Coemergence of the Amphipathic Helix on Ameloblastin With Mammalian Prismatic Enamel

Abstract To investigate correlation between the ameloblastin (Ambn) amino acid sequence and the emergence of prismatic enamel, a notable event in the evolution of ectodermal hard tissues, we analyzed Ambn sequences of 53 species for which enamel microstructures have been previously reported. We found that a potential amphipathic helix (AH) within the sequence encoded by Exon 5 of Ambn appeared in species with prismatic enamel, with a few exceptions. We studied this correlation by investigating synthetic peptides from different species. A blue shift in fluorescence spectroscopy suggested that the peptides derived from mammalian Ambn interacted with liposomes. A downward shift at 222 nm in circular dichroism spectroscopy of the peptides in the presence of liposomes suggested that the peptides of mammals with prismatic enamel underwent a transition from disordered to helical structure. The peptides of species without prismatic enamel did not show similar secondary structural changes in the presence of liposomes. Peptides of mammals with prismatic enamel caused liposome leakage and inhibited LS8 and ALC cell spreading regulated by full-length Ambn. RT-PCR showed that AH is involved in Ambn’s regulation of cell polarization genes: Vangl2, Vangl1, Prickle1, ROCK1, ROCK2, and Par3. Our comprehensive sequence analysis clearly demonstrates that AH motif is closely related to the emergence of enamel prismatic structure, providing insight into the evolution of complex enamel microstructure. We speculate that the AH motif evolved in mammals to interact with cell membrane, triggering signaling pathways required for specific changes in cell morphology associated with the formation of enamel prismatic structure.


Introduction
Enamel, the hardest mineralized tissue found in vertebrates, caps the teeth of almost all sarcopterygians (lobefinned bony fishes and tetrapods) as well as the scales and dermal bones of many extinct lobe-fins (Smith 1978;Satchell et al. 2000;Sire et al. 2009;Qu et al. 2015;Berkovitz and Shellis 2017). Its mechanical properties enable teeth to overcome the mechanical resistance of chewing forces (Berkovitz and Shellis 2017). Mature enamel is composed of hydroxyapatite (HAP) crystallites with an architecture that is hierarchically organized, ranging from nanoscale apatite crystals to microscale enamel prisms (rods) (Cui and Ge 2007). Most mammals have prismatic enamel (Boyde 1997;Wood et al. 1999), except a few species that are toothless, enamelless, or have prismless enamel (Davit-Béal et al. 2009;Loch et al. 2013). In prismatic enamel, the HAP crystallites are grouped in parallel bundles (rods or prisms) bounded by interprismatic crystallites (interrods) oriented at a sharp angle to those in the rods ( fig. 1). This prism decussation helps in resisting the increasing tendency toward fracture as chewing loads increase and thus contributes to mammalian dietary adaptations (Spears et al. 1993;Rensberger 1997;Tabuce et al. 2017). Most nonmammalian vertebrates, including reptiles except for one extant reptile Uromastyx maliensis (Diekwisch 2020), have only prismless enamel (Sander 2000), that is, with HAP crystallites oriented fairly uniformly perpendicular to the enamel surface (Berkovitz and Shellis 2017). Since its origin from the dermal skeleton (Qu et al. 2015), enamel has undergone multiple transitions in its composition and architecture during its evolution (Berkovitz and Shellis 2017). One major architectural transition is from prismless (aprismatic) enamel in reptiles to prismatic enamel in mammals (Sander 1997;Wood et al. 1999). Prismatic enamel is considered a mammalian synapomorphy (Sander 1997;Wood et al. 1999). Fossil evidence suggests that the evolution of prismatic enamel was coincident with that of Tomes' processes of ameloblasts cells, cytoplasmic extensions appearing at the secretoray end of the enamel forming cells during the early stage of enamel formation (amelogenesis) (Lester and Von Koenigswald 1989;Wood et al. 1999).
Formation of tooth enamel mineral occurs in the extracellular matrix (ECM) between secretory ameloblast cells and underlying dentin (Lacruz et al. 2012). This ECM MBE consists of a distinct set of proteins that belong to the family of secretory calcium-binding proteins (SCPPs) and evolved from a common ancestral gene . The main enamel matrix proteins include amelogenin (Amel), ameloblastin (Ambn), enamelin (Enam), and amelotin (Amtn) (Kawasaki and Weiss 2008;Moradian-Oldak 2012). They assemble to create a functional threedimensional (3D) ECM that serves to control the formation of enamel apatite crystals and are eventually degraded and replaced by the mineral (Fincham et al. 1995;Du et al. 2005;Fang et al. 2011;Wald et al. 2017). Mutations, deletions or misexpressions of Ambn, Amel, Enam, and/or Amtn genes cause disturbances in enamel formation affecting HAP crystallite initiation, growth, and organization (Paine et al. 2003;Ozdemir et al. 2005;Jin et al. 2009;Lu et al. 2011;Lacruz et al. 2012;Hu et al. 2014;Poulter et al. 2014;Smith et al. 2016;Wald et al. 2017). In addition to enamel matrix proteins, mutations in genes related to ameloblast cell attachment apparatus also led to enamel malformation, suggesting their possible function in controlling mineral initiation and elongation during the early stages of amelogenesis (Simmer et al. 2021).
Ambn, the second most abundant enamel matrix protein, is tooth-specific (Krebsbach et al. 1996). DNA sequences of Ambn have been reported in the coelacanth, which has enamel, and the spotted gar, which has ganoine, indicating that Ambn appeared during early vertebrate evolution, at least in the last common ancestor of rayfinned fishes (actinopterygians) and lobe-finned fishes (sarcopterygians) 450 Ma (Kawasaki and Amemiya 2014; Braasch et al. 2016). Ambn protein is intrinsically disordered and is enriched in proline, glycine, leucine, and glutamine (Wald et al 2017). The amino acid sequence of Ambn contains positions that have been evolutionarily conserved, indicating their important function in enamel formation (Delsuc et al. 2015). These positions correspond to several cleavage sites and hydroxylated, O-glycosylated, and phosphorylated residues. In addition to the abovementioned conserved residues, Ambn includes domains (or motifs) such as calcium-binding domain (Yamakoshi et al. 2001), and CD63-interaction domain (Iizuka et al. 2011;Delsuc et al 2015) that are partially conserved among species. Cell attachment motifs such as the integrin-binding (Cerny et al. 1996) and heparin-binding motifs (Sonoda et al. 2009), were reported in rodent and are not conserved in mammalian evolution suggesting that these motifs could have a functional role only in rodents. Fibronectin-binding motifs were identified in rat and human Ambn (Beyeler et al. 2010).
Studies based on gain and loss of Ambn function in animal models indicate that Ambn may be involved in cellmatrix adhesion (Fukumoto et al. 2004), the maintenance of the prismatic structure (Mazumder et al. 2016;Wald et al 2017), and the regulation of the mineralization process (Krebsbach et al. 1996;Paine et al 2003;Liang et al 2019). For example, Ambn overexpression in mouse influences the habit of enamel crystallites and the morphology of enamel rods (Paine et al. 2003), which are the basic units of prismatic microstructure. Mutation of the Ambn gene in mouse causes loss of polarity in ameloblast cells and Schematic images of ameloblasts were adapted from Line and Novaes (2005); SEM image of human prismatic enamel (courtesy of Dr Changyu Shao), SEM image of reptile prismless enamel was adapted with permission from Sander (1999). Scale bar = 10 µm.

