Biology subjects

Stetsiv, M.

Publications and source records attributed to Stetsiv, M..

6 recordsLinked to original sources

bioRxiv2026

Inhibition of Dot1L Histone Methyltransferase Expands Bone Injury-Responsive CXCL12⁺ Stromal Progenitors

Adult bone marrow contains a heterogeneous network of skeletal stromal and progenitor cells (SSPC) that maintain bone homeostasis, support the hematopoietic niche, and drive the regenerative responses to injury. Despite their central role in bone regeneration, the mechanisms that regulate injury-induced SSPC activation and fate transitions remain incompletely understood. Here we identify Disruptor of telomeric silencing 1 like (DOT1L), the sole histone methyltransferase responsible for H3K79 methylation, as a critical regulator that restrains stromal activation. Using complementary genetic, pharmacologic, single-cell transcriptomic and injury models, we show that either Prrx1 lineage Dot1L haploinsufficiency or acute pharmacological Dot1L inhibition promote SSPC expansion, but through distinct programs: haploinsufficiency drives the expansion of Cxcl12+ CAR cells, whereas acute pharmacologic inhibition enriches fibroblastic-like stromal states. Single Cell Regulatory Network Inference and Clustering (SCENIC) analysis identifies DOT1L as a stabilizer of homeostatic marrow-supportive transcriptional programs, whose disruption facilitates transition to injury responsive stromal states. Partial loss of Dot1L in the Prrx1 lineage enhances injury-induced intramedullary mineralization in vivo. Collectively, these findings establish DOT1L as a gatekeeper of the stromal progenitor state that restrains lineage commitment and restrains the magnitude of the SSPC response following injury.

cell biology↗

bioRxiv2025

Genetic interaction of Specc1l and Thm1 reveals cytoskeletal - ciliary crosstalk

Cilium formation and actin cytoskeleton dynamics are interconnected, with evidence showing that elevated filamentous actin (F-actin) negatively regulates primary cilia length. Loss of the cytoskeletal protein SPECC1L, which itself does not localize to cilia, leads to increased F-actin and shortened cilia. Depolymerizing F-actin in Specc1l mutant cells restored cilia lengths, substantiating this inverse relationship. In cells harboring a Specc1l allele lacking only the coiled-coil domain 2, intracellular regions with both elevated and reduced F-actin were observed together with cilia shortening. Notably, F-actin was decreased at the ciliary base, suggesting that a different F-actin subpopulation contributes to the inverse relationship. We also identified a genetic interaction between Specc1l and Thm1, which encodes an intraflagellar transport-A (IFT-A) protein. Double or compound heterozygotes for Specc1l and Thm1 exhibited a higher penetrance of cleft palate compared to Specc1l heterozygotes alone. Together, these findings reveal a role for SPECC1L in cytoskeletal regulation of ciliogenesis affecting palate development.

cell biology↗

bioRxiv2024

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using {micro}CT and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stress fractures, cortical defects, trauma from tumor resections, and joint replacements. SUMMARYThe murine bone marrow injury model is a useful tool to study postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.

cell biology↗

bioRxiv2022

Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos

Embryonic palate development involves bilateral vertical growth of palatal shelves - extensions from the maxillary processes - next to the tongue until embryonic day (E) 13.5. Following vertical growth, palatal shelves elevate and adhere above the tongue by E14.5. Current models indicate that this process of elevation involves a complex vertical to horizontal reorienting of the palatal shelves. While earlier studies have implied that this is a rapid process, the precise timing has not been resolved. To understand the dynamics of palatal shelf elevation, we employed time-restricted pregnancies with a one-hour resolution and magnetic resonance imaging of intermediate stages. Our data showed that in almost all C57BL/6J embryos, palatal shelves have not yet elevated by E14.0. However, six hours later at E14.25, palatal shelves have completed elevation in 80% of embryos. Interestingly, all E14.25 embryos with unelevated palatal shelves (20%) were female, suggesting a delay in female embryos. In FVB/NJ embryos, the elevation window started earlier (E13.875-E14.25) without any noticeable sex differences. We frequently captured an intermediate stage with unilateral elevation of either right or left palatal shelf. Magnetic resonance imaging of various stages showed that palatal shelf elevation began with the formation of bilateral bulges in the posterior. These bulges progressed laterally and anteriorly over time. During elevation, we observed increased cell proliferation in the lingual region of the palatal shelf. Within the bulge, cell orientation was acutely tilted towards the tongue and actomyosin activity was increased, which together may participate in the projection of the bulge in the horizontal direction. Thus, our data reveal novel insights into the rapid dynamic changes in palatal shelf elevation that lay the foundation for future studies of normal and abnormal palatogenesis.

developmental biology↗

bioRxiv2021

TWIST1 interacts with adherens junction proteins during neural tube formation and regulates fate transition in cranial neural crest cells

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural fold borders into multiple cell lineages that differentiate into bone, cartilage, neurons, and glial cells. We previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicates that TWIST1 interacts with /{beta}/{gamma}-CATENINS during neural tube closure, and Irf6 is involved in the structural integrity of the neural tube. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects, and CNCC-derived structural abnormalities. Altogether, this study describes an uncharacterized function of Twist1 and Irf6 in the neural tube and CNCCs and provides new target genes of Twist1 involved in cytoskeletal remodeling. Furthermore, the association between DNA variations within TWIST1 putative enhancers and human facial morphology is also investigated. SUMMARY STATEMENTThis study uncovers a new function of Twist1 in neural tube development and epithelial-to-mesenchymal transition in cranial neural crest cells. Data further shows that Twist1-interacting Irf6 is involved in regulating neural tube integrity.

developmental biology↗

bioRxiv2021

In-frame deletion of SPECC1L microtubule binding domain results in embryonic tissue movement and fusion defects

Embryonic morphogenesis of the neural tube, palate, ventral body wall and optic fissure require precise sequence of tissue movement and fusion, which if incomplete, leads to anencephaly/exencephaly, cleft palate, omphalocele and coloboma, respectively. These are genetically heterogeneous birth defects, so there is a continued need to identify etiologic genes. Patients with autosomal dominant SPECC1L mutations show syndromic malformations, including hypertelorism, cleft palate and omphalocele. These SPECC1L mutations cluster in the second coiled-coil domain (CCD2), which facilitates association with microtubules. To study SPECC1L function in mice, we first generated a null allele (Specc1l{Delta}Ex4) lacking the entire SPECC1L protein. Homozygous mutants for these truncations died perinatally without cleft palate or exencephaly. Given the clustering of human mutations in CCD2, we hypothesized that targeted perturbation of CCD2 may be required. Indeed, homozygotes for in-frame deletions involving CCD2 (Specc1l{Delta}CCD2) resulted in ~50% exencephaly and ~50% cleft palate. Interestingly, these two phenotypes are never observed in the same embryo. Examination of embryos with and without exencephaly revealed that the oral cavity was narrower in exencephalic embryos, which allowed palatal shelves to elevate despite their defect. In contrast to an evenly distributed subcellular expression pattern, mutant SPECC1L-{Delta}CCD2 protein showed abnormal subcellular localization, decreased overlap with microtubules, increased actin bundles, and dislocated non-muscle myosin II to the cell cortex. Thus, we show that perturbations of CCD2 in the context of full SPECC1L protein affects tissue fusion dynamics, indicating that human SPECC1L CCD2 mutations are gain-of-function. Improper SPECC1L subcellular localization appears to disrupt connections between actomyosin and microtubule networks, which in turn may affect cell alignment and coordinate movement during tissue morphogenesis.

developmental biology↗