Compound heterozygous variants in MAPK8IP3 were detected in severe congenital hypotonia mimicking lethal spinal muscular atrophy.

Mitogen-activated protein kinase 8-interacting protein 3 gene (MAPK8IP3) encodes the c-Jun-amino-terminal kinase-interacting protein 3 (JIP3) and is involved in retrograde axonal transport. Heterozygous de novo pathogenic variants in MAPK8IP3 result in a neurodevelopmental disorder with or without brain abnormalities and possible axonal peripheral neuropathy. Whole-exome sequencing was performed on an individual presenting with severe congenital muscle hypotonia of neuronal origin mimicking lethal spinal muscular atrophy. Compound heterozygous rare variants (a splice and a missense) were detected in MAPK8IP3, inherited from the healthy parents. Western blot analysis in a muscle biopsy sample showed a more than 60% decrease in JIP3 expression. Here, we suggest a novel autosomal recessive phenotype of a lower motor neuron disease caused by JIP3 deficiency.

Feather arrays are patterned by interacting signalling and cell density waves.

Feathers are arranged in a precise pattern in avian skin. They first arise during development in a row along the dorsal midline, with rows of new feather buds added sequentially in a spreading wave. We show that the patterning of feathers relies on coupled fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling together with mesenchymal cell movement, acting in a coordinated reaction-diffusion-taxis system. This periodic patterning system is partly mechanochemical, with mechanical-chemical integration occurring through a positive feedback loop centred on FGF20, which induces cell aggregation, mechanically compressing the epidermis to rapidly intensify FGF20 expression. The travelling wave of feather formation is imposed by expanding expression of Ectodysplasin A (EDA), which initiates the expression of FGF20. The EDA wave spreads across a mesenchymal cell density gradient, triggering pattern formation by lowering the threshold of mesenchymal cells required to begin to form a feather bud. These waves, and the precise arrangement of feather primordia, are lost in the flightless emu and ostrich, though via different developmental routes. The ostrich retains the tract arrangement characteristic of birds in general but lays down feather primordia without a wave, akin to the process of hair follicle formation in mammalian embryos. The embryonic emu skin lacks sufficient cells to enact feather formation, causing failure of tract formation, and instead the entire skin gains feather primordia through a later process. This work shows that a reaction-diffusion-taxis system, integrated with mechanical processes, generates the feather array. In flighted birds, the key role of the EDA/Ectodysplasin A receptor (EDAR) pathway in vertebrate skin patterning has been recast to activate this process in a quasi-1-dimensional manner, imposing highly ordered pattern formation.

Hierarchical patterning modes orchestrate hair follicle morphogenesis.

Two theories address the origin of repeating patterns, such as hair follicles, limb digits, and intestinal villi, during development. The Turing reaction-diffusion system posits that interacting diffusible signals produced by static cells first define a prepattern that then induces cell rearrangements to produce an anatomical structure. The second theory, that of mesenchymal self-organisation, proposes that mobile cells can form periodic patterns of cell aggregates directly, without reference to any prepattern. Early hair follicle development is characterised by the rapid appearance of periodic arrangements of altered gene expression in the epidermis and prominent clustering of the adjacent dermal mesenchymal cells. We assess the contributions and interplay between reaction-diffusion and mesenchymal self-organisation processes in hair follicle patterning, identifying a network of fibroblast growth factor (FGF), wingless-related integration site (WNT), and bone morphogenetic protein (BMP) signalling interactions capable of spontaneously producing a periodic pattern. Using time-lapse imaging, we find that mesenchymal cell condensation at hair follicles is locally directed by an epidermal prepattern. However, imposing this prepattern's condition of high FGF and low BMP activity across the entire skin reveals a latent dermal capacity to undergo spatially patterned self-organisation in the absence of epithelial direction. This mesenchymal self-organisation relies on restricted transforming growth factor (TGF) ß signalling, which serves to drive chemotactic mesenchymal patterning when reaction-diffusion patterning is suppressed, but, in normal conditions, facilitates cell movement to locally prepatterned sources of FGF. This work illustrates a hierarchy of periodic patterning modes operating in organogenesis.

Unique correlation between GTF2I mutation and spindle cell morphology in thymomas (type A and AB thymomas).