MBE
their detachment from the extracellular matrix leading to severe enamel malformation (Wald et al. 2017).
Recently, we reported that mammalian Ambn has a highly conserved amphipathic helix (AH) motif, located within the sequence encoded by Exon 5 (Su et al. 2019a). AH is an α-helix with opposing polar and nonpolar faces oriented along its long axis (Segrest et al. 1994) . AH motifs are involved in multiple cellular functions, such as anchoring peripheral proteins on membranes (Pataki et al. 2018), deforming lipid bilayers (Brady et al. 2015), and sensing membrane curvature (Hatzakis et al. 2009), as well as in signaling pathways (Carman and Dominguez 2018). We also reported that the Exon 5-encoded region of Ambn contributes to promoting polarization of ameloblast cell lines in a 3D cell culture system (Visakan et al. 2022). Furthermore, in a 2D cell culture model we showed that the AH motif within the same sequence encoded by Exon 5 mediates Ambn-cell adhesion .
Considering that the AH appears only in mammalian Ambn and that prismatic enamel is a mammalian synapomorphy, here we hypothesized a novel correlation between the evolutionary emergence of the AH motif and prismatic enamel within mammals. To test this correlation, we performed AH prediction analysis on Ambn sequences from 53 species and associated these sequences with the enamel microstructures reported in the literature. We then experimentally validated the presence of the functional AH motif in 10 of these species by investigating the interactions of species-specific synthetic peptides with large unilamellar vesicles (LUVs), a model for cell membrane, and by investigating the function of these peptides using cell-based assays. We then investigated the potential function of Ambn in upregulating ameloblast cell polarization genes, which may directly or indirectly affect the development of Tomes' processes in ameloblasts and consequently prismatic enamel formation.

Phylogenetic Tree
The Maximum Likelihood (ML) phylogram inferred from 53 full-length Ambn protein sequences under the JTT + GAMMA model is presented in Figure 2. The Ambn sequences studied here represent 44 families distributed across 28 orders (supplementary table S1, Additional file 1, Supplementary Material online) and were aligned by MAFFT, which is one of the most accurate sequence alignment programs (Additional file 2, Supplementary Material online) (Nuin et al. 2006). The phylogenetic tree showed that Ambn contains a strong phylogenetic signal, consistent with a previous report (Delsuc et al. 2015). The phylogenetic tree supports the three bony vertebrate clades: Actinopterygii, Sarcopterygii, and Tetrapoda; and the major Tetrapoda clades: Amphibia, Reptilia (birds not included), and Mammalia. Within Mammalia, monotremes diverged from the living therians before marsupials and placentals separated from each other. Within the placentals, Xenarthrans and Afrotherians diverged from Boreoeutherians before the latter divided into Euarchontoglires and Laurasiatherians (supplementary fig. S1, Supplementary Material online).

An AH in Ambn is Associated With Prismatic Enamel
The Ambn sequences were screened for the presence of the AH motif using Heliquest and were compared with the enamel microstructures, that is, prismatic or prismless. The predicted AH motif is composed of 18 residues ("RLGFGKALNSLWLHGLLP" in mouse) that form a helix with a hydrophilic face and a hydrophobic one (Su et al. 2019a). Interestingly, the predicted AH sequences frequently coemerge with the prismatic enamel microstructures in mammals ( fig. 2). Out of 42 studied mammals, 33 have a predicted AH motif within the protein sequence encoded by Exon 5 of Ambn. In these 33 mammals with a predicted AH motif, 26 have prismatic enamel and we can infer those 4 others might also have prismatic enamel according to the reported enamel microstructure of other species in the same genus. The only species in the group with a predicted AH motif but confirmed not to have prismatic enamel are whales (Balaenoptera acutorostrata scammoni, Metapenaeus monoceros, and Physeter macrocephalus); we discuss these species further in the next section. A predicted AH motif, however, is not found in mammals without prismatic enamel (except the whales as mentioned above), reptiles, amphibians, sarcopterygians (excluding tetrapods), or actinopterygians. Notably, the Ambn of Ornithorhynchus anatinus, a survivor of an early branch from the mammalian tree (Messer et al. 1998), contains the predicted AH motif, even though only part of its enamel is prismatic and regular prisms are not the primary feature of its enamel structure (Lester and Boyde 1986). O. anatinus has prismatic enamel only in juvenile stages; adults of the species lack enamel (Lester et al. 1987). This might be because O. anatinus has apparently nonfunctional MMP20 and Amtn (Kawasaki and Suzuki 2011;Gasse et al. 2015) but apparently functional Ambn and Enam (Warren et al. 2008). The occurrence of prismatic enamel and Ambn in O. anatinus implies the role of Ambn in the development of prismatic enamel. Ambn protein from two mammalian species without enamel, namely Dasypus novemcinctus and Orycteropus afer afer (Davit-Béal et al. 2009), lost the ability to form an AH motif because the aspartic acid residue, highlighted in red ( fig. 2), reduced the net charge from +2 to +1. This observation supports that there is a close correlation between the conserved AH motif and prismatic enamel.

