The main focus of our lab is the study of Wnt/Frizzled-planar cell polarity (PCP) signaling and associated regulatory and signaling specificity mechanisms. To achieve this we are using a combination of Drosophila in vivo studies, cell culture experiments, and biochemistry and through collaborations we confirm our results in vertebrate models like zebrafish or mouse.
Establishment of planar cell polarity
Epithelial cells, in the epidermis as well as in neural epithelia - and cell in organs in a general context, are polarized in two axes. Epithelial apical-basolateral polarity enables tissues to perform functions like the vectorial transport of fluid or the directed secretion of specialized components. In addition to this ubiquitous axis of polarization, most epithelial tissues acquire a second polarity axis within the plane of the epithelium, commonly referred to as planar cell polarity (PCP). Thus, most cells are polarized with respect to the body axes. This is not only the case in Drosophila (Fig. 1) and other insects but is a widespread feature of epithelia in both invertebrates and vertebrates. In vertebrates for example, tissues requiring PCP signaling include skin development and body hair orientation, polarization of the sensory epithelium in the inner ear, and the directed movement of mesenchymal cell populations during gastrulation (e.g. Fig. 1).
Figure 1. PCP and the organization of several tissues in a variety of systems.
(a-d) Proximal-distal orientation of hairs on appendages in Drosophila and mouse. Drosophila wing cells generate an actin hair that points distally in wild-type (a) or randomly in PCP mutants (b). The pattern of mammalian fur/hairs is also regulated by Fz/PCP signaling: wild-tyep (c) and mfz6- (d; kind gift from J. Nathans). (e-h) PCP aspects of sensory cell orientation. Drosophila eye: the ommatidia, or facets, are composed of photoreceptors, which are arranged in precise trapezoid pattern (e, wild-type). In PCP mutants (f), the arrangement of the photoreceptors and each ommatidium become disorganized. (g, h) Individual sensory hair cells of the mammalian (mouse) choclea generate polarized bundles of actin-based stereocilia (g, wild-type). In Fz/PCP mutants these bundles still form but their orientation becomes randomized (h; gift from M. Kelley). (i-l) PCP effects on convergent extension in vertebrate gastrulation and neurulation (examples from zebrafish): PCP mutants fail to extend their A-P axis properly as cells do not intercalate medially in a coordinated manner, leading to a shorter and broad phenotype; lateral view (i,j) and dorsal view (k,l) of zebrafish embryos.
The Drosophila PCP genes are required for correct establishment of tissue polarity in all tissues analyzed (both neuronal and non-neuronal). Our efforts to understand and molecularly dissect the mechanisms of PCP formation have focused on the wing and the compound eye. In the wing, PCP is reflected in the choice of the site at which an actin-based hair out-growth initiates in each cell and the direction the hair points (Fig. 1). In the eye, PCP is reflected in the mirror-symmetric arrangement of ommatidia relative to the dorso-ventral midline. Mutations in PCP genes result in the loss of wing hair polarity and ommatidial orientation in the eye (Fig. 1). A key feature of PCP signaling is the resulting asymmetric PCP protein localization. For example in the wing in the proximo-distal axis and in the eye in the dorso-ventral axis (Fig. 2). Recent findings indicate that the underlying signaling pathway(s) and molecular features are conserved throughout evolution and regulate related developmental aspects of coordinated cellular polarization in vertebrates. Figure. 2. Core PCP protein localization in
Our lab focuses in particular on the molecular understanding of the regulation of Wnt/Fz signaling pathway(s) that are key regulators of the process (Fig. 3a) and its interactions with other signaling pathways involved, for example Notch signaling in the fly eye (Fig. 3b) or Fat/Dachsous signaling in general PCP establishment.
Drosophila wing and eye cells. (A,B) PCP proteins are asymmetrically localized to proximal and distal cell membranes in pupal wing. Orientation of the gradient indicates the presumed Fz activity. (A) Schematic of localization of PCP proteins; hexagons represent single cells. (B) Confocal image with distal localization of Diego (green), as an example. White outlines: cells not expressing GFP-Diego. (C,D) Eye disc: core PCP proteins are asymmetrically localized. (C) Schematic of R3/R4 cells (colors as in A; purple: colocalization of all factors). (D,D’) Confocal image of eye disc demonstrating localization of Diego (green). White outlines: R3 (bottom) and R4 (top).
Figure 3. The Frizzled/PCP pathway.
(A) Simplified schematic view of the Fz/PCP signaling cascade. The nuclear signaling leading to transcriptional activation is observed in a subset of tissues. Several Rho GTPase family members and JNK/p38 MAPK components act largely redundantly. The membrane proximal components are color coded as in Fig. 2. The Stbm/Vang-Pk complex (blue) antagonizes Fz-Dsh-Dgo (red) signaling. Fmi (purple) stabilizes both complexes. (B) PCP Signaling logic in R3/R4 specification in the Drosophila eye. Fz/Dsh-PCP signaling specifies R3 and induces Delta expression in R3, which in turn activates Notch signaling in R4. Stbm/PK antagonize Dsh in R4 to ensure restricted R3 specification.
Wnt/Frizzled signaling specificity
A second focus of the lab is to understand how signaling specificity is achieved between the canonical Wnt/Fz-beta-Catenin pathway and Fz/PCP signaling (Fig. 4). The two pathways bifurcate at the level of Dishevelled (Dsh) and our efforts are currently concentrated on dissecting the pathway specific modifications of Dsh and the interaction(s) of Dsh with the Fz receptor complexes. Pathway specific Dsh membrane recruitment and Dsh phosphorylation are being studied in detail, both through functional genomics in cell culture assays, biochemical dissection, and in vivo studies. We have identified several genes that help in switching from on pathway to the other and that affect Dsh phosphorylation in distinct ways.
Figure 4. The two main Wnt-Frizzled signaling pathways.
(A) Simplified version of the canonical Wnt/Beta-catenin pathway is shown for comparison with the Fz/PCP cascade (B). The two pathways bifurcate downstream of Dsh. The mechanisms of specific pathway selection are not understood.
Marek Mlodzik, PhD
Professor and Chair
Tel: 212-241-6516 (office), 212-241-7506 (lab), 212-241-6540 (lab)