De novo identification of mammalian ciliary motility proteins using cryo-EM.

Dynein-decorated doublet microtubules (DMTs) are critical components of the oscillatory molecular machine of cilia, the axoneme, and have luminal surfaces patterned periodically by microtubule inner proteins (MIPs). Here we present an atomic model of the 48-nm repeat of a mammalian DMT, derived from a cryoelectron microscopy (cryo-EM) map of the complex isolated from bovine respiratory cilia. The structure uncovers principles of doublet microtubule organization and features specific to vertebrate cilia, including previously unknown MIPs, a luminal bundle of tektin filaments, and a pentameric dynein-docking complex. We identify a mechanism for bridging 48- to 24-nm periodicity across the microtubule wall and show that loss of the proteins involved causes defective ciliary motility and laterality abnormalities in zebrafish and mice. Our structure identifies candidate genes for diagnosis of ciliopathies and provides a framework to understand their functions in driving ciliary motility.

Right, left and cilia: How asymmetry is established.

The initial breaking of left-right (L-R) symmetry in the embryo is controlled by a motile-cilia-driven leftward fluid flow in the left-right organiser (LRO), resulting in L-R asymmetric gene expression flanking the LRO. Ultimately this results in left- but not right-sided activation of the Nodal-Pitx2 pathway in more lateral tissues. While aspects of the initial breaking event clearly vary between vertebrates, events in the Lateral Plate Mesoderm (LPM) are conserved through the vertebrate lineage. Evidence from model systems and humans highlights the role of cilia both in the initial symmetry breaking and in the ability of more lateral tissues to exhibit asymmetric gene expression. In this review we concentrate on the process of L-R determination in mouse and humans.

Biallelic DAW1 variants cause a motile ciliopathy characterized by laterality defects and subtle ciliary beating abnormalities.

PURPOSE: The clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis. METHODS: Clinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants. RESULTS: In this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments. CONCLUSION: Our data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.

High-resolution µCT of a mouse embryo using a compact laser-driven X-ray betatron source.

In the field of X-ray microcomputed tomography (µCT) there is a growing need to reduce acquisition times at high spatial resolution (approximate micrometers) to facilitate in vivo and high-throughput operations. The state of the art represented by synchrotron light sources is not practical for certain applications, and therefore the development of high-brightness laboratory-scale sources is crucial. We present here imaging of a fixed embryonic mouse sample using a compact laser-plasma-based X-ray light source and compare the results to images obtained using a commercial X-ray µCT scanner. The radiation is generated by the betatron motion of electrons inside a dilute and transient plasma, which circumvents the flux limitations imposed by the solid or liquid anodes used in conventional electron-impact X-ray tubes. This X-ray source is pulsed (duration <30 fs), bright (>1010 photons per pulse), small (diameter <1 µm), and has a critical energy >15 keV. Stable X-ray performance enabled tomographic imaging of equivalent quality to that of the µCT scanner, an important confirmation of the suitability of the laser-driven source for applications. The X-ray flux achievable with this approach scales with the laser repetition rate without compromising the source size, which will allow the recording of high-resolution µCT scans in minutes.

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer's vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.

Genetic Analysis Reveals a Hierarchy of Interactions between Polycystin-Encoding Genes and Genes Controlling Cilia Function during Left-Right Determination.

During mammalian development, left-right (L-R) asymmetry is established by a cilia-driven leftward fluid flow within a midline embryonic cavity called the node. This 'nodal flow' is detected by peripherally-located crown cells that each assemble a primary cilium which contain the putative Ca2+ channel PKD2. The interaction of flow and crown cell cilia promotes left side-specific expression of Nodal in the lateral plate mesoderm (LPM). Whilst the PKD2-interacting protein PKD1L1 has also been implicated in L-R patterning, the underlying mechanism by which flow is detected and the genetic relationship between Polycystin function and asymmetric gene expression remains unknown. Here, we characterize a Pkd1l1 mutant line in which Nodal is activated bilaterally, suggesting that PKD1L1 is not required for LPM Nodal pathway activation per se, but rather to restrict Nodal to the left side downstream of nodal flow. Epistasis analysis shows that Pkd1l1 acts as an upstream genetic repressor of Pkd2. This study therefore provides a genetic pathway for the early stages of L-R determination. Moreover, using a system in which cultured cells are supplied artificial flow, we demonstrate that PKD1L1 is sufficient to mediate a Ca2+ signaling response after flow stimulation. Finally, we show that an extracellular PKD domain within PKD1L1 is crucial for PKD1L1 function; as such, destabilizing the domain causes L-R defects in the mouse. Our demonstration that PKD1L1 protein can mediate a response to flow coheres with a mechanosensation model of flow sensation in which the force of fluid flow drives asymmetric gene expression in the embryo.