Exceptions to the Correlation Between AH and Prismatic Enamel
There are a few exceptions to the abovementioned correlation. Two eutherians (Elephantulus edwardii and Fukomys damarensis) and four marsupials (Monodelphis domestica, Sarcophilus harrisii, Dromiciops gliroides, and Phascolarctos cinereus) have prismatic enamel, although MBE theoretically their AH formation may be blocked by a proline residue in the middle of the sequence. In order to show whether there is an AH motif in Ambn in these species, we investigated its presence experimentally using biophysical and cell-based assays, on which we report below.
Another notable exception is related to the three whale species noted above: B. acutorostrata scammoni is edentulous, and M. monoceros and P. macrocephalus have teeth which are enamelless. These three whales have predicted AH motifs and the amino acid sequences of the AH motifs are the same as those of some mammals with prismatic FIG. 2. Evolutionary correlation between predicted amphipathic helix (AH) with enamel prismatic and nonprismatic structures. The helical wheel shows the typical AH of human Ambn, with a large polar face and a small nonpolar face oriented along its long axis. Maximum likelihood tree (JTT + GAMMA model) was constructed using Ambn protein sequences from 53 species. Spotted gar (Lepisosteus oculatus) was used as outgroup. Nodes with more than 70% bootstrap support are indicated with dots. The phylogenetic relationships among placentals are shown in details in supplementary fig. S1, Supplementary Material online. The predicted AH in Ambn sequences are highlighted. In predicted AH column, Y: Ambn has a predicted AH motif in Exon 5; N: Ambn has no predicted AH motif in Exon 5. In Enamel column, * indicates the enamel microstructure was inferred from reported enamel microstructure of species in the same genus.

Grouping of the Species Based on the Bioinformatic Outcomes
The bioinformatic outcomes revealing the presence or absence of predicted AH motifs in the Ambn sequence from different species were used as a basis to create six groups (table 1). Group I: Ambn sequences have predicted AH and the species have prismatic enamel; Group II: The species have prismatic enamel but Ambn sequences have no predicted AH because of the presence of Pro; Group III: The species might have poor prismatic enamel but Ambn sequence has no predicted AH because of the presence of Ser; Group IV: The species have nonprismatic enamel and Ambn sequences have no predicted AH because of the presence of Asp; Group V: The species have nonprismatic enamel, and because of multiple factors that disturb AH folding, Ambn sequences have no predicted AH; Group VI: Ambn sequences have predicted AH but the species are edentulous or enamelless.
Out of a total of 51 species (excluding Ophiophagus hannah and Python bivittatus), 37 species (26 from Group I, 2 from Group IV, and 9 from Group V) support the correlation between the presence of predicted AH on Ambn sequence and prismatic enamel. Four species from Group I may support this correlation but there is some uncertainty six species (Group II) do not support because of the presence of Pro which hinders prediction of AH, one species from Group III may or may not support it, and three species (Group VI, the three whales) are not relevant because they are edentulous or enamelless. Group I contain 30 placentals with AH that have or should have prismatic enamel. Group II contains four marsupials and two placentals with prismatic enamel, but their potential AH may be disrupted by a middle proline residue. Group III consists of only one placental mammal that may have poor prismatic enamel, and its AH hydrophobic face may be disrupted by a serine residue. Group IV contains two enamelless placentals that have their potential AH disrupted by an aspartic acid residue. Group V contains nine species without prismatic enamel that have no predicted AH. Group VI contains three whales with predicted AH that have no enamel.

Design and Characteristics of Ambn-Derived Synthetic Peptides for Biophysical and Cell-based Assays
We designed peptides derived from AH sequences of 10 selected species, which we used for our biophysical and cellbased experiments (table 2). These peptides are from Groups I to V, and include a selection of six placentals, one metatherian, one prototherian, one reptile and one amphibian. These peptides were each at the N-terminus of the sequence encoded by Exon 5 of Ambn. Two extra amino acids were added at the N-terminus to help helical folding (Scholtz and Baldwin 1992). The length of each peptide was 20 residues. Each peptide may represent more than just the species listed in table 2, because different species may have the same amino acid sequence in the selected region.
Peptides from Homo sapiens (Eutheria), Mus musculus (Eutheria), and O. anatinus (Prototheria) are in Group I (table 2). These species have prismatic enamel, and as predicted by Heliquest have a predicted AH. The AH sequence of H. sapiens is also found in Gorilla gorilla gorilla, Pongo abelii, Pan paniscus, Pan troglodytes, Crocuta crocuta, and Galeopterus variegatus. G. gorilla gorilla, P. abelii, P. paniscus, and P. troglodytes are all in the same order as H. sapiens, that is, primates, which have prismatic enamel (Boyde and Martin 1984). C. crocuta also has prismatic enamel (Rensberger and Wang 2005). There is no report on the enamel microstructure of G. variegatus. The AH sequence of M. musculus is also found in Arvicanthis niloticus, Grammomys surdaster, Mastomys coucha, Mesocricetus auratus, Mus caroli, Mus pahari, Rattus norvegicus, and Rattus rattus. All these species are rodents with prismatic enamel (von Koenigswald 1985; Alloing-Séguier et al. Peptides from E. edwardii (Eutheria) and M. domestica (Metatheria) are in Group II, which has prismatic enamel but no predicted AH. This peptide sequence of E. edwardii is not found in any other species. The peptide sequence of M. domestica is also found in P. cinereus, another marsupial with prismatic enamel (Young et al. 1987).
Peptides from Pteropus vampyrus are in Group III and have no predicted AH due the hydrophobic face being disrupted by a polar serine residue (supplementary fig. S2, Supplementary Material online). We were unable to find an SEM image of P. vampyrus enamel in the literature. However, an SEM image of the enamel of Pteropus scapulatus, which is in the same genus, revealed very thin enamel, with just one to two rows of prisms with largely rounded outlines, and an extensive prism-free outer zone of parallel crystallite groups (Lester and Hand 1986). Due to limited access to enamel samples from P. vampyrus, we were unable to confirm our conjecture that P. vampyrus might be poorly prismatic or even prismless. The peptide sequence of P. vampyrus is also found in P. giganteus, another bat in the same genus, whose enamel microstructure has not been reported. P. vampyrus was analyzed in this study because it is a species with a polar serine residue which may disrupt the hydrophobic face of the potential AH motif.
Peptides Peptides from Alligator mississippiensis and Xenopus laevis are in Group V, which are prismless and have no AH. A. mississippiensis is a reptile. The sequence of A. mississippiensis is also found in Alligator sinensis, Caiman crocodilus, Crocodylus porosus, and Gavialis gangeticus, respectively from the families Alligatoridae, Crocodylidae, and Gavialidae of the order Crocodylia, which have prismless enamel (Sander 1997(Sander , 1999. X. laevis is an amphibian, and the peptide sequence of X. laevis is also found in Xenopus tropicalis, another kind of frog with prismless enamel ).

