(Received for publication, November 1, 1995)
From the
In Drosophila melanogaster, the frizzled gene plays an essential role in the development of tissue polarity as assessed by the orientation of cuticular structures. Through a combination of random cDNA sequencing, degenerate polymerase chain reaction amplification, and low stringency hybridization we have identified six novel frizzled homologues from mammals, at least 11 from zebrafish, several from chicken and sea urchin, and one from Caenorhabditis elegans. The complete deduced amino acid sequences of the mammalian and nematode homologues share with the Drosophila frizzled protein a conserved amino-terminal cysteine-rich domain and seven putative transmembrane segments. Each of the mammalian homologues is expressed in a distinctive set of tissues in the adult, and at least three are expressed during embryogenesis. As hypothesized for the Drosophila frizzled protein, the frizzled homologues are likely to act as transmembrane receptors for as yet unidentified ligands. These observations predict the existence of a family of signal transduction pathways that are homologous to the pathway that determines tissue polarity in Drosophila.
Much of animal development is orchestrated through the transmission of intercellular signals. In Drosophila melanogaster, genetic approaches have identified both receptors and ligands that mediate a number of these intercellular signaling processes. Examples include the receptors Sevenless(1) , Notch(2) , torso(3) , and Toll(4) , and the ligands Hedgehog(5) , and wingless(6, 7) . For several of these Drosophila proteins, vertebrate homologues have been identified, and in each case the homologue(s) appear to play important roles in development(8, 9, 10, 11) . These studies demonstrate a remarkable conservation of biochemical mechanisms between vertebrate and invertebrate cell-cell communication pathways despite differences in the morphologies of the corresponding organisms.
In Drosophila, as in other insects, the cuticle contains a
variety of specialized structures, such as bristle sense organs and
hairs. At each surface location these extended structures exhibit a
predetermined orientation with respect to the body axes. For example,
on the wings and legs they point away from the body, and on the thorax
and abdomen they point posteriorly. Mutations that alter the polarity
of these structures define a set of genes that are referred to as
tissue polarity genes(12) . Mutations in frizzled, the
most extensively studied tissue polarity gene, disrupt to various
extents the polarity of hairs on the wing, leg, thorax, and
abdomen(13) . Most alleles also disrupt the orderly arrangement
of eye bristles, giving a rough eye phenotype. Genetic mosaic
experiments in which homozygous frizzled/frizzled
patches are generated in a heterozygous background show that for
some alleles the loss of tissue polarity is confined to the mutant
patch, whereas for other alleles the loss of tissue polarity also
spreads to adjacent cells distal to the mutant patch(14) .
These experiments have led Adler and colleagues (14) to propose
that the frizzled gene product may be involved in both sending
and receiving polarity information.
Strong support for a model in which frizzled acts directly in tissue polarity signaling comes from the sequence of the frizzled gene(15, 16) . This sequence reveals a deduced protein product of 587 amino acids that begins with a putative signal peptide and contains seven putative transmembrane segments. Immunostaining of cells expressing frizzled cDNA shows that the protein accumulates in the plasma membrane and that the amino and carboxyl termini face the extracellular and intracellular spaces, respectively(17) . Although the frizzled protein shares no primary sequence homology with any known receptor, its role in development and its plasma membrane location strongly suggest that it acts as a receptor, a ligand, or both in the transmission of tissue polarity information.
The goal of the present work is to determine the extent to which frizzled-like proteins exist outside of Drosophila. Previous work by Chan et al.(18) identified two closely related rat genes, the protein products of which share approximately 40% amino acid identity with the Drosophila frizzled protein. These genes were identified serendipitously, and their biological functions are currently unknown. In the present work, we show that at least one frizzled-like gene exists in the nematode, Caenorhabditis elegans, and that a large number of frizzled-like genes exist in vertebrates. These observations predict the existence of a family of signal transduction pathways homologous to the pathway that determines tissue polarity in Drosophila.
