Cell-signaling, pattern formation, growth control, developmental glycobiology
Control of Tissue Patterning and Growth During Development
Understanding how growth is controlled is a major goal of developmental biology.
Decades ago, regeneration experiments revealed an intimate relationship between
organ patterning and organ growth, but the molecular basis for this relationship
has remained elusive. We are engaged in projects whose long-term goal is to
define relationships between patterning and growth in developing and
regenerating organs. Much of our research takes advantage of the powerful
genetic, molecular, and cellular techniques available in Drosophila
melanogaster, which facilitate both gene discovery and the analysis of gene
function. Our current research focusses on a novel signaling pathway, the
Fat-Hippo pathway, which play important roles in growth control from
Drosophila to humans. We study both the molecular mechanism of Fat-Hippo
signaling, and its developmental functions.
Mechanism of Fat-Hippo Signaling
The Fat signaling pathway is named for a Drosophila gene, fat, which
encodes a cadherin protein that acts as a transmembrane receptor for this
pathway. Fat signaling influences both gene expression and planar cell polarity
(PCP). Several years ago, we discovered that the influence of Fat signaling on
gene expression is effected through an intersection with the Hippo-Warts
signaling pathway. Several components of this pathway act as tumor suppressors
or oncogenes, both in Drosophila and in mammals. Our studies have analyzed
multiple steps of Fat signaling, from regulation of the Fat receptor at the
membrane, to the intersection with Warts-Hippo signaling, to the regulation of
gene expression through the transcriptional coactivator Yorkie.
Genetic studies in Drosophila identified the four-jointed gene (fj) as a
regulator of Fat signaling. More recently, we found that fj encodes a novel
protein kinase that phosphorylates specific Ser or Thr residues within
cadherin domains of Fat and its transmembrane ligand, Dachsous (Ds). Fj
functions in the Golgi to phosphorylate Fat and Ds, and was the first
molecularly-identified Golgi kinase. Modification of Fat by Fj promotes
Fat-Ds binding, whereas modification of Ds by Fj inhibits Fat-Ds binding. As
a consequence of these opposing effects, juxtaposition of cells differing in
their levels Fj results in asymmetric Fat-Ds binding, which we think helps
to polarize cells.
We have also investigated the nature of Fat receptor activation. discs
overgrown (dco) encodes Drosophila CKI, which we placed into the Fat-Hippo
pathway through genetic experiments. More recently, we determined that the
cytoplasmic domain of Fat is phosphorylated by Dco. As this Dco-mediated Fat
phosphorylation is promoted by Ds, it provides the first biochemical marker
of Fat receptor activation. Our evaluation of dco mutants indicated that
they affect Fat’s influence on growth and gene expression, but not its
influence on PCP, which implies that Fat is activated in distinct ways for
Hippo versus PCP signaling.
The dachs gene occupies a central position within the Fat signaling pathway,
as dachs influences both the transcriptional and planar cell polarity
outputs of Fat signaling. dachs encodes a myosin-related protein, and Fat
controls the subcellular localization of Dachs protein: absence of Fat
allows Dachs to accumulate on the membrane, whereas active Fat forces Dachs
off the membrane. These observations imply that Fat signaling is transmitted
through its influence on Dachs localization.
Yorkie, a transcription factor of the Fat and Hippo signaling pathways, is
negatively regulated by the Warts kinase. Characterization of
Warts-dependent phosphorylation of Yorkie in vivo revealed that Warts
promotes phosphorylation of Yorkie at multiple sites, and we characterized
their contributions to Yorkie regulation. Investigations of Yorkie that
couldn't be phosphorylated on these sites led us and others to characterize
another type of Yorkie regulation, in which Warts or Expanded can repress
Yorkie by directly binding to it.
In ongoing experiments, we are continuing to investigate multiple steps in
Fat-Hippo signaling using a combination of genetic, histological and
biochemical approaches. In parallel with this, we are pursuing
investigations of how the pathway is used and regulated in different
developmental contexts.
Developmental roles of Fat-Hippo Signaling
Many signaling pathways are regulated by ligands expressed in gradients.
However, conventionally, a ligand gradient is interpreted according to the
intensity of the signal, ie higher levels elicit a stronger signal, and
lower levels elicit a weaker signal. One of the remarkable features of Fat
signaling is that rather than receptor activity being governed simply by the
amount of ligand, Fat signaling can be influenced by the vector and slope of
the Ds and Fj gradients, with the vector influencing PCP, and the slope
influencing transcription. The influence of the Fj and Ds gradients on
Fat-Hippo signaling was established through experiments in which we
demonstrated that juxtaposition of cells that express different levels of fj
or ds stimulates expression of Fat/Hippo pathway target genes and cell
proliferation, whereas uniform expression of fj and ds in the wing inhibits
cell proliferation. We also linked the graded expression of fj and ds to the
Dpp morphogen gradient, and showed that the localization and activity of Fat
pathway components, and Fat signaling through Dachs, is required for the
influence of the Dpp gradient on cell proliferation. We think the ability of
cells to respond to the Fj and Ds gradients can be explained by their
ability to polarize Fat activity within cells. Dachs protein is normally
asymmetrically localized in the developing wing, and manipulations of Ds and
Fj expression revealed that this asymmetry is directed by the Ds and Fj
gradients.
Studies of Fat-Hippo signaling in Drosophila initially focussed on
imaginal discs. More recently, we have investigated roles of the pathway in
other organs. Neuroepithelial cells are neural progenitor cells that
function during early neural development as symmetrically dividing neural
stem cells. Fat-Hippo signaling controls both the proliferation and the
differentiation of optic neuroepithelial cells. Analysis of the
differentiation requirement led us to discover that they need to undergo a
cell cycle pause to transition from neuroepithelial cells into neuroblasts.
Activation of Yorkie impedes this cell cycle pause. The cell cycle pause
appears to be needed because it influences regulation of Notch signaling.
Our studies identified a mechanism through which the action of multiple
signaling pathways is coordinated during neuronal differentiation by their
relation to the cell cycle.
The adult midgut has emerged as a Drosophila model for analysis of somatic
stem cells. Midgut intestinal stem cells (ISCs) maintain homeostasis, and if
the midgut is damaged, then ISC proliferation increases. We have found that
this increase is mediated by the Hippo pathway, but in a non-autonomous
fashion. Yorkie is activated in differentiated cells in response to tissue
damage. Activation of Yorkie then promotes expression of cytokines, which
increase proliferation of nearby ISCs through the Jak-Stat pathway. We are
also exploring the role and regulation of the pathway in regeneration and
response to tissue damage in other organs.
Homologues of many genes in Fat and Hippo signaling are conserved in
mammals, but it was not clear whether mammals have a Fat signaling pathway
equivalent to that in Drosophila, nor what its roles were. To investigate
this, we created a mutation in a murine ds homologue, Dchs1, and have
characterized it, together with mutations in a murine fat homologue, Fat4
Our analysis thus far has identified novel requirements for Dchs1-Fat4
signaling in multiple organs.
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