ATMIN is a transcriptional regulator of both lung morphogenesis and ciliogenesis.

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmin(gpg6/gpg6), Atmin(H210Q/H210Q) and Dynll1(GT/GT), revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1(GT/GT) embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.

Atmin mediates kidney morphogenesis by modulating Wnt signaling.

The DNA damage protein and transcription factor Atmin (Asciz) is required for both lung tubulogenesis and ciliogenesis. Like the lungs, kidneys contain a tubular network that is critical for their function and in addition, renal ciliary dysfunction has been implicated in the pathogenesis of cystic kidney disease. Using the Atmin mouse mutant Gasping6 (Gpg6), we investigated kidney development and found it severely disrupted with reduced branching morphogenesis, resulting in fewer epithelial structures being formed. Unexpectedly, transcriptional levels of key cilia associated genes were not altered in Atmin(Gpg6/Gpg6) kidneys. Instead, Gpg6 homozygous kidneys exhibited altered cytoskeletal organization and modulation of Wnt signaling pathway molecules, including ß-catenin and non-canonical Wnt/planar cell polarity (PCP) pathway factors, such as Daam2 and Vangl2. Wnt signaling is important for kidney development and perturbation of Wnt signaling pathways can result in cystic, and other, renal abnormalities. In common with other PCP pathway mutants, Atmin(Gpg6/Gpg6) mice displayed a shortened rostral-caudal axis and mis-oriented cell division. Moreover, intercrosses between Atmin(Gpg6/+) and Vangl2(Lp/+) mice revealed a genetic interaction between Atmin and Vangl2. Thus we show for the first time that Atmin is critical for normal kidney development and we present evidence that mechanistically, Atmin modifies Wnt signaling pathways, specifically placing it as a novel effector molecule in the non-canonical Wnt/PCP pathway. The identification of a novel modulator of Wnt signaling has important implications for understanding the pathobiology of renal disease.

Pkd1l1 establishes left-right asymmetry and physically interacts with Pkd2.

In mammals, left-right (L-R) asymmetry is established by posteriorly oriented cilia driving a leftwards laminar flow in the embryonic node, thereby activating asymmetric gene expression. The two-cilia hypothesis argues that immotile cilia detect and respond to this flow through a Pkd2-mediated mechanism; a putative sensory partner protein has, however, remained unidentified. We have identified the Pkd1-related locus Pkd1l1 as a crucial component of L-R patterning in mouse. Systematic comparison of Pkd1l1 and Pkd2 point mutants reveals strong phenocopying, evidenced by both morphological and molecular markers of sidedness; both mutants fail to activate asymmetric gene expression at the node or in the lateral plate and exhibit right isomerism of the lungs. Node and cilia morphology were normal in mutants and cilia demonstrated typical motility, consistent with Pkd1l1 and Pkd2 activity downstream of nodal flow. Cell biological analysis reveals that Pkd1l1 and Pkd2 localise to the cilium and biochemical experiments demonstrate that they can physically interact. Together with co-expression in the node, these data argue that Pkd1l1 is the elusive Pkd2 binding partner required for L-R patterning and support the two-cilia hypothesis.

Transport and barrier mechanisms that regulate ciliary compartmentalization and ciliopathies.

Primary cilia act as cell surface antennae, coordinating cellular responses to sensory inputs and signalling molecules that regulate developmental and homeostatic pathways. Cilia are therefore critical to physiological processes, and defects in ciliary components are associated with a large group of inherited pleiotropic disorders - known collectively as ciliopathies - that have a broad spectrum of phenotypes and affect many or most tissues, including the kidney. A central feature of the cilium is its compartmentalized structure, which imparts its unique molecular composition and signalling environment despite its membrane and cytosol being contiguous with those of the cell. Such compartmentalization is achieved via active transport pathways that bring protein cargoes to and from the cilium, as well as gating pathways at the ciliary base that establish diffusion barriers to protein exchange into and out of the organelle. Many ciliopathy-linked proteins, including those involved in kidney development and homeostasis, are components of the compartmentalizing machinery. New insights into the major compartmentalizing pathways at the cilium, namely, ciliary gating, intraflagellar transport, lipidated protein flagellar transport and ciliary extracellular vesicle release pathways, have improved our understanding of the mechanisms that underpin ciliary disease and associated renal disorders.