AIM: Recent study has revealed frequent GTF2I mutation in thymomas, with the frequency being highest in types A and AB, followed by B1, B2, B3 and thymic carcinoma. This has led to the conclusion that GTF2I mutation correlates with more indolent histology subtype and better prognosis. In our study, the GTF2I mutation was tested in thymic epithelial tumours to investigate the relation between the mutation status and histology subtype. METHODS: The GTF2I mutation was tested in 111 thymic epithelial tumours by Sanger sequencing. Correlations between GTF2I mutation status and clinicopathological parameters were evaluated. RESULTS: There were 16 cases of type A, including atypical type, 37 type AB, 13 B1, 23 B2, 9 B3, 6 micronodular type, 2 metaplastic type and 5 thymic carcinomas. GTF2I mutation was seen in 78.6% of type A and 83.9% of type AB, while it was not expressed in type B, metaplastic type or thymic carcinoma (p<0.001). 75% of micronodular type also showed the mutation. Both thymoma histotype and stage were significantly associated with GTF2I mutation by univariate analysis. The presence of GTF2I mutation showed a trend towards a favourable prognosis, but this is likely due to their strong association with more indolent histologic subtypes (types A and AB). CONCLUSIONS: GTF2I mutation appears unique in type A and AB thymomas, including those with atypical features and micronodular type, all of which share spindle cell morphology, indicating they represent a group biologically distinct from type B thymomas.

Genome-wide SNP scan of pooled DNA reveals nonsense mutation in FGF20 in the scaleless line of featherless chickens.

BACKGROUND: Scaleless (sc/sc) chickens carry a single recessive mutation that causes a lack of almost all body feathers, as well as foot scales and spurs, due to a failure of skin patterning during embryogenesis. This spontaneous mutant line, first described in the 1950s, has been used extensively to explore the tissue interactions involved in ectodermal appendage formation in embryonic skin. Moreover, the trait is potentially useful in tropical agriculture due to the ability of featherless chickens to tolerate heat, which is at present a major constraint to efficient poultry meat production in hot climates. In the interests of enhancing our understanding of feather placode development, and to provide the poultry industry with a strategy to breed heat-tolerant meat-type chickens (broilers), we mapped and identified the sc mutation. RESULTS: Through a cost-effective and labour-efficient SNP array mapping approach using DNA from sc/sc and sc/+ blood sample pools, we map the sc trait to chromosome 4 and show that a nonsense mutation in FGF20 is completely associated with the sc/sc phenotype. This mutation, common to all sc/sc individuals and absent from wild type, is predicted to lead to loss of a highly conserved region of the FGF20 protein important for FGF signalling. In situ hybridisation and quantitative RT-PCR studies reveal that FGF20 is epidermally expressed during the early stages of feather placode patterning. In addition, we describe a dCAPS genotyping assay based on the mutation, developed to facilitate discrimination between wild type and sc alleles. CONCLUSIONS: This work represents the first loss of function genetic evidence supporting a role for FGF ligand signalling in feather development, and suggests FGF20 as a novel central player in the development of vertebrate skin appendages, including hair follicles and exocrine glands. In addition, this is to our knowledge the first report describing the use of the chicken SNP array to map genes based on genotyping of DNA samples from pooled whole blood. The identification of the sc mutation has important implications for the future breeding of this potentially useful trait for the poultry industry, and our genotyping assay can facilitate its rapid introgression into production lines.

Prevalence of RPGR-Mediated Retinal Dystrophy in an Unselected Cohort of Over 5000 Patients.

Purpose: Comprehensive genetic testing for inherited retinal dystrophy (IRD) is challenged by difficult-to-sequence genomic regions, which are often mutational hotspots, such as RPGR ORF15. The purpose of this study was to evaluate the diagnostic contribution of RPGR variants in an unselected IRD patient cohort referred for testing in a clinical diagnostic laboratory. Methods: A total of 5201 consecutive patients were analyzed with a clinically validated next-generation sequencing (NGS)-based assay, including the difficult-to-sequence RPGR ORF15 region. Copy number variant (CNV) detection from NGS data was included. Variant interpretation was performed per the American College of Medical Genetics and Genomics guidelines. Results: A confirmed molecular diagnosis in RPGR was found in 4.5% of patients, 24.0% of whom were females. Variants in ORF15 accounted for 74% of the diagnoses; 29% of the diagnostic variants were in the most difficult-to-sequence central region of ORF15 (c.2470-3230). Truncating variants made up the majority (91%) of the diagnostic variants. CNVs explained 2% of the diagnostic cases, of which 80% were one- or two-exon deletions outside of ORF15. Conclusions: Our findings indicate that high-throughput, clinically validated NGS-based testing covering the difficult-to-sequence region of ORF15, in combination with high-resolution CNV detection, can help to maximize the diagnostic yield for patients with IRD. Translational Relevance: These results demonstrate an accurate and scalable method for the detection of RPGR-related variants, including the difficult-to-sequence ORF15 hotspot, which is relevant given current and emerging therapeutic opportunities.