Peptides Derived From Mammalian Ambn Interact With LUV
LUV have been demonstrated to be an appropriate model to mimic cell membrane regions involved in epithelial cell-ECM adhesion (Su et al. 2019a). We used intrinsic fluorescence spectroscopy to test the electrostatic attraction of the peptides to LUV by investigating the blue shift of the peptides upon the addition of LUV ( fig. 3). The peptides in Groups I-IV showed significant blue shift with the addition of LUV ( fig. 3 A-H), indicating that the added LUV interacted with the peptides and made the local environments of the tryptophan residues more hydrophobic. Considering that the liposome used in this assay was negatively charged, and that the peptides were positively charged, it is likely that the charged peptides of mammals were attracted electrostatically to the LUV surface. In contrast, there was no visible blue shift for the peptides of Group V, indicating that the added LUV did not come close to the peptides in solution, and suggesting that LUV did not interact with these peptides of reptiles or amphibians ( fig. 3I, J).

Helical Content of Peptides of Mammals With Prismatic Enamel Increases With LUV
We then used circular dichroism (CD) spectroscopy to track helix formation by investigating the secondary structural changes of the peptides upon the addition of LUV. The peptide concentration for CD spectroscopy was 100 µM, 10 times that used for fluorescence spectroscopy. This high peptide concentration led to slight aggregation in the CD cell for the peptides from H. sapiens, O. anatinus, E. edwardii, and O. afer afer; and moderate aggregation in the CD cell for the peptides from M. domestica, P. vampyrus, and D. novemcinctus. All these aggregate peptides showed a maximum peak between 220 and 230 nm in the CD spectrum ( fig. 4). In Predicted AH column, Y indicates the sequence of the peptide has a predicted amphipathic helix; Nindicates the sequence of the peptide has no predicted amphipathic helix. Residues which may impede the formation of an amphipathic helix are highlighted in red. In Group column, the peptides were grouped according to table 1.

MBE
Before adding LUV, all the peptides except that of O. afer afer showed a minimum peak between 200 and 210 nm, and a value close to zero or below zero at around 195 nm, suggesting that these peptides lacked welldefined structures at pH 7.4. This result is consistent with the random coil secondary structure predicted with high confidence by I-TASSER (supplementary fig. S3, Supplementary Material online). Lacking well-defined structures might be a reason why the peptides aggregate at 100 µM. O. afer afer showed a minimum peak at around 218 nm and a positive value at around 195 nm, suggesting that O. afer afer has preformed secondary structures.
In Group I, the behavior of the peptide from M. musculus was consistent with our previous report (Su et al. 2019a), that is, there was a random coil-to-helix conformational transition ( fig. 4B), which is a typical behavior of the AH motif (Seelig 2004). The CD spectra of the peptides from H. sapiens and O. anatinus were affected by peptide aggregation. However, minimum peaks appeared at around 222 nm and the CD value at 195 nm increased ( fig. 4A and C), suggesting that the two peptides gained some welldefined structures when LUV were added, and this secondary structure was predominantly helical. A random coil-to-α-helix conformational transition also happened to the peptides from H. sapiens and O. anatinus.
In Group II, peptide aggregation affected the conformational changes detected by CD. With the addition of LUV, the CD value decreased at around 222 nm ( fig. 4D and F), suggesting that the peptides from E. edwardii and M. domestica underwent detectable secondary conformational changes.
In Groups III and IV, the behavior of peptides from P. vampyrus, D. novemcinctus, and O. afer afer was similar to that of the peptide from E. edwardii ( fig. 4F-H). Peptides from P. vampyrus and D. novemcinctus showed a slight decrease at around 222 nm, suggesting an increase of secondary structural content, but the results did not indicate an increase of helical content because there was no minimum peak at around 222 nm. O. afer afer showed slight changes, but the increase was at around 200 nm, implying that a smaller amount of well-defined structure was forming.
In Group V, peptides from A. mississippiensis and X. laevis did not show significant CD value changes ( fig. 4I and J), suggesting that these two peptides did not undergo any conformational changes. A slight decrease between 195 and 210 nm was observed for the peptide from X. laevis but may have been the result of background noise and does not suggest any structural change.

Peptides of Mammals With Prismatic Enamel Anchor to Lipid Membrane
A membrane leakage assay was used to test for membrane insertion, a typical behavior which is important for the  In Groups I and II, the fluorescence signals increased significantly ( fig. 5A-C), suggesting that all these peptides caused leakage of the lipid membrane of liposomes to some extent. Consistent with our previous report (Su et al. 2019a), the peptide from M. musculus always caused a large amount of liposome leakage. The peptide from H. sapiens caused liposome leakage, while not always as efficiently as that of M. musculus. In some cases, the peptide from H. sapiens caused moderate leakage, like O. anatinus. The variability between different replicates of this assay might be due to the different solubility of the peptide from H. sapiens. Peptides from O. anatinus always caused moderate leakage. Despite our in silico analysis suggesting that E. edwardii and M. domestica do not have a predicted AH, these peptides always caused moderate leakage as well. We therefore speculate that, despite their potentially disruptive proline residues, these two peptides formed functional amphipathic helices which could be inserted into the liposome lipid membrane.
In Groups III and IV, the fluorescence signals barely increased, suggesting that peptides from P. vampyrus, D. novemcinctus, and O. afer afer did not cause the leakage of the liposomes ( fig. 5D and E). From this it can be inferred that these peptides did not insert into the lipid membrane, even if they interacted with the liposomes as shown by fluorescence spectra and underwent slight structural changes as shown by CD.
In Group V, the fluorescence signals did not increase ( fig. 5F), suggesting that peptides from A. mississippiensis and X. laevis did not cause leakage of the liposomes at all. This is consistent with the fluorescence spectroscopy which showed that they were not adsorbed by the liposomes, and CD which showed that they did not undergo conformational changes with the addition of liposomes.

