Chapter Seven - Frizzled Receptors in Development and Disease

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Abstract

Frizzled proteins are the principal receptors for the Wnt family of ligands. They mediate canonical Wnt signaling together with Lrp5 and Lrp6 coreceptors. In conjunction with Celsr, Vangl, and a small number of additional membrane and membrane-associated proteins, they also play a central role in tissue polarity/planar cell polarity (PCP) signaling. Targeted mutations in 9 of the 10 mammalian Frizzled genes have revealed their roles in an extraordinarily diverse set of developmental and homeostatic processes, including morphogenetic movements responsible for palate, ventricular septum, ocular furrow, and neural tube closure; survival of thalamic neurons; bone formation; central nervous system (CNS) angiogenesis and blood–brain barrier formation and maintenance; and a wide variety of processes that orient subcellular, cellular, and multicellular structures relative to the body axes. The last group likely reflects the mammalian equivalent of tissue polarity/PCP signaling, as defined in Drosophila, and it includes CNS axon guidance, hair follicle and tongue papilla orientation, and inner ear sensory hair bundle orientation. Frizzled receptors are ubiquitous among multicellular animals and, with other signaling molecules, they very likely evolved to permit the development of the complex tissue architectures that provide multicellular animals with their enormous selective advantage.

Introduction

Frizzled proteins are the principal receptors for the Wnt family of signaling molecules, and they are found throughout the animal kingdom, including in the most primitive metazoa, but they are not present in plants or in simpler (single cell) eukaryotes, such as yeast (Schenkelaars, Fierro-Constain, Renard, Hill, & Borchiellini, 2015). Frizzled proteins share a common architecture: a conserved extracellular cysteine-rich domain (CRD) is followed by a domain with seven presumptive transmembrane segments. The genetic dissection of Frizzled function began in Drosophila, with the characterization of the frizzled (fz) phenotype by Gubb and Garcia-Bellido (1982). Loss-of-function mutations in fz lead to a tissue polarity phenotype, defined as a misorientation of surface structures, such as cuticular bristles and wing hairs, which normally exhibit a precise orientation relative to the body axes. This pathway is generally referred to by the not entirely accurate designation “planar cell polarity” (PCP).

The Drosophila fz gene was isolated by Vinson, Conover, and Adler (1989), and the first two vertebrate Frizzled homologues were identified several years later (Chan et al., 1992). A search for additional vertebrate Frizzled family members using degenerate PCR identified eight family members in mammals, as well as multiple family members in birds and fish (Wang et al., 1996). Two additional mammalian Frizzled genes were identified subsequently, bringing the total to 10 (Koike et al., 1999, Wang et al., 1997). The large number of vertebrate Frizzled genes together with their receptor-like structure led Wang et al. (1996) to suggest that “If Frizzled proteins act as receptors, that would imply the existence of a corresponding family of ligands. … Currently the only family of ligands known to be of this size and for which no receptors have been identified are the Wnt proteins. The availability of a large number of Frizzled genes should facilitate a biochemical test of the possibility that these two families of proteins interact directly.” Within a year, that suggestion was confirmed with the demonstration that Drosophila Wingless binds to and promotes beta-catenin stabilization via Frizzled2 (Bhanot et al., 1996). These experiments also identified the Frizzled CRD as the site of Wnt binding (Bhanot et al., 1996, Hsieh et al., 1999). The recently determined structure of a Wnt–Frizzled CRD complex shows an unusual ligand–receptor interaction characterized by a relatively small protein–protein interface and a relatively large interface between the CRD and a lipid that is covalently attached to the Wnt (Janda, Waghray, Levin, Thomas, & Garcia, 2012).

The identification of Frizzleds as Wnt receptors implied that Frizzleds act in at least two distinct signaling pathways: tissue polarity/PCP signaling (which may or may not involve a Wnt ligand) and canonical Wnt signaling. The former pathway appears to involve reorganization of the cytoskeleton and is characterized by a small set of asymmetrically localized plasma membrane proteins (including Frizzled) and membrane-associated cytosolic proteins (Goodrich & Strutt, 2011). The latter pathway involves Wnt–Frizzled-dependent disinhibition of a single-pass transmembrane coreceptor (Lrp5/Lrp6 in mammals; arrow in Drosophila) leading to inhibition of beta-catenin phosphorylation and proteolysis, with a resultant migration of beta-catenin into the nucleus to regulate transcription in combination with Lef/Tcf transcription factors (MacDonald et al., 2009, Nusse, 2012). A third pathway, the Wnt-calcium pathway, is less well defined and has been suggested to involve a G-protein-based transduction system (Wang & Malbon, 2003).