A description of the probes and RFLPs for nearly all the loci linked to the frizzled genes has been reported. These include: Gls, Ctla4, and Fn1 on chromosome 1(26, 27) ; Gnai1 and En2 on chromosome 5(28) ; Tyr on chromosome 7(29) ; Csfg and Wnt3 on chromosome 11(30) ; Ctla1, Gja3, and Nfl on chromosome 14(31, 32) ; and Hspg1 and Myc on chromosome 15 (33) . One locus, olfactory marker protein (Omp), has not been reported previously for the Frederick IB. The probe was kindly provided by Dr. Randall Reed and it detected a 2.2-kb BamHI fragment in C57BL/6J DNA and 3.3-kb BamHI fragment in M. spretus DNA. Recombination distances were calculated as described (34) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
To search for additional frizzled family members, we used PCR with different pairs of degenerate primers (Table 1) to amplify genomic DNA and cDNA from both vertebrates and invertebrates. Alignment of the Drosophila, rat, and partial human sequences revealed two regions of amino acid identity that were used to design a first set of degenerate PCR primers, YW-157 and YW-158. PCR amplification of mouse genomic DNA and human retina cDNA revealed several novel frizzled family members: Mfz2, Mfz3, Mfz4, Mfz6, Mfz7, and Hfz3, and Hfz5, respectively. A mixture of these cloned PCR products were used in a low stringency screen of day 8.5 and day 12.5 mouse embryo cDNA libraries, a day 18.5 embryonic mouse brain library, and a mouse genomic DNA library. Multiple independent cDNA clones were obtained that fell into five classes: Mfz2, Mfz3, Mfz4, Mfz6, and Mfz7. One-half of the Mfz2 coding region was sequenced and was found to be greater than 96% identical at the amino acid level to Rfz2. Mfz2 is therefore likely to represent the orthologue of Rfz2 (Chan et al., 1992). From the Mfz3 and Mfz4 cDNA clones the complete coding region was sequenced (Fig. 1). For Mfz6, the complete coding region was sequenced from a combination of RACE-PCR products and cDNA clones (Fig. 1).
Figure 1: Amino acid sequences of mammalian and nematode frizzled homologues. The conserved cysteine-rich domains and the transmembrane domains were aligned using GeneWorks software; the divergent signal sequences, linker regions (connecting the cysteine-rich domain and the transmembrane domain), and carboxyl-terminal tails were aligned manually to minimize the total number of gaps. The region of collagen XVIII (37) that has homology to the cysteine-rich domain is aligned beneath that segment. The consensus sequence indicates those residues that occur in five or more of the 10 frizzled family members. Lines above the sequences indicate the locations of putative membrane spanning segments. Dfz1, the frizzled gene of Drosophila melanogaster(15, 16) ; Rfz1 and Rfz2, two rat homologues of frizzled(18) ; M, mouse; H, human; C, C. elegans; coll XVIII, collagen XVIII.
Mouse genomic clones encompassing part or all of the coding region were isolated for Mfz2, Mfz3, Mfz4, Mfz6, Mfz7, and Mfz8; partial genomic DNA sequences were obtained from the Mfz3, Mfz4, and Mfz6 clones and these sequences confirm the cDNA and RACE PCR sequences in the regions in which they overlap. The Mfz7 and Mfz8 coding regions were deduced from genomic DNA sequence as no Mfz7 cDNA clones obtained thus far contain the 5` end of the coding region and Mfz8 cDNA clones have not yet been identified. Conceptual translation of 2.3 and 2.4 kb of contiguous sequence from Mfz7 and Mfz8 genomic clones, respectively, predicts in each case a single long open reading frame that begins with a putative signal sequence and includes all of the characteristic features of a frizzled family member (Fig. 1). Based upon this contiguous homology we tentatively propose that the Mfz7 and Mfz8 genes lack introns within the coding region.
