UC Berkeley

The size of an animal is determined by the growth, proliferation, and survival of every cell in its body. Individual cells must respond to local cues and properly adjust their growth behavior to collectively generate tissues and organs of the proper size. How cells regulate their growth, and how they coordinate to regulate the size of the organs, are two fundamental questions which have long captivated developmental biologists, and which remain incompletely understood.

Much of what is already known about growth regulation has been learned from the study of mutants. Mutations affecting multiple signaling pathways can lead to overgrowth or undergrowth. However, the study of genetic mosaics has shown that outcome of growth-disrupting mutations depends not only on the properties of affected cells, but also on the interactions those cells have with their neighbors. For example, cells heterozygous for a class of mutations called Minute are slow growing but viable when all cells carry the mutation, but are eliminated in the presence of wild type neighbors. This phenomenon, termed cell competition, highlights how cells can non-autonomously influence growth and survival within a mosaic tissue.

I conducted a genetic screen in Drosophila to identify new genes which regulate growth in mosaics, focusing specifically on cell adhesion genes. The screen identified 9 genes which, when knocked down in clones in the wing imaginal disc, alter the size, shape, or number of clones which are recovered later in development. Of particular interest was the gene echinoid (ed): clones of cells lacking ed are small, round, and underrepresented, but ed mutations affecting large populations of cells can cause tissue overgrowth. My dissertation work is aimed at understanding the mechanistic basis of these seemingly contradictory phenotypes and to clarify the role of ed in regulating cellular and tissue growth. I demonstrate that cells lacking ed die by apoptosis at increased rates, at least in part because they express lower levels of the anti-apoptotic protein Diap1. This contributes to the elimination of ed-deficient clones in mosaic tissues. I also confirm that organs which are mostly or entirely deficient of ed overgrow because of a failure to terminate growth when the organ has reached its appropriate final size.

Ed has been previously shown to have a function in restricting growth via its interactions with the Hippo pathway, which regulates the expression of pro-growth and anti-apoptotic genes downstream of the transcription factor Yorkie (Yki). I show that this prevailing model cannot account for many of the phenotypes observed in ed-depleted tissues. Contrary to other reports, I found that many—but not all—Yki target genes are expressed at lower levels in ed-depleted cells. In mosaics, many of these same Yki targets are expressed at higher levels in the wild-type neighbors of ed clones. These observations are consistent with clonal elimination but inconsistent ed having a simple, growth-inhibitory effect via the Hippo pathway.

To understand why ed mutant organs overgrow, I screened for dominant modifiers of the overgrowth phenotype caused by Gal4-driven knockdown of ed in the wing. The top hit in this screen was upd2Δ, which suppressed overgrowth and enhanced cell death. While characterizing these modification phenotypes, I determined that they were ed-independent artifacts of a UAS construct present in the upd2Δ allele which interacts with Gal4. Although ultimately uninformative to the overgrowth phenotype associated with ed, this line of inquiry uncovered important information about the properties of the upd2Δ allele which may be of interest to researchers who use this allele, or other alleles generated in a similar manner.

Overall, this work advances our understanding of how growth is regulated by echinoid, highlights the context-dependence of ed’s effects on growth, and paints a more complicated picture of how ed interacts with the Hippo pathway than previously appreciated.