In this review, we focus on the roles of mammalian Frizzled genes in vivo, which have been largely defined by studying the effects of targeted mutations in mice. As seen in Fig. 1, a comparison of the amino acid sequences and intron–exon structures among the 10 mammalian Frizzled family members reveals branches consisting of Fz1, Fz2, and Fz7 (subfamily 1); Fz5 and Fz8 (subfamily 2); Fz9 and Fz10 (subfamily 3); Fz4 (subfamily 4); and Fz3 and Fz6 (subfamily 5, the most distant branch). Substantial genetic redundancy has been observed between pairs of Frizzled genes within subfamilies 1, 2, and 5, but, thus far, there appears to be far less redundancy between Frizzled genes in different subfamilies. However, these data should be interpreted cautiously because the redundancy data are still incomplete: many of the 45 possible pairwise combinations of Frizzled gene knockouts—especially with Frizzleds from different subfamilies—have not been tested. Moreover, in many tissues, there is overlapping expression of multiple Frizzleds, as seen, for example, in the spiral ganglion in the inner ear (Shah, Kang, Christensen, Feng, & Kollmar, 2009), so that functional redundancy may involve more than two genes. The patterns of sequence relatedness also correlate with function: Fz3 and Fz6 appear to be devoted largely or exclusively to tissue polarity signaling, whereas Fz4 is devoted largely or exclusively to canonical Wnt signaling. Some mammalian Frizzleds could signal via more than one pathway, as shown for Drosophila Frizzled, which signals through both the canonical Wnt and PCP pathways (Bhanot et al., 1999, Bhat, 1998, Muller et al., 1999).

In the text that follows, we have organized the descriptions of Frizzled gene function by subfamily. We note that most of the phenotypic analyses in mice were performed on a mixed genetic background.

Section snippets

Frizzled1, Frizzled2, and Frizzled7

Fz1−/− mice show no apparent phenotype, whereas loss of Fz2 leads to cleft palate and neonatal lethality in ~ 50% of mice and runting associated with reduced olfactory function in the other ~ 50% (Yu et al., 2010). Approximately 15% of Fz7−/− fetuses have a ventricular septal defect (VSD), a common cardiac anomaly that results in mixing of blood between the left and the right ventricular chambers, while the remaining ~ 85% of Fz7−/− mice have no apparent phenotype except for a kinked tail (Yu, Ye,

Frizzled5 and Frizzled8

Homozygous deletion of Fz5 leads to embryonic lethality at midgestation secondary to placental insufficiency (Ishikawa et al., 2001). When the placental phenotype is bypassed by conditionally ablating Fz5 in embryonic but not extraembryonic tissues (Fz5CKO/CKO;Sox2-Cre), the resulting mice survive to adulthood and exhibit interesting phenotypes in the eye and the thalamus (CKO, conditional knockout). The ocular phenotype is ~ 50% penetrant and is characterized by increased cell death in the

Frizzled9 and Frizzled10

Fz9 has long intrigued human geneticists because it is one of ~ 20 genes in the chromosome 7 region that is present in a hemizygous state in Williams–Beuren syndrome, a complex multisystem disorder (OMIM 194050). Although Fz9−/− mice are viable and fertile, in-depth studies by three research groups have identified: (1) a subtle abnormality in B-cell development (Ranheim et al., 2005), (2) an increase in cell death in the dentate gyrus and a defect in visuo-spatial learning (Zhao et al., 2005),

Frizzled4

Among the Frizzleds that are known or suspected to activate the canonical Wnt pathway, Fz4 is the best understood in terms of its biological function, its interactions with ligands and other receptor components, and its role in human disease. Fz4−/− mice exhibit a severe defect in retinal vascularization, progressive hearing loss, and a slowly progressive cerebellar degeneration that is associated with loss of the blood–brain barrier (BBB) in the cerebellum (Wang et al., 2001, Wang et al., 2012

Frizzled3 and Frizzled6

Among mammalian Frizzled genes, Fz6 is the family member that most closely resembles Drosophila fz, as determined by the macroscopic appearance of its loss-of-function phenotype (Guo, Hawkins, & Nathans, 2004).

Fz6 is expressed in the epidermis and in hair follicles starting at ~ E13. Fz6−/− mice are healthy and fertile, with skin and hair follicles that appear microscopically normal. However, at the earliest stages of their development, the orientations of Fz6−/− hair follicles are aberrant and

Conclusion

The past three decades represent the first phase of exploration of the Frizzled family. At least some of the functions of most mammalian Frizzled family members are now known at a descriptive level, and for three family members the signaling pathways in which they participate have been identified. Despite the enormous progress made during this time, our current level of understanding remains rudimentary. To cite three examples: (1) the determinants of Wnt–Frizzled specificity are still unknown,

Acknowledgments

Supported by the Howard Hughes Medical Institute, the Ellison Medical Foundation, and the National Eye institute (NIH).

Conflict of interest: The authors have declared that no conflict of interest exists.

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