In a parallel screen of an adult human retina cDNA library using PCR products derived from Hfz3 and Hfz5, multiple cDNA clones encoding Hfz3, Hfz5, and Hfz7 were identified. From this collection, the complete sequence of the Hfz5 coding region was determined (Fig. 1). Surprisingly, neither genomic nor cDNA clones were obtained for fz5 orthologues from the mouse library screens described above. However, PCR amplification of mouse genomic DNA using Hfz5-specific primers generated a product, the translated sequence of which is distinct from other mouse frizzled sequences and is 95% identical to Hfz5 over 143 amino acids, indicating that the mouse genome contains a Hfz5 orthologue. This Mfz5 PCR product was used for subsequent RNase protection and in situ hybridization studies (see below).
To examine more fully the diversity of the frizzled family, we searched the NCBI data base of partial cDNA sequences (expressed sequence tags, ESTs) for frizzled homologues. Statistically significant homology was detected in a C. elegans partial cDNA sequence, cm08b2(21) . This cDNA clone was used to identify the corresponding genomic segment from a C. elegans genomic DNA library, and was mapped on the C. elegans genome to yeast artificial chromosomes Y95B8 and Y34D9 on chromosome I. The complete sequence of the mRNA was determined by sequencing this cDNA clone, 5` RACE PCR products, and the exon regions of the corresponding genomic clone (Fig. 1). These sequences reveal a highly divergent frizzled family member that is encoded by nine exons distributed over 8 kb of genomic DNA (Fig. 2). This gene will be referred to as Cfz1.
Figure 2:
Intron-exon structure of the nematode
frizzled gene, Cfz1. A restriction map derived from a cloned 15.2-kb
segment of C. elegans genomic DNA in EMBL3 is shown
above the intron-exon structure of the Cfz1 gene. Sequences were
obtained across each intron-exon boundary in the cloned genomic DNA,
and exon locations were determined by restriction mapping and by PCR
amplification of plasmid subclones using exon-specific primers and
flanking vector primers. Introns 1-8 begin after the following
base pairs in the cDNA (numbered relative to the first nucleotide in
the coding region): 128, 270, 483, 624, 716, 825, 1002, and 1457. The
message contains a trans-spliced leader joined to exon 1 at position
-29. B, BamHI; E, EcoRI; H, HindIII; S, SalI.
As another approach to exploring the diversity of the frizzled family, a set of seven degenerate PCR primers were designed based on an alignment of the sequences of the human and mouse clones described above and the known Drosophila and rat sequences (Table 1; YW164-YW285). These, as well as the original pair of degenerate PCR primers were used for PCR amplification of chicken, sea urchin, and zebrafish genomic DNA. PCR products of the predicted size were obtained from a subset of the PCR reactions, and these were subcloned and sequenced. The resulting sequences revealed two, three, and 13 distinct frizzled-like sequences from chicken, sea urchin, and zebrafish, respectively (Fig. 3). The 13 zebrafish PCR products are partially overlapping and define at least 11 distinct genes.
Figure 3: Partial amino acid sequences of zebrafish, chicken, and sea urchin frizzled homologues. Frizzled-related sequences were PCR amplified from genomic DNA using the degenerate primers listed in Table 1. The names of the PCR products indicate the source of the genomic DNA templates: ZG, zebrafish; ChG, chicken; SG, sea urchin. Individual subcloned PCR products were sequenced on both strands; each sequence shown was confirmed from two or more independent PCR products. The consensus shown below lists those amino acids that occur in the majority of the PCR products in those regions where six or more sequences are available. The aligned sequences begin in transmembrane segment 2 (top left) and end in transmembrane segment 7 (lower right).
The
original pair of degenerate PCR primers, YW157 and YW158, was also used
to amplify Drosophila genomic DNA. This PCR reaction was
designed to be selective for novel frizzled-like sequences, as this
region in the Drosophila frizzled gene is known to be
interrupted by a large intron(16) . This reaction generated a
single PCR product that matched the expected size of a contiguous
frizzled coding region. Subsequent work has shown that the
corresponding gene, which we will refer to as Dfz2, encodes a
frizzled-like protein with less than 35% amino acid identity with the
original Drosophila frizzled gene product. ()
The sequences of the six novel mouse and human frizzled genes reported here brings to eight the number of distinct frizzled-like sequences identified in mammalian genomes. This probably represents an incomplete sampling of frizzled family members because: 1) the PCR reactions selected for family members with high homology to the original group of frizzled sequences used in primer design, 2) PCR amplification on genomic DNA selected for family members that did not contain introns between the primers, and 3) cleavage of PCR products with EcoRI and HindIII followed by preparative gel electrophoresis eliminated products with internal EcoRI or HindIII sites. In support of this idea, whole genome Southern blot hybridization with ZG05, one of the zebrafish PCR products, reveals a large number of hybridizing fragments in chicken, mouse, and human genomic DNA (Fig. 4). As this region shows significant divergence among the cloned frizzled family members at the DNA level, it is likely that some of the hybridizing fragments are derived from frizzled sequences other than the ones reported here.
