Identification of Astrotactin2 as a Genetic Modifier That Regulates the Global Orientation of Mammalian Hair Follicles

Planar cell polarity (PCP) signaling controls the global orientation of surface structures, such as hairs and bristles, in both vertebrates and invertebrates. In Frizzled6 -/- (Fz6 -/-) mice, hair follicle orientations on the head and back are nearly random at birth, but reorient during early postnatal development to eventually generate a nearly parallel anterior-to-posterior array. We report the identification of a naturally occurring exon 5 deletion in Astrotactin2 (Astn2) that acts as a recessive genetic modifier of the Fz6 -/- hair patterning phenotype. A genetically engineered Astn2 exon 5 deletion recapitulates the modifier phenotype. In Fz6 -/- ;Astn2 ex5del/del mice, hair orientation on the back is subtly biased from posterior-to-anterior, leading to a 180-degree orientation reversal in mature mice. These experiments suggest that Astn2, an endosomal membrane protein, modulates PCP signaling.


Introduction
In complex multi-cellular organisms, individual surface structures such as hairs, feathers, scales, and bristles typically exhibit a high degree of spatial order. In birds and mammals, the stereotyped orientations of feathers and hairs reflect the underlying orientations of follicles within the dermis. Hair follicle orientation is controlled by planar cell polarity (PCP) signaling, as determined by the changes in follicle orientation associated with mutations in the core PCP genes Frizzled6 (Fz6), Celsr1, and Van Gogh-like2 (Vangl2) in mice [1][2][3][4][5]. In the absence of Fz6, the initial orientations of hair follicles on the head and back appear to be largely randomized, in contrast to the nearly parallel orientations of follicles on most of the body surface of wild type (WT) mice.
During the first postnatal week, hair follicles in WT mice undergo a subtle reorientation, referred to as "refinement", which minimizes angular differences among neighboring follicles. This process also leads to a more precise alignment of follicles with the body axes (on the back) or with local anatomic structures (on the limbs) [2,3]. In Fz6 -/mice, the refinement process is associated with far larger angular reorientations than in WT mice, presumably because Fz6 -/follicles exhibit a greater diversity of initial orientations [2,3]. In Fz6 -/back skin, this process leads initially to a series of large-scale patterns, such as whorls, most of which disappear by postnatal day (P)10-P15 as the field of follicle vectors progressively aligns along an anterior-toposterior direction.
Current evidence suggests that PCP proteins are essential for cell-to-cell propagation and intracellular interpretation of polarity information, but the molecules and mechanisms responsible for setting up the initial asymmetry in spatial information remain unknown [6]. In the present study, we identify a genetic modifier of the PCP hair patterning phenotype that imposes a large-scale asymmetry on hair follicle orientation.

