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Research Description:
Developmental control of Organogenesis in C. elegans Embryos
How are cells specified and assembled into an organ? Organ formation depends on the coordinate development of multiple cell types to produce an integrated structure such as a heart, a kidney, or an eye. Our goal is to identify the genes that regulate organ development and understand how they function. We have chosen the nematode Caenorhabditis elegans for this purpose because this animal is easily manipulated by genetic and molecular techniques. In addition, sequencing of the genome sequence is virtually complete.
Our focus is the worm pharynx, a muscular organ used for feeding. It is composed of a bilobed tube that siphons food (bacteria), grinds it up and passes it on to the midgut. Morphologically, pharynx formation resembles organ development in more complex animals. However, the worm pharynx is much simpler than most organs. For example, it is composed of only eighty cells and seven cell types. This simplicity enables us to study the process of pharyngeal development at the resolution of single cells.
One key regulator of pharynx development is pha-4, a FoxA transcription factor homolog that establishes pharyngeal identity during early embryogenesis (Horner et al.). In the absence of pha-4, pharyngeal precursor cells are transformed into ectodermal cells. Conversely, ectopic pha-4 is sufficient to redirect other cell types to become pharyngeal cells. How does pha-4 perform this function? To answer this question we need to understand how the pha-4 protein works - something that is not well understood for any FoxA homolog in any organism. We have undertaken three approaches to figure out the role of PHA-4 during pharynx development. 1. First, we identified direct targets of PHA-4 using a microarray approach (Gaudet and Mango). We used microarray to identify 338 genes selectively expressed in the developing pharynx. Surprisingly, we found that PHA-4 directly activates most or all of these pharyngeal genes directly. Furthermore, the relative affinity of PHA-4 for its binding site (TRTTKRY with R = A/G, K = T/G, Y = T/C) modulates the onset of gene expression, with high affinity sites associated with earlier expression than lower affinity sites. This global role in pharyngeal gene expression provides a model for how pha-4 specifies organ identity and offers a mechanism to coordinate the expression of hundreds of pharyngeal genes. Second, we undertook a genetic screen for suppressors of pha-4 loss-of-function mutations and identified potential negative regulators of PHA-4. We have also discovered a novel protein, CHW-1, that interacts with PHA-4 in a yeast two hybrid screen. Preliminary data suggest a genetic interaction between pha-4 and chw-1 loss of function. These studies may reveal the different roles of pha-4 in different tissues. Third, we are developing assays to visualize PHA-4 protein bound to target DNA in vivo. The goal is to understand the relationship between PHA-4 binding and target gene expression in living embryos.
In addition to our studies focused on pha-4, we are also working towards dissecting the complex regulatory network that governs pharyngeal development. We are using a computational approach to discover new transcriptional regulators that govern cell type or temporal gene expression within pharyngeal cells. We are also conducting an RNAi screen with our microarrray positives to identify those genes important for cell fate decisions or morphogenesis during pharynx development.
Pharyngeal morphogenesis
During wildtype development, a ball of pharyngeal precursors is converted into a linear tube connected to the buccal cavity anteriorly and the midgut posteriorly. We call this process pharyngeal extension. We have used time-lapse videomicroscopy and GFP reporter constructs to follow the behavior of the pharyngeal precursors during pharyngeal extension (Portereiko and Mango). Our studies demonstrate that pharyngeal extension can be loosely divided into three stages. i) reorganization of cellular polarity within anterior pharyngeal cells, which opens the pharyngeal lumen towards the exterior, ii) formation of an epithelium that mechanically couples the pharynx with the nascent buccal cavity, and iii) an apparent contraction that pulls the pharynx anteriorly and the buccal cavity posteriorly, similar to a 'purse-string' mechanism. Our findings suggest that pharyngeal extension occurs by 'pulling' rather than 'pushing' and that cells within the pharynx and buccal cavity are under tension during the third stage. We have confirmed these predictions by destroying different populations of pharyngeal or buccal cavity cells using a laser microbeam.
To identify the molecules that regulate pharyngeal morphogenesis, we conducted a genetic screen for worms that die because the pharynx is not attached to the mouth. We identified 14 mutants that we are beginning to characterize in depth. One of these mutations is allelic to the MKLP homologue zen-4. zen-4 plays a role in bundling midzone microtubules during cytokinesis, but had not been implicated in morphogenesis before. Our findings suggest a new role for MKLP and microtubules to polarize the cell during epithelium formation.
Epigenetic silencing
Double-stranded RNA (dsRNA) injected into C. elegans inhibits expression of genes homologous to the injected RNA. This phenomenon, called RNA interference, may be related to homology-dependent gene silencing observed in other organisms including vertebrates. In a third project unrelated to organogenesis, we are analyzing what happens during RNAi and what genes are required for this process. We have shown that genes previously implicated in nonsense-mediated decay are also required for persistence of RNAi (the smg genes; Domeier et al.). Therefore, these two RNA degradation pathways may use some of the same factors to recognize and degrade their targets. We have also discovered that dsRNA is amplified during RNAi, to propagate silencing during the lifetime of the worm. Our current goal is to understand the role of the smg factors during RNAi and in particular, during amplification.
Research Keywords:
organogenesis, embryogenesis, genomics, genetics, development, transcription, morphogenesis