Peptides of Mammals With Prismatic Enamel Inhibit Cell Spreading on Ambn-Coated Plates
Cell spreading assays were conducted to test the biological functions of the AH motif. Two ameloblast-like cells, LS8 and ALC, which represent early and later stages of ameloblast cells, respectively (Sarkar et al. 2014), were mixed with the AH-derived peptides and then cultured on Ambn-coated plates. The relative numbers of spreading cells were then calculated by counting both the spreading cells and nonspreading cells. Peptide AB2C, which is located at the C-terminal of the sequence encoded by Exon 5, adjacent to the AH motif of M. musculus Ambn,

MBE
does not have an AH motif and therefore was used as negative control in addition to PBS alone. In Group I, the relative numbers of spreading cells with the peptide from M. musculus were 0.04 ± 0.04 for LS8 ( fig. 6A) and 0.67 ± 0.19 for ALC ( fig. 6B), both of which were lower than the AB2C and PBS controls, suggesting that the peptide from M. musculus inhibited the spreading of both types of cells. The relative numbers of spreading cells with the peptide from H. sapiens were 0.30 ± 0.03 for LS8 and 0.70 ± 0.03 for ALC, and with the peptide from O. anatinus was 0.33 ± 0.03 for LS8 and 0.69 ± 0.08 for ALC. All these numbers were lower than those of controls, suggesting that the peptides in Group I inhibited cell spreading by competing with full-length recombinant mouse Ambn coated on the plates.
In Group II, the relative numbers of spreading cells with the peptide from E. edwardii were 0.10 ± 0.05 for LS8 and 0.59 ± 0.04 for ALC, and with the peptide from M. domestica were 0.46 ± 0.07 for LS8 and 0.75 ± 0.02 for ALC. These values were lower than those of controls, suggesting that the peptides from E. edwardii and M. domestica inhibited cell spreading by competing with full-length mouse Ambn coated on the plate, that is, the two peptides were functional in inhibiting cell spreading despite the proline in a potentially disruptive position.
In Groups III, IV, and V, the relative numbers of spreading cells were not significantly different from those of controls, suggesting that the peptides from P. vampyrus, D. novemcinctus, O. afer afer, A. mississippiensis, and X. laevis did not inhibit spreading of LS8 and ALC on Ambn-coated plates.

Ambn Increases Expression of Genes Related to Cell Polarization
It has been reported that prismatic structure is closely related to Tomes' processes, which are present at the apical or secretory end of mammalian ameloblast cells (Moss-Salentijn et al. 1997). Thus, we examined whether Ambn can regulate the expression of genes which are involved in ameloblast polarity and Tomes' process development (Nishikawa and Kawamoto 2015;Otsu and Harada 2016) (fig. 7). After treating LS8 cells with Ambn for 4 h, the mRNA level of Vangl2, a planar cell polarity gene which is abundantly expressed at Tomes' processes, was increased by 2.8 times compared to the untreated group. mRNA levels of Vangl1 and Prickle1, two planar cell polarity genes which are abundant at the distal junctional area of secretory ameloblasts forming the inner layer of enamel (Nishikawa and Kawamoto 2015), were respectively increased by 3.5 times and 4.1 times after treatment with Ambn. mRNA levels of ROCK1 and ROCK2, two genes in RhoA-ROCK signaling pathway which are involved in actomyosin assembly contraction and/or actin depolymerization (Otsu and Harada 2016), were respectively increased by 6.9 times and 3.7 times. The mRNA level of Par3, a gene in Cdc42 signaling pathway which is involved in cell polarization/cell-cell adhesion (Chiba et al. 2020), was increased by 3.8 times. Our recent report that Ambn promotes polarization of ameloblast cell lines further supports that Ambn might be involved in determining enamel microstructure through regulating ameloblast polarity or Tomes' process development (Visakan et al. 2022).

FIG. 5.
Membrane leakage assay with 1 µM Ambn-derived peptides. The peptides of Homo sapiens, Mus musculus, Ornithorhynchus anatinus, Elephantulus edwardii, and Monodelphis domestica resulted in a significant increase in fluorescence signal (A-C), suggesting that these peptides caused significant membrane leakage. In contrast, the peptides of Pteropus vampyrus, Dasypus novemcinctus, and Orycteropus afer afer showed a slight increase in fluorescence signal (D and E), suggesting that these peptides caused minor membrane leakage. The peptides from Alligator mississippiensis and Xenopus laevis showed no increase (F), suggesting that these peptides did not cause leakage.

MBE
To examine the function of the AH motif in regulating ameloblast differentiation and/or cell polarity, expression of Vangl2 was analyzed in LS8 cells treated with heat denatured Ambn and AmbnΔ5, which were used as negative controls. AmbnΔ5 is the variant missing the entire sequence encoded by Exon 5, and thus without the AH motif. The heat denatured Ambn and AmbnΔ5 groups showed no significant difference from the control without treatment, and Vangl2 gene expression in the group with full-length Ambn was about twice that of the other groups ( fig. 7B), suggesting that the AH motif is involved in Ambn's function in ameloblast development and/or cell polarity.

Discussion
Prismatic enamel is vital to mammalian functional adaptation in response to the increased biomechanical stress loaded on teeth (Spears et al. 1993;Rensberger 1997;Sander 1997;Wood et al. 1999) and is a result of the shape and function of the Tomes' processes of the secretory ameloblasts (Nanci and Warshawsky 1984). Ameloblasts secrete enamel SCPPs including Amel, Ambn, Enam, Amtn and Odam, which are all critical for normal enamel development and mineralization (Kawasaki and Weiss 2008). While certain regions in these proteins are highly conserved across all species with enamel, indicating their common functional significance in enamel formation, other regions have distinctive differences between mammals and nonmammals. For example, only 77 out of 1,550 positions in Enam (5%) and 80 out of 447 (18%) positions in Ambn have remained unchanged during the approximately 220 million years of mammalian evolution (Al-Hashimi et al. 2009;Delsuc et al. 2015). Examples of differences in common motifs across various species include the mammalian Amel, which has a longer polyproline tripeptide repeat sequence than nonmammalian Amel (Delgado et al. 2005;Diekwisch et al. 2009). The elongated polyproline tandem repeat is associated with enamel apatite crystal length ). Another example is the structure of nonmammalian Amtn, which differs from mammalian Amtn, with a RGD motif at its C-terminus. Furthermore, Amtn is expressed throughout all stages of amelogenesis in nonmammalian tetrapods, while in rodents its expression is limited to the maturation stage of enamel . The above differences between mammalian and nonmammalian enamel matrix proteins have been linked to the evolution of prismatic enamel by many investigators, but the genetic and molecular basis for the emergence of prismatic microstructure has remained unclear.
In the present study we link the evolutionary changes in the AH motif of Ambn sequence to the emergence of prismatic enamel in mammals. Below we provide explanation for the lack of such association in a few exceptional cases noted in our investigation. Among the 53 species we analyzed using bioinformatic methods, we observed that the mammals with predicted AH on Ambn (except the three whales B. acutorostrata scammoni, M. monoceros, and P. macrocephalus) have prismatic enamel or at least some degree of prism development (Group I, table 1). The animals without prismatic enamel, except the whales, lack a predicted AH in Ambn (Groups IV and V, table 1). The two eutherians (E. edwardii and F. damarensis) and the four marsupials (M. domestica, S. harrisii, D. gliroides, and P. cinereus) do not have a predicted AH motif but have prismatic enamel (Group II, table 1).
The experimental data summarized in table 3 confirmed the presence of the predicted AH motif in Group I, and the presence of the functional but nonpredicted AH motif in Group II. In contrast, those species without prismatic enamel, Groups IV and V, do not have a functional AH, conforming to our predictions. Typically, an FIG. 6. Cell spreading assay on plates coated with 10 µg/ml recombinant mouse Ambn and 40 µg/ml Ambn-derived peptides. The peptides of Homo sapiens, Mus musculus, Ornithorhynchus anatinus, Elephantulus edwardii, and Monodelphis domestica inhibited the cell spreading of LS8 cells (A) and ALC cells (B), while other peptides did not. *P < 0.05; **P < 0.01; ***P < 0.001.