Results and Discussion
This work began with the chance discovery of an unusual and stereotyped hair pattern among siblings in a Fz6 -/intercross, referred to hereafter as the ridge phenotype. This phenotype is characterized by a transverse ridge across the back, which arises when hairs in the upper back that are oriented in an anterior-to-posterior direction encounter hairs on the lower back that are oriented in a posterior-to-anterior direction (Figs 1 and S1). The ridge pattern is not observed in typical Fz6 -/mice. As seen in Figs 1 and S1, typical Fz6 -/back skins at P8 exhibit limited deviations from the strictly anterior-to-posterior follicle orientation of WT follicles.
Additional crosses established that the ridge phenotype segregates as a recessive trait and is only observed in the absence of Fz6. As the genetic background of our Fz6 -/line consisted of contributions from C57Bl6/J and SV129, as well as an indeterminate contribution from a Flpexpressing line, we guessed that the Fz6 -/line might harbor sufficient genetic diversity that a genome-wide SNP screen could identify the locus responsible for the ridge phenotype. This strategy revealed a single linkage peak based on typing of 1,449 loci across the genome in 43 ridge+ and 39 ridge-progeny from a Fz6 -/intercross that was segregating the ridge phenotype. The peak resides on chromosome 4 and has a multipoint LOD score of 30 (Fig 2A). To narrow the region within which the ridge locus resides, we scored hair patterns and polymorphic markers flanking the critical interval in >1,500 progeny of Fz6 -/-;ridge/ridge x Fz6 -/-; ridge/+ parents, and then fine-mapped the recombination breakpoints in the subset of progeny that exhibited a recombination event within the critical interval ( Fig 2B). This analysis narrowed the critical interval to a 2.3 Mb segment encompassing or adjacent to the genes for Tolllike receptor4 (Tlr4), Astrotactin2 (Astn2), and Trim32, a gene embedded within intron 16 of the Astn2 gene ( Fig 2C). A 74.7 kb spontaneous deletion that overlaps the Tlr4 gene, and that eliminates Tlr4 mRNA and protein production [7,8], was crossed into the Fz6 -/line and found to have no effect on hair patterning, indicating that the ridge phenotype does not arise from a loss of Tlr4 function. PCR amplification and sequencing of all of the exons of these three genes showed only one difference between control C57Bl6/J and ridge chromosomes: a consistent right of each flat mount correspond to the boxed regions labeled a-c and illustrate the correlation between vector scoring (red arrows) and the raw data (montage images showing follicle orientations). Rostral is at the top; caudal is at the bottom. The narrow slits and oval holes correspond to the locations of the eyes and ears, respectively. WT follicles are almost perfectly aligned in an anterior-to-posterior direction (B). Most Fz6 -/follicles are aligned in an anterior-toposterior direction, except for a region in the mid-back (C). Fz6 -/-;ridge/ridge follicles in the caudal half of the back exhibit a uniformly reversed (i.e. posteriorto-anterior) orientation (D). White scale bars, 1 mm. doi:10.1371/journal.pgen.1005532.g001 failure to amplify Astn2 exon 5 from ridge chromosomes (Figs 2D and S2). Tests with ten additional PCR primer pairs in the flanking introns revealed a~30 kb deletion that encompasses Astn2 exon 5 and has endpoints within a pair of LINE elements (Fig 2E).
To search for the origin of the Astn2 exon 5 deletion, we analyzed the ES cells that were used to generate the targeted Fz6 null allele [1]. This ES cell line ("R1") was derived by Nagy et al [9] from a cross between two 129/Sv lines [10][11][12]. PCR typing showed that the~30 kb deletion is present in both R1 ES cells and in 129X1/SvJ mice, but not in the closely related 129S1/SvlmJ or 129S6/SvEvTac lines ( Fig 3A). NextGen sequencing of genomic DNA from the critical interval confirmed the presence of this deletion in 129X1/SvJ and Fz6 -/-;ridge/ridge lines but not in the 129S1/SvlmJ or 129S6/SvEvTac lines (S3 Fig). Importantly, when the Astn2 allele present in each of the three 129 lines was crossed into the Fz6 -/background and assessed in the homozygous state, only the 129X1/SvJ-derived Astn2 allele produced the ridge phenotype ( Fig 3B). The data presented thus far provide strong correlative evidence that the Astn2 exon 5 deletion causes the ridge phenotype. To definitively test this hypothesis, we used gene targeting in ES cells derived from 129S6/SvEvTac mice to generate a conditional allele in which Astn2 exon 5 is flanked by loxP sites (S4 Fig). When this conditional allele is made homozygous in a Fz6 -/background (Fz6 -/-;Astn2 ex5fl/fl ) the hair pattern is indistinguishable from that seen in Fz6 -/mice, i.e. it lacks a ridge. By contrast, Fz6 -/-;Astn2 ex5del/del mice, which lack Astn2 exon 5 (following Cre-mediated germ-line recombination of the Astn2 ex5fl allele), show a ridge phenotype indistinguishable from that seen in the original Fz6 -/-;ridge/ridge mice ( Fig 4A). As with the naturally occurring ridge genotype, the Astn2 ex5del/del genotype does not produce a hair patterning phenotype on a Fz6 +/background. These experiments demonstrate that a small deletion encompassing Astn2 exon 5 is responsible for the ridge phenotype, thereby identifying a mammalian modifier locus and revealing its origin as a recent spontaneous deletion.
It is possible that the naturally occurring~30 kb ridge deletion eliminates transcriptional regulatory sequences in addition to eliminating Astn2 exon 5. Such a possibility is less likely for the engineered exon 5 deletion (Astn2 ex5del ), which is only 1.07 kb in length. The transcription start sites of the Trim32 and Astn2 genes are at distances of 443 kb and 345 kb from Astn2 exon 5 ( Fig 2C). RT-PCR analysis of Trim32 transcripts in embryonic day (E)15.5 Fz6 -/and Fz6 -/-; ridge/ridge skin showed qualitatively similar expression levels (S5 Fig). A similar RT-PCR analysis of Astn2 transcripts in E15.5 Fz6 -/-, Fz6 -/-;ridge/ridge, and WT skin also showed qualitatively similar expression levels (S6A and S6B Fig). Although we cannot exclude the formal possibility that sequences in or immediately adjacent to Astn2 exon 5 regulate the expression of a more distant gene and that the ridge phenotype reflects perturbations in that regulation, the weight of the evidence supports the conclusion that the ridge phenotype reflects the absence of Astn2 exon 5 coding sequences.
