Frantisek Spoutil

EHMT1 and EHMT2 genes encode human euchromatin histone lysine methyltransferase 1 and 2 (EHMT1 alias GLP; EHMT2 alias G9a) that form heteromeric GLP/G9a complexes with essential roles in epigenetic regulation of gene expression. While EHMT1 haploinsufficiency has been established as the cause of Kleefstra syndrome 1, the pathogenesis of G9a dysfunction in human disease remains largely unknown. We identified seven de novo EHMT2 variants in patients with clinical presentation, episignatures, histone modifications and transcriptomic profiles similar to those of Kleefstra syndrome 1. In vitro studies revealed that these variants encode for structurally stable G9a proteins that are catalytically incompetent due to aberrant interactions either with histone H3 tail or with S-adenosylmethionine. Heterozygous mice carrying a patient-derived variant exhibited growth retardation, facial/skull dysmorphia and aberrant behavior. Here we report pathogenic EHMT2 variants that likely exert dominant-negative effect on GLP/G9a complexes and thus genocopy the EHMT1 haploinsufficiency via a distinct molecular mechanism, defining an autosomal dominant EHMT2 -related Kleefstra syndrome.

Family of Sequence Similarity 83H (FAM83H/ SACK1H) is primarily expressed in epithelial cells, where it interacts with casein kinase 1 (CK1) and keratins to regulate cytoskeletal organization, cell proliferation, and vesicular trafficking. Mutations in FAM83H are known to cause amelogenesis imperfecta, highlighting its critical role in enamel formation. We generated Fam83h -deficient mice ( Fam83h ^{em2(IMPC)Ccpcz} , Fam83h ^-/- ) and mice lacking a part of the N-terminal CK1-binding domain ( Fam83h ^{Δ 87/ Δ 87} ). Consistent with other Fam83h -deficient models, these mice are subviable, smaller in size, and exhibit a sparse, scruffy coat, scaly skin, general weakness, and hypoactivity. Notably, both strains show impaired lymphoid cell development in early postnatal life. In the thymus, Fam83h expression is confined to thymic epithelial cells (TECs), and its deficiency in stromal cells results in disrupted thymic architecture and severe block in the expansion of DN3 (double-negative stage 3) T cells, ultimately leading to insufficient T cell production. Single-cell transcriptomic analysis reveals that Fam83h ^-/- cortical TECs (cTECs) express reduced levels of the TEC master regulator Foxn1 , and its multiple downstream target genes, suggesting a critical role for FAM83H likely in coordination with CK1in cTEC maturation. Highlights: Fam83h -deficient mice exhibit multiple epithelial defects, but no obvious enamel defects Fam83h deficiency disrupts lymphocyte development Fam83h deficiency impairs thymic epithelial cell maturation and thymus function

Several rodent genera radiated in Ethiopian highlands during Pleistocene, giving rise to species specialized to different elevation zones. The zonation is accompanied by vegetation change from savannahs or forests in lower elevations, through the mid-elevation belt of ericaceous shrublands to Afroalpine moorlands on the mountain tops. Such evolution on elevation gradient is expected to promote phenotypic adaptations and several genera are known to include highly distinctive high-elevation specialists. In this study, we systematically examined shape and size differentiation of skull and mandible in three genera, Lophuromys, Arvicanthis and Stenocephalemys, each including from six to eight species inhabiting different elevation zones on both sides of the Great Rift Valley. Employing computed tomography, three-dimensional virtual models, landmark-based geometric morphometrics and a powerful toolkit of partial least squares analysis, we demonstrated the elevation-related differentiation is a prominent feature of skull and mandible shape variation in all three genera. We identified the parts of skull and mandible which change most between the successive zones. These include, notably, orbits and insertion areas of masticatory muscles, which suggests adaptive shifts in visual perception and diet. We also showed that while the same parts are modified in all three genera, they are modified in different ways and to a different degree. The evidence for size differentiation was mixed despite the universal trend of larger size in higher elevations. Our study confirms the high-elevation specialists represent the most distinctive forms, which is especially true for the species from Bale and Arsi Mountains, East of the Great Rift Valley.

Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium‐carbon ratio. However, the methods mentioned above only show the result—already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels—starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high‐resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC.Basic Protocol 1: In vivo MicroCT scanning of mouseBasic Protocol 2: Ex vivo HR‐MicroCT of the teethBasic Protocol 3: SEM for teeth microstructureBasic Protocol 4: Stimulated emission depletion (STED) microscopy

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2^cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1β by neutrophil granulocytes. In this study, we show that in addition to IL-1β, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2^cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2^cmo mice did not affect IL-1β levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2^cmo mice. Key Points NADPH oxidase hyperactivity accompanies autoinflammation in Pstpip2^cmo mice. This hyperactivity is cell intrinsic and not caused by an inflamed environment. NADPH oxidase deletion prevents autoinflammatory bone damage in Pstpip2^cmo mice.

The Tasmanian tiger or thylacine (Thylacinus cynocephalus) was an iconic Australian marsupial predator that was hunted to extinction in the early 1900s. Despite sharing striking similarities with canids, they failed to evolve many of the specialized anatomical features that characterize carnivorous placental mammals. These evolutionary limitations are thought to arise from functional constraints associated with the marsupial mode of reproduction, in which otherwise highly altricial young use their well-developed forelimbs to climb to the pouch and mouth to suckle. Here we present the first three-dimensional digital developmental series of the thylacine throughout its pouch life using X-ray computed tomography on all known ethanol-preserved specimens. Based on detailed skeletal measurements, we refine the species growth curve to improve age estimates for the individuals. Comparison of allometric growth trends in the appendicular skeleton (fore- and hindlimbs) with that of other placental and marsupial mammals revealed that despite their unique adult morphologies, thylacines retained a generalized early marsupial ontogeny. Our approach also revealed mislabelled specimens that possessed large epipubic bones (vestigial in thylacine) and differing vertebral numbers. All of our generated CT models are publicly available, preserving their developmental morphology and providing a novel digital resource for future studies of this unique marsupial.

Significance: Formation of the hardest mineralized tissue in vertebrates, tooth enamel, relies on a unique set of enamel matrix proteins (EMPs). These EMPs assemble into a 3D extracellular organic matrix that directs the deposition of calcium and phosphate ions into hydroxyapatite crystallites. However, the molecular basis of EMP assembly into the organic matrix remains poorly understood. This study shows that self-assembly of the key EMPs, ameloblastin and amelogenin, involves a short linear amino acid motif that is evolutionarily conserved from the first tetrapods to man. Functionality of this motif in ameloblastin is shown to be essential for organization of the enamel organic matrix and for proper organization of hydroxyapatite crystallites into the compact bundles that determine the structure and mechanical resistance of enamel.

The patterning of repeated structures is a major theme in developmental biology, and the interrelationship between spacing and size of such structures is an open question. Fungiform papillae are repeated epithelial structures that house taste buds on the anterior tongue. Here, we report that FGF signaling is a critical regulator of fungiform papillae development. We found that mesenchymal FGF10 controls the size of the papillary area, while overall patterning remains unchanged. Our results show that FGF signaling negatively affects the extent of canonical Wnt signaling, which is the main activation pathway during fungiform papillae development, but this effect does not occur at the level of gene transcription. Rather, our experimental data together with computational modeling indicate that FGF10 modulates the range of Wnt effects, likely via induction of Sostdc1 expression. We suggest that modification of the reach of Wnt signaling could be due to local changes in morphogen diffusion, representing a novel mechanism in this tissue context, and we propose that this phenomenon might be involved in a broader array of mammalian developmental processes.