable, or within a small constant factor, with respect to the C/C++ libraries. Key words: real solving, polynomial systems, complexity, MAPLE softwareComment: 17 pages, 4 algorithms, 1 table, and 1 figure with 2 sub-figure

Multiple enhancers contribute to spatial but not temporal complexity in the expression of the proneural gene, amos

BACKGROUND: The regulation of proneural gene expression is an important aspect of neurogenesis. In the study of the Drosophila proneural genes, scute and atonal, several themes have emerged that contribute to our understanding of the mechanism of neurogenesis. First, spatial complexity in proneural expression results from regulation by arrays of enhancer elements. Secondly, regulation of proneural gene expression occurs in distinct temporal phases, which tend to be under the control of separate enhancers. Thirdly, the later phase of proneural expression often relies on positive autoregulation. The control of these phases and the transition between them appear to be central to the mechanism of neurogenesis. We present the first investigation of the regulation of the proneural gene, amos. RESULTS: Amos protein expression has a complex pattern and shows temporally distinct phases, in common with previously characterised proneural genes. GFP reporter gene constructs were used to demonstrate that amos has an array of enhancer elements up- and downstream of the gene, which are required for different locations of amos expression. However, unlike other proneural genes, there is no evidence for separable enhancers for the different temporal phases of amos expression. Using mutant analysis and site-directed mutagenesis of potential Amos binding sites, we find no evidence for positive autoregulation as an important part of amos control during neurogenesis. CONCLUSION: For amos, as for other proneural genes, a complex expression pattern results from the sum of a number of simpler sub-patterns driven by specific enhancers. There is, however, no apparent separation of enhancers for distinct temporal phases of expression, and this correlates with a lack of positive autoregulation. For scute and atonal, both these features are thought to be important in the mechanism of neurogenesis. Despite similarities in function and expression between the Drosophila proneural genes, amos is regulated in a fundamentally different way from scute and atonal