Figure 4:
Southern blot hybridization of chicken,
mouse, and human genomic DNA probed with the zebrafish frizzled PCR
product ZG05. The filter was hybridized at 37 °C in 40% formamide
and 5 SSC, and washed at 50 °C in 0.5
SSC. The
amino acid sequence of ZG05 is shown in Fig. 3. H,
human; Ch, chicken; M1, mouse cell line BW; M2, mouse Balb/c. Size standards (in kb) are shown to the left.
Figure 5: Hydropathy profiles of mammalian and nematode frizzled homologues. Solid lines indicate the putative signal peptide and transmembrane domains. Hydropathy values were calculated using MacVector version 3.5 software according to the Kyte-Doolittle algorithm with a window size of 12 amino acids.
The carboxyl-terminal half of each frizzled protein consists
primarily of a 220-250 amino acid region in which seven
hydrophobic segments of approximately 20-25 amino acids each are
joined by short hydrophilic segments ( Fig. 1and Fig. 5).
Pairwise comparisons among mammalian frizzled sequences show that the
overall amino acid sequence identity in this region ranges from 37% to
85% (Table 2). Interestingly, several small stretches of amino
acid sequence in this region are highly conserved among all of the
frizzled family members (Fig. 1). Although this region in the
nematode Cfz1 sequence shows only 13-16% identity in any pairwise
comparison, its hydropathy profile closely matches that of the other
family members. Among the mammalian and Drosophila frizzled
sequences, gaps in the alignment in this region are confined to the
hydrophilic segments; alignment of the more divergent Cfz1 sequence
requires several small gaps in the hydrophobic segments. These
characteristics are reminiscent of those found in sequence comparisons
among G-protein coupled receptors, and suggest that, like G-protein
coupled receptors, the hydrophobic segments of the frizzled proteins
form seven membrane spanning -helices linked by hydrophilic
loops(38, 39) . In the discussion that follows, we
will refer to this part of the protein as the transmembrane region. The
transmembrane regions show no primary sequence homology with G-protein
coupled receptors, and they lack the most conserved motifs, such as the
(Glu/Asp)-Arg-(Tyr/Phe) triplet at the carboxyl terminus of the third
transmembrane segment, that are found in nearly all G-protein coupled
receptors(40) .
Carboxyl-terminal to the transmembrane region, the frizzled proteins show little or no homology. In Drosophila frizzled this region has been shown to reside on the cytosolic face of the plasma membrane, consistent with the extracellular location of the amino terminus and the assignment of seven transmembrane segments based on the hydropathy profile(17) . The lengths of the carboxyl-terminal regions range from 25 amino acids in Mfz7 to 200 amino acids in Mfz6.