Astn2 and its close homologue Astn1 have been implicated in neuronal migration along glial scaffolds [13,14]. Astn1 and Astn2 are predicted to have a signal peptide, two transmembrane domains, and an unusual transmembrane topography in which both N-and C-termini reside on the extracellular face of the membrane (S6C and S6D Fig). Both proteins localize to endosomes, and are expressed in multiple tissues during development [14]. By in situ hybridization, we observed Astn2 expression in hair follicles starting at the earliest stage of their development ( Fig 4B). Although the precise mechanism of action of the Astrotactins is still unclear, their endosomal localization suggests that they might be involved in recycling of plasma membrane proteins [14]. Interestingly, Devenport et al. [15] observed that PCP protein complexes in the developing epidermis are internalized into endosomes and then reassembled at the plasma membrane with every cell division. These observations suggest the possibility that an alteration in Astn2 might modify the Fz6 -/phenotype by affecting PCP protein trafficking. If correct, this hypothesis would also imply that Fz6 -/embryos retain some level of PCP signaling in the skin.
Deletion of Astn2 exon 5 leads to an in-frame deletion of 36 amino acids in the predicted cytosolic domain, a region with no homology to any proteins other than Astn1. Interestingly, constitutive alternative splicing leads to frequent skipping of Astn2 exon 4, which leads to an in-frame deletion of 52 amino acids, also in the predicted cytosolic domain (Figs S6 and S7). Constitutive exon 4 skipping implies that large changes in the putative intracellular domain are compatible with protein stability and function, which suggests that deletion of exon 5 may alter but not abolish Astn2 function.
How might deletion of Astn2 exon 5 influence hair follicle development to uniformly reverse the orientations of thousands of follicles in Fz6 -/-;ridge/ridge mice? The answer to this question could be related to the striking changes in orientation that occurs among Fz6 -/follicles during early postnatal development. As noted in the Introduction, at birth, Fz6 -/mice show hair follicle orientations on the back that appear to be approximately random, but over the first 1-2 postnatal weeks, these follicles reorient to generate a series of increasingly organized macroscopic patterns, eventually reorienting in an anterior-to-posterior direction. Our earlier work suggested that the reorientation process obeys a local consensus rule that minimizes angular differences among neighboring follicles [2,3]. Computer simulations demonstrated that this process efficiently enhances the amplitude of any global bias in initial orientation while simultaneously suppressing random orientation noise, with the result that a small initial orientation bias produces a uniform reorientation of all follicles along the direction defined by that bias [2].
These quantitative analyses suggest that loss of Astn2 exon 5 either (1) continuously acts to reorient follicles in the lower back in a posterior-to-anterior direction, or (2) creates an initial posterior-to-anterior orientation bias, which is subsequently enhanced by the local refinement process. Although we cannot, at present, distinguish between these alternative models, the morphologic data imply that dramatically different follicle orientation patterns in mature skin can be consistently generated from subtly different patterns in immature skin. The performance of this system is all-the-more-remarkable because, in the Fz6 -/background, the field of immature follicle vectors has a very low signal-to-noise ratio.
PCP signaling plays a central role in a wide variety of developmental processes. In addition to hair follicle orientation, these include neural tube closure, the orientation of motile cilia and of vestibular and auditory hair cells, and axon guidance [6]. The shared dependence of these processes on PCP signaling suggests that insights obtained from studying any one of them may shed light on the others. The relationship between hair follicle orientation and axon guidance is especially intriguing and is emphasized by the requirement for Celsr and Frizzled family members in both processes [5,16,17] and by the partial interchangeability of Fz6 and its close homologue Fz3, which controls axon guidance [18]. The role of Astrotactins in both neuronal mount images (n = 3 mice per genotype). For each skin, 81 follicle angles were determined for a set of follicles closest to the grid points on a 9 x 9 grid (see Methods for further details). Zero degrees corresponds to anterior-to-posterior; 180 and -180 degrees corresponds to posterior-to-anterior. Scale bars, 0.5 mm. Astn2 exon 5 deletion has no effect on follicle orientation in a Fz6 +/background. Fz6 -/-;Astn2 ex5del/del is indistinguishable from Fz6 -/-;ridge/ridge. (D) Quantification of follicle orientations on the lower backs of eight Fz6 -/-, five Fz6 -/-;ridge/ridge, and nine Fz6 -/-;Astn2 ex5del/del mice at P3. Left, histograms shows all of the follicles quantified per genotype (n). Follicles with an anterior-to-posterior direction are shown in black; follicles with a posterior-to-anterior direction are shown in red. The ratio of the two classes is indicated above each histogram. Right, scatter plot showing the percent of follicles with a reversed (i.e. posterior-to-anterior) orientation for each skin. P-value was calculated with a student's t-test.
doi:10.1371/journal.pgen.1005532.g004 migration and hair follicle orientation suggests an even closer connection between patterning mechanisms in skin and brain.