MBE
AH motif interacts with a lipid membrane through three steps: electrostatic attraction, adsorption, and conformational change (Seelig 2004). The experimental data clearly show that the peptides of Group I, which have prismatic enamel, bind to liposomes as shown by intrinsic fluorescence spectra, and then exhibit the typical behavior of an AH, that is, random coil-to-helix transition (Seelig 2004), as shown by CD spectra. These peptides were adsorbed onto liposomes (LUV) and inserted into the bilayer lipid membrane, as shown by membrane leakage assay. They also actively competed with the full-length Ambn coated on a plate to inhibit the spreading of LS8 and ALC cells. These data showed that peptides of Group I have typical AH behaviors and are functional. The peptides derived from E. edwardii and M. domestica of Group II, both of which come from species that have prismatic enamel, were attracted to liposomes and exhibited a conformational transition evidenced by the decrease of an upward peak at around 222 nm in the CD spectrum. This upward peak was related to protein aggregation (Benjwal et al. 2006), likely because of the high peptide concentration required by the CD experiments and was consistent with the observed turbidity in a cuvette (data not shown). The observation that these peptides inserted into the bilayer lipid membrane (leaked the LUV) and inhibited cell spreading indicated that they form a functional AH, even though the random coil-to-helix transition could not be observed by CD. The peptide from P. vampyrus of Group III was attracted by liposomes, underwent slight conformational transition, caused slight liposome leakage, and did not inhibit the spreading of LS8 or ALC cells. P. vampyrus lacks an AH, and thus might have poorly prismatic enamel or might even be prismless. In contrast to the peptides of Groups I and II, which have prismatic enamel, the peptides of Group IV and Group V did not form a functional AH, even though the peptides of Group IV could be attracted to liposomes. The three whales in Group VI have an AH motif but these species have no prismatic enamel. This might be because the B. acutorostrata scammoni has inactivated Enam, Mmp20, and Amel genes (Yim et al. 2014); M. monoceros has a premature stop codon in exon 13 of Ambn (Meredith et al. 2013); and P. macrocephalus has inactivated ACP4 (Randall et al. 2021). The correlation between the AH and prismatic structure therefore is not relevant for these three whales.
Interestingly, the peptides of Group II have no predicted AH due to a proline residue in the middle, but the experimental data showed that these peptides form a functional AH. A proline residue in the middle of an amino acid sequence is generally supposed to break α-helical and β-sheet structures in water-soluble proteins because its side chain cyclizes back to the backbone amide, leaving one of its dihedral angles (φ) fixed at ca. −65° (Kumeta et al. 2018;Richardson and Richardson 1988). However, proline is not an intrinsic α -helix breaker when the protein is associated with a membrane (Williamson 1994;Li et al. 1996). Peptides from E. edwardii and M. domestica, each of which have a proline in the middle of their amino acid sequence, were thus not expected to form helices based on predictions in silico. When these peptides were adsorbed onto liposomes, the residues facing the membrane were in a membrane-mimetic environment instead of an aqueous one, which would greatly enhance the   FIG. 7. Expression of Vangl1, ROCK1, ROCK2, PAR3, Vangl2, and PRICKLE1 in LS8 cells treated with recombinant Ambn and its variants. A1, Relative RNA levels of VANGL1, ROCK1, ROCK2, and PAR3 in LS8 cells treated with 10 µg/ ml Ambn for 4 h. A2, Relative RNA levels of VANGL2 and PRICKLE1 in LS8 cells treated with 10 µg/ml Ambn for 4 h. B, Relative RNA levels of VANGL2 in LS8 cells treated with 20 µg/ml heat-denatured Ambn (negative control), AmbnΔ5 and Ambn for 7 h. *P < 0.05; **P < 0.01; ***P < 0.001.