The mapping results are summarized in Fig. 6. Eight loci were detected on seven mouse autosomes. From 130 N2 mice examined, no recombinants were identified that separate Mfz5 and Mfz7, on mouse chromosome 1. Interestingly, Mfz5 and Mfz7 share a degree of similarity that is no greater than that seen in an average pairwise comparison of the available mammalian frizzled genes. The remaining family members are all unlinked. In addition, two loci were detected with a probe derived from Mfz2 (the mouse orthologue of Rfz2): one on chromosome 5 (Mfz2-rs1) and one on 11 (Mfz2-rs2). The human orthologue of Rfz1 has recently been mapped to human chromosome 17q21.1(41) . Mouse chromosome 11 shares a large region of homology with the long arm of human chromosome 17q (Fig. 6; data not shown). The syntenic relationship between these chromosomes and the high degree of homology between Rfz1 and Rfz2 (18) suggests that Mfz2-rs2 is the mouse orthologue of Rfz1 and that the authentic Mfz2 locus (represented by Mfz2-rs1) resides in the proximal region of mouse chromosome 5. The remaining frizzled family members have not been mapped in humans. However, based on synteny, we predict that the human orthologues of Mfz2 will map to human chromosome 7q, Mfz3 to 14q, 13, or 8p, Mfz4 to 11q, Mfz5 to 2q, Mfz6 to 8q, Mfz7 to 2q, and Mfz8 to 10p or 18q (Fig. 6).
Figure 6: Partial chromosome linkage maps showing the locations of the frizzled (Fz) family members in mouse. The genes were mapped by interspecific backcross analysis. Partial chromosome linkage maps showing the locations of the frizzled genes in relation to linked loci are shown. The number of recombinant N2 animals over the total number of N2 animals typed plus the recombination frequencies, expressed as genetic distance in centiMorgans (+/-1 S.E.), is shown for each pair of loci on the left of the chromosome maps. Where no recombinants were found between loci, the upper 95% confidence limit of the recombination distance is given in parentheses. The positions of loci in human chromosomes, where known, are shown to the right of the maps. References for the map positions of human loci described in this study can be obtained from GDB (Genome Data Base), a computerized data base of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).
Finally, we have compared our interspecific maps with composite mouse linkage maps that report the map location of many uncloned mouse mutations (provided from Mouse Genome Data base, a computerized data base maintained at The Jackson Laboratory, Bar Harbor, ME). The frizzled genes mapped in regions of the composite maps that contain a number of mouse mutations (data not shown). It will be of interest to determine whether any of these mutations are candidates for alterations in a frizzled locus.
Figure 7: Tissue distribution of frizzled transcripts in the adult mouse. Ten micrograms of RNA was used for RNase protection with each of the mouse frizzled probes. The RNA samples are as follows: 1, brain; 2, eye; 3, heart; 4, kidney; 5, liver; 6, lung; 7, spleen; 8, testis; 9, yeast tRNA. A control reaction with a mouse RNA polymerase II probe is shown at the bottom.
An anatomic examination at higher resolution was undertaken by in situ hybridization of mouse embryos at days 11.5 and 17.5, of postnatal day 1 animals, and of adult brains. While a detailed account of these experiments is beyond the scope of the present paper, Fig. 8shows a sample of some of the in situ hybridization patterns for Mfz2, Mfz3, Mfz4, and Mfz5 that suggest in several cases a higher degree of tissue specificity than was indicated by the RNase protection analysis. At embryonic day 17.5, Mfz2 transcripts are most abundant in the ventricular zone in the developing central nervous system, the region that contains actively dividing neuroblasts (Fig. 8C). At all stages tested, Mfz3 transcripts are present at high levels throughout the central nervous system, consistent with the pattern seen by RNase protection (Fig. 8, B, E, F, and H). Mfz4, which was observed by RNase protection to be relatively abundant in the adult central nervous systems, is found by in situ hybridization to be present exclusively within the choroid plexus (Fig. 8G); strong Mfz4 hybridization is seen in the lateral ventricles and along the midline in the third ventricle. At embryonic day 17.5 and postnatal day 1, Mfz5 transcripts are most abundant in the developing retina and/or the underlying choroid, consistent with the strong signal obtained with eye RNA by RNase protection (Fig. 8, A and D).
Figure 8: In situ hybridization to Mfz3, Mfz4, and Mfz5 transcripts during mouse development and in the adult mouse central nervous system. A-C, embryonic day 17.5 saggital sections probed with Mfz5 (A), Mfz3 (B), and Mfz2 (C). D and E, postnatal day 1 coronal section of the head probed with Mfz5 (D) and Mfz3 (E). F, embryonic day 11.5 saggital section probed with Mfz3. G and H, adult brain probed with Mfz4 (G) and Mfz3 (H).
