Evaluation of methods for one-dimensional spatial analysis of two-dimensional patterns in mouse chimaeras

The relative extent of cell mixing in tissues of mouse chimaeras or mosaics can be studied by comparing the distributions of the two cell populations in the tissues. However, the mean patch size is misleading because it is affected by both the extent of cell mixing and the relative contributions of the two cell populations. Previous work suggested that effects attributable to differences in tissue composition among chimaeras can be factored out either by correcting the mean patch size or by using the median patch size for the minority cell population and restricting the analysis to grossly unbalanced chimaeras. In the present study, computer simulations of two-dimensional mosaic arrays of black and white squares (representing cells) were used to simulate chimaeric tissues. Random arrays simulated tissues with extensive cell mixing, arrays of cell clumps (representing coherent clones) simulated less mixed tissues, and striped arrays simulated tissues with elongated but fragmented descendent clones. The computer simulations predicted that (i) the median patch length (minority cell population) and the corrected mean patch length would both distinguish between random and clumped patterns and (ii) differences in the variation of the composition of two perpendicular series of one-dimensional transects would distinguished between stripes and randomly orientated patches. Both predictions were confirmed by analysis of histological sections of the retinal pigment epithelium from fetal and adult mouse chimaeras. This study demonstrates that two types of non-random two-dimensional variegated patterns (clumps and stripes) can be identified in chimaeras without two-dimensional reconstruction of serial sections

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß′ COPI coatomer subunits and demonstrate an accumulation of ‘coat-less’ vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation

Dopachrome tautomerase variants in patients with oculocutaneous albinism

Purpose: Albinism is a clinically and genetically heterogeneous condition. Despite analysis of the 20 known genes, ~30% patients remain unsolved. We aimed to identify new genes involved in albinism. Methods: We sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. Results: We identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14-bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR-Cas9 was used in C57BL/6J mice to create mutations identical to the missense variants carried by the patients, along with one loss-of-function indel. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared with Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanophores and RPE cells. Conclusion: DCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8

Robust genetic analysis of the X-linked anophthalmic (Ie) mouse

Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000–58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism

ZMYND10 functions in a chaperone relay during axonemal dynein assembly

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of ‘client’ proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease

Reconciling diverse mammalian pigmentation patterns with a fundamental mathematical model

Bands of colour extending laterally from the dorsal to ventral trunk are a common feature of mouse chimeras. These stripes were originally taken as evidence of the directed dorsoventral migration of melanoblasts (the embryonic precursors of melanocytes) as they colonize the developing skin. Depigmented 'belly spots' in mice with mutations in the receptor tyrosine kinase Kit are thought to represent a failure of this colonization, either due to impaired migration or proliferation. Tracing of single melanoblast clones, however, has revealed a diffuse distribution with high levels of axial mixing--hard to reconcile with directed migration. Here we construct an agent-based stochastic model calibrated by experimental measurements to investigate the formation of diffuse clones, chimeric stripes and belly spots. Our observations indicate that melanoblast colonization likely proceeds through a process of undirected migration, proliferation and tissue expansion, and that reduced proliferation is the cause of the belly spots in Kit mutants

A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice

The cilia and cell cycles are inextricably linked. Centrioles in the basal body of cilia nucleate the ciliary axoneme and sequester pericentriolar matrix (PCM) at the centrosome to organize the mitotic spindle. Cilia themselves respond to growth signals, prompting cilia resorption and cell cycle re-entry. We describe a fluorescent cilia and cell cycle biosensor allowing live imaging of cell cycle progression and cilia assembly and disassembly kinetics in cells and inducible mice. We define assembly and disassembly in relation to cell cycle stage with single-cell resolution and explore the intercellular heterogeneity in cilia kinetics. In all cells and tissues analyzed, we observed cilia that persist through the G1/S transition and into S/G2/M-phase. We conclude that persistence of cilia after the G1/S transition is a general property. This resource will shed light at an individual cell level on the interplay between the cilia and cell cycles in development, regeneration, and disease. The cilia and cell cycles are fundamental processes coupled through shared machinery. Ford et al. develop and characterize a multicistronic biosensor that can simultaneously label the cell and cilia cycles in mice, enabling live imaging studies of their kinetics

Palmitoylation Regulates Epidermal Homeostasis and Hair Follicle Differentiation

Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear β-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis

Human and mouse mutations in WDR35 cause short-rib polydactyly syndromes due to abnormal ciliogenesis

Defects in cilia formation and function result in a range of human skeletal and visceral abnormalities. Mutations in several genes have been identified to cause a proportion of these disorders, some of which display genetic (locus) heterogeneity. Mouse models are valuable for dissecting the function of these genes, as well as for more detailed analysis of the underlying developmental defects. The short-rib polydactyly (SRP) group of disorders are among the most severe human phenotypes caused by cilia dysfunction. We mapped the disease locus from two siblings affected by a severe form of SRP to 2p24, where we identified an in-frame homozygous deletion of exon 5 in WDR35. We subsequently found compound heterozygous missense and nonsense mutations in WDR35 in an independent second case with a similar, severe SRP phenotype. In a mouse mutation screen for developmental phenotypes, we identified a mutation in Wdr35 as the cause of midgestation lethality, with abnormalities characteristic of defects in the Hedgehog signaling pathway. We show that endogenous WDR35 localizes to cilia and centrosomes throughout the developing embryo and that human and mouse fibroblasts lacking the protein fail to produce cilia. Through structural modeling, we show that WDR35 has strong homology to the COPI coatamers involved in vesicular trafficking and that human SRP mutations affect key structural elements in WDR35. Our report expands, and sheds new light on, the pathogenesis of the SRP spectrum of ciliopathies