MBE
helical propensity of proline, and the proline could help to stabilize the α-helical conformation (Williamson 1994;Li et al. 1996). Prolines in α-helices can confer structural flexibility and functional integrity to a protein (Kumeta et al. 2018). Thus, peptides from E. edwardii and M. domestica could form a functional AH when they were adsorbed onto liposomes. Leakage and cell spreading assays with the peptide of S. harrisii (FSRFGFGKQFNPLWMHGLLP) (supplementary fig. S4, Supplementary Material online) further supported this observation. Thus, the two studied eutherians (E. edwardii and F. damarensis) and four marsupials (M. domestica, S. harrisii, D. gliroides, and P. cinereus) have a functional AH with a medial proline.
Our detailed in silico sequence analysis together with experimental data using synthetic peptides and liposomes collectively showed that, out of a total of 51 species (excluding O. hannah and P. bivittatus), 43 species (86.3% of the total, including 26 from Group I, 6 from Group II, 2 from Group IV, and 9 from Group V) support the correlation between the presence of AH and emergence of prismatic enamel. Four additional species (7.8%) from Group I may support the link, one species (2.0%) from Group III may or may not support it, and three species (5.9%, Group VI, the three whales) are not relevant. Notably, the sequences of the synthetic peptides can in many cases be found in more than just the one representative species listed in table 2. The species with sequences identical to the synthetic peptides derived from AH motifs have prismatic enamel, while the species with sequences identical to the synthetic peptides derived from equivalent regions without AH lack prismatic enamel. Therefore, the correlation between AH and prismatic enamel can be expanded to more than the 53 species analyzed in this study. The observation that an AH motif was found in O. anatinus is another strong support that the AH motif coemerged with prismatic enamel. It is noteworthy that one extant reptile, U. maliensis, has prismatic or prism-like enamel structures (Diekwisch 2020). However, when examined in detail, the prisms are not like those in mammalian prismatic enamel (Sander 1997;Wood et al. 1999). Because Tomes' processes (or Tomes' process-like structures) are formed in Uromastyx, there might be a similar correlation between its prismatic structure, Tomes' process, and an AH motif in some matrix protein of U. maliensis.
During enamel formation, ameloblasts retract from the extracellular space within which enamel is formed. Precise and coordinated directional movement of ameloblasts together with extracellular matrix secretion dictate the prismatic architecture of enamel. Tomes' processes of ameloblasts have been associated with the secretion and organization of enamel matrix proteins, the alignment of hydroxyapatite crystallites and ultimately the prismatic patterns of mature enamel (Moss-Salentijn et al. 1997). Ambn was shown to colocalize with the cell membrane at the Tomes' process . The novel association between the AH domain in Ambn sequence and enamel prismatic structure in this study is proposed to be related to a few extracellular molecular events involving Ambn protein interacting with different targets. These events include matrix-cell adhesion, control of enamel apatite crystal initiation and growth , as well as formation of enamel prismatic structure. The interacting targets may include other enamel matrix proteins (Su et al. 2016;Bapat et al 2020), the cell surface (via the AH domain) , and the mineral (Shao et al 2022). Our present findings suggest that in addition to cell membrane binding and enamel matrix association, Ambn may be involved in signaling pathways related to ameloblast differentiation and/or cell polarization during early stages of enamel development. We speculate that after being anchored to ameloblasts' cell membrane, Ambn might cooperate with other enamel matrix proteins such as amelogenin, enamelin, laminins, and integrins to trigger signaling pathways related to cell movement/polarization and perhaps to regulate the development of Tomes' process (Simmer et al. 2021). In Fluo(rescence) column, "+" indicates a blue shift in the intrinsic fluorescence spectra, suggesting there is a interaction; "-" indicates an absence of blue shift, suggesting there is no interaction. In CD column, "+" indicates a helix is formed in the presence of LUV, "-" indicates no increase in helical content, "?" indicates inconclusive results. In Membrane leakage column, "++" indicates the peptides cause severe LUV leakage, "+" indicates the peptides cause mild leakage, "?" indicates the peptides cause slight leakage and inconclusive results, "-" indicates no leakage. In LS8 and ALC column, "++" indicates the peptides inhibit cell spreading severely, "+" indicates the peptides inhibit cell spreading moderately, "-" indicates no detectable inhibition. In Predicted AH and Experimental validated AH; "Y" indicates a predicted or validated AH, "N" indicates indicates no predicted or validated AH. a Enamel microstructure was inferred from that of other species in the same genus.

MBE
Consistent with the above hypothesis, in a recent study from our laboratory we reported that ameloblastin causes cell elongation and polarization in 3D cultures of ameloblast cell lines (Visakan et al. 2022). Preferential cell elongation was characterized by increased cell aspect ratio, and polarization by the asymmetrical distribution of cell junctional complex and polarity protein markers such as E-cadherin, Par3, and Claudin-1 within the elongated cells. Furthermore, it was observed that the AH-containing Exon 5-encoded region of Ambn was largely responsible for these cell effects. Heat-denaturing or deletion of Exon 5 led to the dysfunction of Ambn in promoting gene expression associated to cell polarization. These observations suggested that the AH motif encoded by the sequence in Exon 5 is involved in molecular events to initiate cell polarization or morphological changes. The present RT-PCR data showed that addition of Ambn to ameloblast-like LS8 cells regulates the expression of cell polarization genes including Vangl2, Vangl1, Prickle1, ROCK1, and ROCK2. Ambn not only influences polarization, differentiation and proliferation of ameloblasts, but it also affects extracellular matrix signaling pathways in cell culture systems. It has been reported that Ambn modulates osteoclastogenesis through the integrin/ERK pathway (Lu et al. 2013), may indirectly suppress RANKL-induced osteoclastogenesis via MAPK signaling pathway (Chaweewannakorn et al. 2017), and may affect cell adhesion via RhoA signaling pathway and cell cycle progression through p27 (Zhang et al. 2011). In the case of enamel, like BAR domain proteins, which also have amphipathic helices (Carman and Dominguez 2018), Ambn may serve as a multi-functional hub that adheres to the cells while initiating signaling pathways, and thus may be involved in the development of Tomes' processes.
In summary, we present a correlation between the emergence of the AH motif of Ambn and the evolution of prismatic enamel in mammals. Our thorough Ambn sequence analysis provides insight into the origin of prismatic enamel, which is one of the central questions in the evolution of ectodermal hard tissues. We speculate that the AH motif evolved to trigger signaling pathways for initiation of cell polarization or morphological changes including Tomes' processes of ameloblasts. Future investigation is needed to explore the mechanism of action of such signaling pathways.

Bioinformatic and Molecular Evolution Analysis of Ambn in Relation to Enamel Structure
A total of 53 full-length Ambn protein sequences, representative of major lineages, were extracted from the GenBank or UniProt databases (supplementary table S1 (Katoh et al. 2005). The time-tree was generated using the RelTime method. Divergence times for all branching points in the topology were calculated using the Maximum Likelihood method and JTT matrix-based model (Jones et al. 1992). The estimated log likelihood value of the topology shown is −12469.17. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 2.5577]). The tree is drawn to scale, with branch lengths measured in the relative number of substitutions per site. All positions with less than 95% site coverage were eliminated, that is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There was a total of 298 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 (Tamura et al. 2021), and the number of bootstrap replications was 1000. Lepisosteus oculatus was selected as an outgroup species because it is a species of ray-finned bony fish, which do not share the common ancestor of tetrapods (Braasch et al. 2016). The bootstrap probability (Pb) of 32 interior branches of the tree was higher than 70; Pb of 11 interior branches was higher than 50; and Pb of 7 interior branches was less than 50.
The projection of Ambn protein sequences in a helical wheel ( fig. 2) was calculated by Heliquest service at http://heliquest.ipmc.cnrs.fr/ (Gautier et al. 2008). Hydrophobic moment (<μH>) and net charge (z) of every 18 residues were applied to calculate the discriminant factor (D factor) by the following equation: D = 0.944(<µH>) + 0.33 (z) (https://heliquest.ipmc.cnrs.fr/ HelpProcedure.htm#heading2). If the D factor is smaller than 0.68, the sequence is assigned to be "not an AH". If the D factor is greater than 1.34 and there is a hydrophobic surface, the sequence is assigned to be "an AH". If the D factor is between 0.68 and 1.34 and there is a hydrophobic surface, the sequence may be "an AH". If there is a proline residue in the middle, the sequence was assumed to be "not an AH" before being validated by experimental data.