Morphogenesis and bone integration of the mouse mandibular third molar.

The mouse third molar (M3) develops postnatally and is thus a unique model for studying the integration of a non-mineralized tooth with mineralized bone. This study assessed the morphogenesis of the mouse M3, related to the alveolar bone, comparing M3 development with that of the first molar (M1), the most common model in odontogenesis. The mandibular M3 was evaluated from initiation to eruption by morphology and by assessing patterns of proliferation, apoptosis, osteoclast distribution, and gene expression. Three-dimensional reconstruction and explant cultures were also used. Initiation of M3 occurred perinatally, as an extension of the second molar (M2) which grew into a region of soft mesenchymal tissue above the M2, still far away from the alveolar bone. The bone-free M3 bud gradually became encapsulated by bone at the cap stage at postnatal day 3. Osteoclasts were first visible at postnatal day 4 when the M3 came into close contact with the bone. The number of osteoclasts increased from postnatal day 8 to postnatal day 12 to form a space for the growing tooth. The M3 had erupted by postnatal day 26. The M3, although smaller than the M1, passed through the same developmental stages over a similar time span but showed differences in initiation and in the timing of bone encapsulation.

Maintenance of distinct melanocyte populations in the interfollicular epidermis.

Hair follicles and sweat glands are recognized as reservoirs of melanocyte stem cells (MSCs). Unlike differentiated melanocytes, undifferentiated MSCs do not produce melanin. They serve as a source of differentiated melanocytes for the hair follicle and contribute to the interfollicular epidermis upon wounding, exposure to ultraviolet irradiation or in remission from vitiligo, where repigmentation often spreads outwards from the hair follicles. It is unknown whether these observations reflect the normal homoeostatic mechanism of melanocyte renewal or whether unperturbed interfollicular epidermis can maintain a melanocyte population that is independent of the skin's appendages. Here, we show that mouse tail skin lacking appendages does maintain a stable melanocyte number, including a low frequency of amelanotic melanocytes, into adult life. Furthermore, we show that actively cycling differentiated melanocytes are present in postnatal skin, indicating that amelanotic melanocytes are not uniquely relied on for melanocyte homoeostasis.

Dynamic relationship of the epithelium and mesenchyme during salivary gland initiation: the role of Fgf10.

Salivary glands provide an excellent model for the study of epithelial-mesenchymal interactions. We have looked at the interactions involved in the early initiation and development of murine salivary glands using classic recombination experiments and knockout mice. We show that salivary gland epithelium, at thickening and initial bud stages, is able to direct salivary gland development in non-gland pharyngeal arch mesenchyme at early stages. The early salivary gland epithelium is therefore able to induce gland development in non-gland tissue. This ability later shifts to the mesenchyme, with non-gland epithelium, such as from the limb bud, able to form a branching gland when combined with pseudoglandular stage gland mesenchyme. This shift appears to involve Fgf signalling, with signals from the epithelium inducing Fgf10 in the mesenchyme. Fgf10 then signals back to the epithelium to direct gland down-growth and bud development. These experiments highlight the importance of epithelial-mesenchymal signalling in gland initiation, controlling where, when and how many salivary glands form.

Lumen formation in salivary gland development.

During salivary gland morphogenesis, the developing ducts and acini must hollow out to form lumina which will eventually allow the free passage and modification of saliva on its journey from acini to oral cavity. The molecular mechanisms that participate in the creation of this tubular structure are of great research interest. Histological studies show that lumen formation begins during the mid stages of branching morphogenesis. At this stage, apoptotic cells are detectable in the developing salivary ducts at sites where lumina are forming, suggesting that programmed cell death is instrumental in clearing the luminal space. The formation of cell-cell junctions between the epithelial cells lining the space is also an integral part of lumen formation, since these junctions form a barrier around the lumen and allow the surfaces of the lumen-lining cells to become specialized. This chapter will discuss the mechanisms involved in salivary gland lumen formation during development, and draw on the most recent research in this interesting field.