The experiments reported here reveal a large family of putative transmembrane receptors that are related to the Drosophila tissue polarity gene frizzled. The identification of eight frizzled genes in mammals and at least 11 in zebrafish indicates that members of this family are likely to play multiple roles in vertebrate development and/or physiology. The finding of frizzled family members in organisms as diverse as humans and nematodes indicates that the frizzled proteins are likely to be part of a universal signaling system in the animal kingdom. Moreover, the expression of frizzled family members in many different tissues and during embryonic development suggests that they are involved in a wide variety of developmental and/or homeostatic processes. We consider below the structures and possible functions of the frizzled proteins in light of our current knowledge regarding Drosophila frizzled.
The aberrant orientation of surface structures seen in Drosophila frizzled mutants indicates that frizzled is involved in transmitting or interpreting a signal that determines the polarity of the bristle and hair cell precursors. Interestingly, in weak frizzled alleles hairs remain locally aligned but show global disorientation, resulting in whorls composed of several dozen hairs. As discussed in Adler et al.(13) , one interpretation of this observation is that frizzled receives distinct global and local polarity signals, with receipt of the latter signal being more robust. Alternately, frizzled may be involved in the maintenance of a single external polarity signal which becomes globally disorganized but remains locally intact when frizzled activity is diminished. The latter model would also account for the observed local spread of the altered tissue polarity phenotype in genetic mosaics. Although these models differ in the hypothesized role for frizzled, they both postulate the existence of an extracellular ligand-receptor system that must ultimately organize the cytoskeleton of the hair and bristle cells to direct their growth.
The frizzled sequences reported in Vinson et al.(14) , Chan et al.(18) , and the present work bring to 10 the
number of full-length frizzled sequences known. All frizzled sequences
share the same overall domain structure. Sequence comparisons show that
the cysteine-rich domains and the transmembrane domains are strongly
conserved, although in the case of Cfz1 the conservation in the
transmembrane domain is principally at the level of hydropathy. These
sequence analyses suggest a working model in which a cysteine-rich
extracellular domain is tethered by a variable linker region to a
bundle of seven membrane spanning -helices (Fig. 9). If it
is assumed for this discussion that frizzled proteins act as receptors,
then this structural model suggests a dynamic model of transmembrane
signal transduction analogous to that proposed for the subfamily of
G-protein coupled receptors with amino-terminal ligand binding
domains(42, 43) . Specifically, we envision frizzled
signal transduction to proceed through the following steps: 1)
extracellular ligand binds to the cysteine-rich domain, 2) ligand
binding changes the interaction of the cysteine-rich region with the
transmembrane domain (possibly as a result of direct contact between
the ligand and the transmembrane domains), 3) changes in this
interaction on the extracellular face of the protein result in a
rearrangement of transmembrane
-helices, and 4) rearrangement of
transmembrane helices alters the cytosolic face of the protein which
either promotes or inhibits interactions with second messenger
components. Elaborations or variations on this model are readily
envisioned, including the possibility that in vivo the
frizzled proteins function not as monomers but as part of a homo- or
heteromultimeric complex.
Figure 9:
Working model of frizzled proteins. The
amino terminus is located on the extracellular face of the plasma
membrane and the carboxyl terminus on the cytosolic face. Seven
-helical segments span the membrane.
If frizzled proteins act as receptors, that would imply the existence of a corresponding family of ligands. In the simplest case, if there is one ligand for each receptor, then mammals would be expected to have at least eight frizzled ligands, and zebrafish at least 11. Currently, the only family of ligands known to be of this size and for which no receptors have been identified are the Wnt proteins(10) . 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. Finally, the lack of primary sequence homology between the frizzled family and known receptor families suggests that these proteins may couple to a novel effector pathway. The existence of multiple frizzled proteins implies either the existence of a large number of homologous effector pathways or a convergence on a smaller number of pathways, as seen in G-protein coupled signal transduction.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U43205 [GenBank]and U43316[GenBank]-U43321[GenBank].