Testing Morphogen Hypotheses: One of the most enduring concepts in the field of developmental biology is the idea that expression gradients contain multiple thresholds that control and position different cell fates. To rigorously test this idea, we are carefully measuring the spatial relationships between potentially morphogenetic gradients and the positions of their target gene expression patterns. In parallel, we use genetic experiments and transgenic technologies to precisely manipulate these gradients. In our first experiments, we have focused on the Bicoid gradient (Figure 2) and on gradients of transcription factors encoded by the gap genes hunchback, Kruppel, knirps, and giant. These experiments are focused on three simple questions: First, do gradients really function as morphogens? Second, is there a specific range of concentrations that contains the important thresholds of the morphogen? Third, how many discrete thresholds can be set by a single gradient?
In our first experiments, we have used genetic experiments to flatten the Bcd gradient, and then measured the amounts of Bcd required for activating specific target genes. Surprisingly, we have found that all tested target genes can be activated by lower concentrations than those present in the wild type gradient where boundaries for those genes are formed. These results suggest that Bcd concentrations are in excess at each position within the wild type gradient, which challenges the strict interpretation of the morphogen hypothesis. Our working hypothesis is that there are repressors that interfere with Bcd-dependent activation, and that combinations of activating and repressing proteins position and register multiple target gene expression boundaries.
Defining the Bicoid-dependent transcription network. Bicoid is a homeodomain-containing transcription factor that is expressed in a long-range anterior gradient in the early embryo (Figure 2). Loss of Bicoid function leads to a mutant embryo that lacks all head and thoracic structures. Previous studies and our recent work have identified more than 50 target genes that are directly activated by Bicoid. Activation of each target gene involves direct binding of Bicoid to one or more enhancers that appear as rather tightly linked clusters of Bicoid-binding sites. These enhancers direct expression patterns at different positions along the anterior posterior axis (Figure 3), and a major goal is to understand the cis-regulatory logic that controls the differential positioning of different target genes.
We are using an integrated approach in pursuit of this goal. First, we use bio-informatics methods and published ChIP-Chip data to identify all clusters of Bicoid-binding sites that are similar to those in the known target genes. Candidate clusters are cloned into reporter genes, transformed into the genome, and tested for in vivo activity by in situ hybridization experiments.
While collecting an ever-growing number of Bcd-dependent elements, we are using data mining techniques to identify sequence motifs or binding site arrangements that correlate with target gene positioning. Among the most over-represented sites in a specific group of enhancers appears to be a binding site for the Run transcription factor, which is expressed in a gradient that spatially opposes the Bcd gradient. We are currently testing this hypothesis using genetic, biochemical, and transgenic assays.
Defining Functions of Individual Bicoid Target Genes. How individual Bicoid target genes function in anterior patterning has been impossible to address because removal of Bicoid function abolishes the expression of all target genes. However, in recent years, we have developed a transgenic system for expressing an anterior gradient of any protein in embryos lacking Bicoid altogether. This approach is being used to systematically assay the activities of the known target genes. Our results suggest that some genes are involved primarily in repressing posterior organizing genes in the presumptive head regions, while others are involved in transducing the specific instructive activities that lead to the formation of individual anterior segments. These studies are particular interesting in light of the fact that the bicoid gene is not well conserved, even among insects.
The Nitty-Gritty of Enhancer-mediated Transcriptional Mechanisms: In our previous work, we have defined a detailed model for the regulation of a single stripe of expression in the early embryo (even-skipped (eve) stripe 2). The basic strategy is to use DNA-binding experiments and evolutionary conservation to define all sequence motifs in an individual enhancer. These motifs are tested by in vitro mutagenesis in the context of reporter genes. Transacting factors that bind to novel motifs are identified using yeast one-hybrid screens.
We are currently focused on a 500 bp enhancer element that forms two stripes (eve 3 and eve 7) in the early embryo (Figure 4). So far we have identified more than 25 binding sites in this enhancer. By manipulating these sequences and the spacing between them in detail, we hope to further augment our understanding of how these DNA sequences integrate the combined effects of different combinations of regulatory factors.
These projects are currently funded by grants from the NIH and NYU’s Research Challenge Fund.