PKD1L1 Is Involved in Congenital Chylothorax.

Besides visceral heterotaxia, Pkd1l1 null mouse embryos exhibit general edema and perinatal lethality. In humans, congenital chylothorax (CCT) is a frequent cause of fetal hydrops. In 2021, Correa and colleagues reported ultrarare compound heterozygous variants in PKD1L1 exhibiting in two consecutive fetuses with severe hydrops, implicating a direct role of PKD1L1 in fetal hydrops formation. Here, we performed an exome survey and identified ultrarare compound heterozygous variants in PKD1L1 in two of the five case-parent trios with CCT. In one family, the affected carried the ultrarare missense variants c.1543G>A(p.Gly515Arg) and c.3845T>A(p.Val1282Glu). In the other family, the affected carried the ultrarare loss-of-function variant (LoF) c.863delA(p.Asn288Thrfs*3) and the ultrarare missense variant c.6549G>T(p.Gln2183His). Investigation of the variants' impact on PKD1L1 protein localization suggests the missense variants cause protein dysfunction and the LoF variant causes protein mislocalization. Further analysis of Pkd1l1 mutant mouse embryos revealed about 20% of Pkd1l1-/- embryos display general edema and pleural effusion at 14.5 dpc. Immunofluorescence staining at 14.5 dpc in Pkd1l1-/- embryos displayed both normal and massively altered lymphatic vessel morphologies. Together, our studies suggest the implication of PKD1L1 in congenital lymphatic anomalies, including CCTs.

Foxj1 regulates floor plate cilia architecture and modifies the response of cells to sonic hedgehog signalling.

Sonic hedgehog signalling is essential for the embryonic development of many tissues including the central nervous system, where it controls the pattern of cellular differentiation. A genome-wide screen of neural progenitor cells to evaluate the Shh signalling-regulated transcriptome identified the forkhead transcription factor Foxj1. In both chick and mouse Foxj1 is expressed in the ventral midline of the neural tube in cells that make up the floor plate. Consistent with the role of Foxj1 in the formation of long motile cilia, floor plate cells produce cilia that are longer than the primary cilia found elsewhere in the neural tube, and forced expression of Foxj1 in neuroepithelial cells is sufficient to increase cilia length. In addition, the expression of Foxj1 in the neural tube and in an Shh-responsive cell line attenuates intracellular signalling by decreasing the activity of Gli proteins, the transcriptional mediators of Shh signalling. We show that this function of Foxj1 depends on cilia. Nevertheless, floor plate identity and ciliogenesis are unaffected in mouse embryos lacking Foxj1 and we provide evidence that additional transcription factors expressed in the floor plate share overlapping functions with Foxj1. Together, these findings identify a novel mechanism that modifies the cellular response to Shh signalling and reveal morphological and functional features of the amniote floor plate that distinguish these cells from the rest of the neuroepithelium.

A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy.

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.

Cilia, calcium and the basis of left-right asymmetry.

The clockwise rotation of cilia in the developing mammalian embryo drives a leftward flow of liquid; this genetically regulated biophysical force specifies left-right asymmetry of the mammalian body. How leftward flow is interpreted and information propagated to other tissues is the subject of debate. Four recent papers have shed fresh light on the possible mechanisms.

Cited2 controls left-right patterning and heart development through a Nodal-Pitx2c pathway.

Malformations of the septum, outflow tract and aortic arch are the most common congenital cardiovascular defects and occur in mice lacking Cited2, a transcriptional coactivator of TFAP2. Here we show that Cited2(-/-) mice also develop laterality defects, including right isomerism, abnormal cardiac looping and hyposplenia, which are suppressed on a mixed genetic background. Cited2(-/-) mice lack expression of the Nodal target genes Pitx2c, Nodal and Ebaf in the left lateral plate mesoderm, where they are required for establishing laterality and cardiovascular development. CITED2 and TFAP2 were detected at the Pitx2c promoter in embryonic hearts, and they activate Pitx2c transcription in transient transfection assays. We propose that an abnormal Nodal-Pitx2c pathway represents a unifying mechanism for the cardiovascular malformations observed in Cited2(-/-) mice, and that such malformations may be the sole manifestation of a laterality defect.

