Abstracts
Quantitative comparison of gene expression at cellular resolution in Drosophilids
Angela DePace, PhD, Harvard Medical School
Understanding how gene regulatory networks evolve requires us to measure the functional consequences of even small changes in sequence. We have applied high-resolution microscopy and image processing methods to blastoderm embryos of multiple species of Drosophila to determine the expression patterns of key transcriptional regulators of segmentation and a subset of their targets in their native context. These species span a range of evolutionary distances, and comparative sequence analysis reveals a wide variety of changes in relevant cis-regulatory elements. Our imaging techniques allow multiple types of statistically rigorous inter-species comparisons to be made, both between individual embryos and between composite multi-gene models, revealing widespread quantitative changes in expression patterns. We measure multiple types of gene-specific variation, including changes in spatial position, number of cells comprising a pattern, and the dynamics of expression. Our comparative analyses aim to put these differences in the context of complete developing embryos. Which changes are due to differences in the geometry of the embryos and which are due to genetic differences in the transcriptional networks? Furthermore, which are changes initiated by variation in the trans-network, and which are due to changes in how cis-regulatory sequences interpret that network? Differentiating these types of variation will allow us to interpret which specific sequence changes have functional consequences for gene expression, and provide insights into the molecular mechanism of gene regulation and the functional constraints under which regulatory networks evolve.
Enzyme substrate competition in a developing embryo
Stanislav Y Shvartsman, Princeton University
Enzymes can have remarkable substrate specificity. Fructokinase, for example, phosphorylates only one substrate, fructose. Protein-modifying enzymes, however, can have hundreds of substrates. Is processing of multiple substrates ordered in space and time, or are they processed concurrently and compete for a common enzyme? Ordered modification of multiple substrates by a single enzyme is essential for cell cycle control, where it can be mediated by kinetic proofreading mechanisms. Here we show that enzymes /in vivo/ can also operate in a strongly competitive regime. Based on genetic and imaging approaches, we propose that in the early /Drosophila/ embryo multiple substrates of the highly conserved Mitogen Activated Protein Kinase (MAPK) compete for this enzyme. As a consequence, the signaling activity of MAPK becomes dependent on the levels and spatial distributions of its substrates. The resulting substrate competition network integrates the actions of maternal morphogen gradients that pattern the embryo.
The master transcriptional networks controlling proliferation and differentiation in the developing brain
Antonio Iavarone, Columbia University
Self-renewal and proliferation of neural stem cells and the decision to initiate neurogenesis are crucial events directing brain development. Here we show that the ubiquitin ligase Huwe1 operates upstream of the N-Myc-DLL3-Notch pathway to control neural stem cell activity and promote neurogenesis. Conditional inactivation of the Huwe1 gene in the mouse brain caused neonatal lethality associated with disorganization of the laminar patterning of the cortex. These defects stemmed from severe impairment of neurogenesis associated with uncontrolled expansion of the neural stem cell compartment. Loss and gain of function experiments in the mouse cortex demonstrated that Huwe1 restrains proliferation and enables neuronal differentiation by suppressing the N-Myc-DLL3 cascade. Notably, human high-grade gliomas carry focal hemizygous deletions of the X-linked Huwe1 gene in association with amplification of the N-myc locus. Our results indicate that Huwe1 balances proliferation and neurogenesis in the developing brain and this pathway is subverted in malignant brain tumors.