Ethics statement
This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved Institutional Animal Care and Use Committee (IACUC) protocol MO13M469 of the Johns Hopkins Medical Institutions.

Production of Astn2 ex5fl/fl mice
The Astn2 floxed exon5 targeting construct was electroporated into MC1 ES cells (from 129S6/ SvEvTac-mice; a kind gift from Mitra Cowan) and plated in G418 and ganciclovir for positive and negative selection. Colonies were screened by Southern blotting, and clones carrying the targeted allele were injected into C57BL/6 blastocysts. Chimeras were bred to C57BL/6, and the FRT-flanked PGK-neo cassette was removed by crossing to germline Flp mice to generate the Astn2 ex5fl allele. The Astn2 ex5del allele was generated by crossing mice carrying Astn2 ex5fl to mice carrying germline Sox2-Cre [19] (Tg(Sox2-Cre)1Amc/J; from Jackson Laboratories).

Phenotyping, genotyping, and mouse husbandry
For meiotic mapping, Fz6 -/-;ridge/+ and Fz6 -/-;ridge/ridge progeny of Fz6 -/-;ridge/+ x Fz6 -/-; ridge/ridge parents were phenotyped at~P8-P10 by visual inspection of the hair pattern (i.e. examined for the presence or absence of the transverse ridge), and genotyped by scoring microsatellite insertion/deletion variants with the PCR primers listed in S1 Table. High resolution mapping of the critical interval was performed by SNP genotyping with the PCR primers listed in S2 Table. PCR primers for amplifying the 23 Astn2 exons are listed in S3 Table. PCR primers for mapping the~30 kb deletion encompassing Astn2 exon 5 ( Fig 2E) are listed in S4 Table. RT-PCR primers for amplifying Dbc1 and Trim32 are listed in S5 Table. Genome-wide SNP screen and LOD score calculation An Illumina mouse SNP array with 1,449 loci was used to type 43 ridge+ and 39 ridge-progeny from a Fz6 -/intercross that was segregating the ridge phenotype. The multipoint LOD score was calculated using R software with the quantitative trait locus (QTL) bioinformatics add-on package Version 1.21-2 (release March 18, 2011; http://www.rqtl.org). The calculation used a hidden Markov model with the Haley-Knott regression. The highest LOD score was 29.7 on Chromosome 4, with the peak at position 63.65.