Protein Expression and Purification
Recombinant mouse ameloblastin (rAmbn) and the variant delta 5 (AmbnΔ5), which lacks the sequence encoded by Exon 5, were expressed and purified following the method described in our recent publication (Su et al. 2019b). Briefly, Ambn was expressed in Escherichia coli with Histidine (His), Thioredoxin (Trx), and S-tags in BL21, using pET-32a plasmid (Novagen) with inserted mouse Ambn gene (GenBank No. AAB93765.1). The expressed protein was purified using Nickel affinity chromatography (Ni-NTA Agarose, Qiagen), followed by dialysis through 10K MWCO dialysis membrane (SnakeSkin™ Dialysis Tubing, ThermoFisher). His-, Trx-, and S-tags were cleaved by Enterokinase (light chain, New England Biolabs), and were removed by a reversed phase High Performance Liquid Chromatography (HPLC) system (Varian Prostar system with Agilent OpenLab CDS software). Phenomenex C4 column (10 × 250 mm, 5 μm) was used, and proteins were MBE eluted with an increasing gradient of 32-72% acetonitrile over 80 min, at a flow rate of 1.5 ml/min. Collected proteins were lyophilized and characterized with SDS-PAGE. The heat denaturation of rAmbn was performed by heating rAmbn at 95°C for 10 min.

Peptide Synthesis
The peptides were designed based on the potential amphipathic helices in Ambn, and were synthesized by Biomer Technology (CA, USA) with a purity of 95%. The rational for the design and details of their sequences are listed in tables 1 and 2.
Intrinsic Tryptophan Fluorescence Spectroscopy 100 μM peptides were prepared in pH 7.4, 10.0 mM Tris-HCl buffer, and kept at 4°C overnight. The peptides were diluted to 10 μM in pH 7.4, 10.0 mM Tris-HCl buffer, and then mixed with 300 μM, 100 nm LUVs and kept at room temperature for 5.0 min before measurements. Tryptophan fluorescence spectra were recorded on a Quanta Master 4 fluorescence spectro-fluorometer (HORIBA Scientific). The excitation wavelength was 295 nm, the emission wavelength range was 300-400 nm, and all the split widths were 5 nm.
CD Spectroscopy 100 μM peptides were prepared in pH 7.4, 10.0 mM Tris-HCl buffer, and kept at 4°C overnight. Then, 100 μM peptides were mixed with 300 μM, 100 nm LUVs in pH 7.4, 10.0 mM Tris-HCl buffer and kept at room temperature for 5.0 min before measurements. Far-UV CD spectra were recorded on a J-815 circular dichroism spectrometer (JASCO) over a wavelength range of 190-260 nm using a 1 mm path length quartz cell.

Membrane Leakage Assay
For membrane leakage assays, 400 nm LUVs were prepared encapsulating 9.0 mM ANTS (8-aminonaphthalene-1,3,6trisulfonic acid) and 25.0 mM DPX (p-xylene-bis-pyridinium bromide), following previous methods (Su et al. 2019a). 300 μM 100 nm LUVs encapsulating 9.0 mM ANTS and 25.0 mM DPX were mixed separately with 1 μM peptides. 0.05% Triton X-100 were used as controls. Release of fluorophore ANTS was monitored by measuring the increase of ANTS fluorescence intensity using a Quanta Master 4 fluorescence spectro-fluorometer (HORIBA Scientific). Excitation and emission wavelengths were 380 and 520 nm. All data were normalized to the fluorescence intensity when all the vesicles were disrupted by Triton X-100.

Cell Spreading Assay
Cell spreading assays were conducted following published protocols (Humphries 2001;Sonoda et al. 2009). LS8 (Chen et al. 1992) and ALC cells (Nakata et al. 2003) representing different stages of ameloblast cells were cultured in low glucose Dulbecco's modified Eagle's Medium (DMEM) (Corning) containing 100 U/L penicillin and 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (Corning) in a 5.0% CO 2 atmosphere at 37°C. The assay was performed in 96-well cell culture plates (Cellstar), which were coated with 100 μl 5 μg/ml recombinant mouse Ambn in pH 7.4 PBS, and then were blocked with 50 μl 10 mg/ml BSA in in divalent cation-free PBS for 1-2 h at room temperature. The cells were treated with peptides. For a single well, about 1.0 × 10 4 cells were added. After incubation for 2 h for LS8 cells or 1.5 h for ALC cells, the percentage of cells that adopted spread morphology was determined using a microscope.

RT-PCR
Confluent LS8 cells in a 12-well plate were treated with 10 µg/ml Ambn for 4 h, or with 20 µg/ml Ambn, heatdenatured Ambn, or AmbnΔ5 for 7 h. After treatment, the RNA was purified by RNeasy Plus Mini Kit (Qiagen) following the kit's instructions. Then, reverse transcription of RNA into cDNA was conducted using iScript Reverse Transcription Supermix for RT-qPCR (Bio-rad). Finally, the relative RNA levels of Vangl1, Vangl2, ROCK1, ROCK2, Par3, and Prickle1 were detected by CFX96 Real-Time System (Bio-rad) using iQ SYBR Green Supermix and the primers listed in supplementary table S3, Supplementary Material online.

Statistical Analysis
Statistical analysis was carried out using one-tailed Student's t-tests in Microsoft Excel. The differences were considered significant if P < 0.05. All data were representative of at least three independent experiments using different batches of purified proteins.

Supplementary Material
Supplementary data are available at Molecular Biology and Evolution online.