Static respiratory cilia associated with mutations in Dnahc11/DNAH11: a mouse model of PCD.

Primary ciliary dyskinesia (PCD) is an inherited disorder causing significant upper and lower respiratory tract morbidity and impaired fertility. Half of PCD patients show abnormal situs. Human disease loci have been identified but a mouse model without additional deleterious defects is elusive. The inversus viscerum mouse, mutated at the outer arm dynein heavy chain 11 locus (Dnahc11) is a known model of heterotaxy. We demonstrated immotile tracheal cilia with normal ultrastructure and reduced sperm motility in the Dnahc11(iv) mouse. This is accompanied by gross rhinitis, sinusitis, and otitis media, all indicators of human PCD. Strikingly, age-related progression of the disease is evident. The Dnahc11(iv) mouse is robust, lacks secondary defects, and requires no intervention to precipitate the phenotype. Together these findings show the Dnahc11(iv) mouse to be an excellent model of many aspects of human PCD. Mutation of the homologous human locus has previously been associated with hyperkinetic tracheal cilia in PCD. Two PCD patients with normal ciliary ultrastructure, one with immotile and one with hyperkinetic cilia were found to carry DNAH11 mutations. Three novel DNAH11 mutations were detected indicating that this gene should be investigated in patients with normal ciliary ultrastructure and static, as well as hyperkinetic cilia.

TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy.

Super-resolution microscopy reveals the molecular organization of biological structures down to the nanoscale. While it allows the study of protein complexes in single cells, small organisms, or thin tissue sections, there is currently no versatile approach for ultrastructural analysis compatible with whole vertebrate embryos. Here, we present tissue ultrastructure expansion microscopy (TissUExM), a method to expand millimeter-scale and mechanically heterogeneous whole embryonic tissues, including Drosophila wing discs, whole zebrafish, and mouse embryos. TissUExM is designed for the observation of endogenous proteins. It permits quantitative characterization of protein complexes in various organelles at super-resolution in a range of ∼3 mm-sized tissues using conventional microscopes. We demonstrate its strength by investigating tissue-specific ciliary architecture heterogeneity and ultrastructural defects observed upon ciliary protein overexpression. Overall, TissUExM is ideal for performing ultrastructural studies and molecular mapping in situ in whole embryos.

Analysis of the asymmetrically expressed Ablim1 locus reveals existence of a lateral plate Nodal-independent left sided signal and an early, left-right independent role for nodal flow.

BACKGROUND: Vertebrates show clear asymmetry in left-right (L-R) patterning of their organs and associated vasculature. During mammalian development a cilia driven leftwards flow of liquid leads to the left-sided expression of Nodal, which in turn activates asymmetric expression of the transcription factor Pitx2. While Pitx2 asymmetry drives many aspects of asymmetric morphogenesis, it is clear from published data that additional asymmetrically expressed loci must exist. RESULTS: A L-R expression screen identified the cytoskeletally-associated gene, actin binding lim protein 1 (Ablim1), as asymmetrically expressed in both the node and left lateral plate mesoderm (LPM). LPM expression closely mirrors that of Nodal. Significantly, Ablim1 LPM asymmetry was detected in the absence of detectable Nodal. In the node, Ablim1 was initially expressed symmetrically across the entire structure, resolving to give a peri-nodal ring at the headfold stage in a flow and Pkd2-dependent manner. The peri-nodal ring of Ablim1 expression became asymmetric by the mid-headfold stage, showing stronger right than left-sided expression. Node asymmetry became more apparent as development proceeded; expression retreated in an anticlockwise direction, disappearing first from the left anterior node. Indeed, at early somite stages Ablim1 shows a unique asymmetric expression pattern, in the left lateral plate and to the right side of the node. CONCLUSION: Left LPM Ablim1 is expressed in the absence of detectable LPM Nodal, clearly revealing existence of a Pitx2 and Nodal-independent left-sided signal in mammals. At the node, a previously unrecognised action of early nodal flow and Pkd2 activity, within the pit of the node, influences gene expression in a symmetric manner. Subsequent Ablim1 expression in the peri-nodal ring reveals a very early indication of L-R asymmetry. Ablim1 expression analysis at the node acts as an indicator of nodal flow. Together these results make Ablim1 a candidate for controlling aspects of L-R identity and patterning.

The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear.

BACKGROUND: The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. RESULTS: We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. CONCLUSIONS: The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7.