Hybridization capture and NextGen sequencing
Genomic DNA from 129S1/SvlmJ, 129S6/SvEvTac, 129X1/SvJ, and Fz6 -/-;ridge/ridge mice was purified from brain tissue by proteinase K digestion and CsCl centrifugation, fragmented to a mean size of~350 bp, captured on a custom designed Agilent SureSelect oligonucleotide array that covered all non-repetitive sequences in the interval 6.58-6.72 Mb on chromosome 4 (mouse genome build 38), and subjected to 150 base paired-end sequencing on an Illumina MySeq to a mean coverage depth of~50X.

Skin flat mount analysis
Skin flat mounts were prepared as described in Chang et al [20]. To visualize follicles using the endogenous melanin pigment, the dorsal back skin (at P3 and P8) was dissected and flattened by pinning its edges to a flat Sylguard surface, fixed overnight in 4% paraformaldehyde in PBS, dehydrated through a graded alcohol series, and then clarified with benzyl benzoate:benzyl alcohol (BBBA) in a glass dish. Images were collected with a dissecting microscope.

Quantification of follicle orientations
Hair follicle orientations were scored one at a time by placing the image of a freely rotatable vector over the skin flatmount image (3.2 mm x 2.5 mm), superimposing the vector on the follicle of interest, and assessing the best fitting vector orientation by visual inspection. Two sampling strategies were used. For low-density sampling (Fig 4C), orientations were determined only for the 81 follicles closest to each point of intersection of the nine vertical and nine horizontal lines in a 2.88 mm x 2.25 mm grid overlaid on each image. For high-density sampling (Fig 4D), all follicles within each image were scored. Vector orientations were measured in Photoshop and ImageJ. Statistical comparisons were performed in Microsoft Excel. To assess the reproducibility of the scoring method, images of two P3 back skin flat mounts (one Fz6 -/and the other Fz6 -/-;Astn2 ex5del/del ) were rotated 180 degrees, and follicle orientations for all four images (two original and two rotated; n is approximately 700 follicles per image) were determined by an individual who was blinded to the genotypes and to the relatedness of the images. As shown in S8 Fig, when corrected for the 180 degree rotation, the distributions of follicle angles in the two rotated images were found to be nearly identical to the distributions in the original images, and each image reproduced the distinctive genotype-specific patterns shown in Fig 4D, which were based on quantification of 22 back skin images.

In situ hybridization
In situ hybridization was performed as described [21]. Digoxigenin-labeled riboprobes were transcribed using T7 RNA polymerase from the Astn2 cDNA (coding regions within exons 19-23), which was cloned from WT mouse E15.5 skin by RT-PCR. Images were captured on an Imager Z1 microscope (Zeiss) using Openlab software. Images of two P3 back skin flat mounts (one Fz6 -/and the other Fz6 -/-;Astn2 ex5del/del ) were rotated 180 degrees, and the orientations of all follicles within the four images (two original and two rotated) were determined as described in Methods. The scorer was blinded to the genotypes and to the relatedness of the images. A, B, D, E, Follicle orientation histograms are shown for the two original (A and D) and two rotated (B and E) images. For each image, the number of follicles scored and the ratio of left-to-right (black) and right-to-left (red) vectors is shown. In the original images, anterior was to the left and posterior was to the right. B' and E', Histograms of the rotated data set after correction for the 180-degree rotation. C and F, Distributions of follicle angles for the original images (blue lines) and the two rotated images after correction for the 180-degree rotation (red lines). (TIF) S1