Biology subjects

Pagella, P.

Publications and source records attributed to Pagella, P..

5 recordsLinked to original sources

bioRxiv2022

Notch signaling pathway in tooth shape variations

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth-crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth-crown shape modifications convergent to those observed along Muridae evolution. These alterations are due to the modulation of numerous genes, where Notch signaling is a hub for essential morphogenetic networks. A three-dimensional metamorphosis approach allowed tooth morphology prediction in individuals carrying Jagged1 mutations. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution. Significance statementDental microevolution changes in vertebrates are regulated by the Notch signaling pathway.

developmental biology↗

bioRxiv2022

Nogo-A regulates the fate of human dental pulp stem cells towards osteogenic, adipogenic, and neurogenic differentiation

Human teeth are highly innervated organs that contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. Specific molecules are often used in these treatments to favorably modulate stem cells function and fate. Nogo-A, a key regulator of neuronal growth and differentiation, is already used in clinical tissue regeneration trials. While the functions of Nogo-A in neuronal tissues are extensively explored, its role in teeth still remains unknown. In this work, we first immunohistochemically analyzed the distribution of Nogo-A protein in the dental pulp of human teeth. Nogo-A is localized in a variety of cellular and structural components of the dental pulp, including odontoblasts, fibroblasts, neurons and vessels. We also cross-examined Nogo expression in the various pulp cell clusters in a single cell RNA sequencing dataset of human dental pulp, which showed high levels of expression in all cell clusters, including that of stem cells. We then assessed the role of Nogo-A on the fate of human dental pulp stem cells and their differentiation capacity in vitro. Using immunostaining, Alizarin Red S and Oil Red O staining we showed that Nogo-A delayed the differentiation of cultured dental pulp stem cells towards the osteogenic, adipogenic and neurogenic lineages, while addition of the blocking anti-Nogo-A antibody had opposite effects. These results were further confirmed by qRT-PCR, which demonstrated overexpression of genes involved in osteogenic (RUNX2, ALP, SP7/OSX), adipogenic (PPAR-{gamma}2, LPL) and neurogenic (DCX, TUBB3, NEFL) differentiation in presence of the anti-Nogo-A antibody. Conversely, the osteogenic and adipogenic genes were downregulated by Nogo-A. Taken together, our results show that the functions of Nogo-A are not restricted to neuronal cells, but are extended to other cell populations, including dental pulp stem cells. We show that Nogo-A regulates their fates towards osteogenic, adipogenic and neurogenic differentiation, thus indicating its potential use in the clinics.

cell biology↗

bioRxiv2022

Time-resolved analysis of Wnt-signaling reveals β-catenin temporal genomic repositioning and cell type-specific plastic or elastic chromatin responses

Wnt signaling orchestrates gene expression via its effector {beta}-catenin. Whether {beta}-catenin targets genomic regions simultaneously or in a temporal fashion, and how this impacts the chromatin dynamics to modulate cell behavior, is currently unknown. Here we find that {beta}-catenin binds different loci at each time-point after stimulation, implying that the definition of Wnt-targets is fundamentally temporal. This process is intrinsically cell-type specific. In fact, Wnt/{beta}-catenin progressively shapes the chromatin of human embryonic stem cells consistent with their mesodermal differentiation: we call this genomic response plastic. In embryonic kidney cells, on the other hand, Wnt/{beta}-catenin drives a transient chromatin opening, followed by a re-establishment of the pre-stimulation state: a response that we define elastic. Finally, the Wnt-induced transient chromatin opening requires {beta}-catenin, suggesting a previously unappreciated pioneering role for this molecule. We submit that the plastic-vs-elastic behavior constitutes part of the mechanism explaining how Wnt/{beta}-catenin drives divergent cell-fate decisions during development and homeostasis.

genomics↗

bioRxiv2022

A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets

Upon WNT/{beta}-catenin pathway activation, stabilized {beta}-catenin travels to the nucleus where it associates with the TCF/LEF family of transcription factors, which constitutively bind to genomic Wnt Responsive Elements (WREs), to activate transcription of target genes. Discovering the binding profile of {beta}-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage Under Target and Release Using Nuclease (CUT&RUN) has recently emerged as a prime technique for mapping the binding profile of chromatin interacting proteins. In our attempts to profile different regulators of the WNT/{beta}-catenin transcriptional complex, CUT&RUN performed reliably when targeting transcription factors such as TCF/LEF, but it failed to produce consistent binding patterns of the non-DNA-binding {beta}-catenin. Here, we present a biochemical modification of the CUT&RUN protocol, which we refer to as LoV-U (Low Volume and Urea), that enables the generation of robust and reproducible {beta}-catenin binding profiles. CUT&RUN-LoV-U uncovers direct WNT/{beta}-catenin target genes in human cells, as well as in ex vivo cells isolated from developing mouse tissue. CUT&RUN-LoV-U can profile all classes of chromatin regulators tested and is well suited for simultaneous processing of several samples. We submit that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/{beta}-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades.

developmental biology↗

bioRxiv2021

A single cell atlas of human teeth

Teeth exert fundamental functions related to mastication and speech. Despite their great biomedical importance, an overall picture of their cellular and molecular composition is still missing. In this study, we have mapped the transcriptional landscape of the various cell populations that compose human teeth at single-cell resolution, and we analyzed in deeper detail their stem cell populations and their microenvironment. Our study identified great cellular heterogeneity in the dental pulp and the periodontium. Unexpectedly, we found that the molecular signatures of the stem cell populations were very similar, and that their distinctive behavior could be due to substantial differences between their microenvironments. Our findings suggest that the microenvironmental specificity is the potential source for the major functional differences of the stem cells located in the various tooth compartments and open new perspectives towards cell-based dental therapeutic approaches.

cell biology↗