Biology subjects

Bazzi, H.

Publications and source records attributed to Bazzi, H..

8 recordsLinked to original sources

bioRxiv2025

Specific and redundant roles for Gli2 and Gli3 in establishing cell fate during hair follicle development

Ectodermal appendage formation requires hedgehog (HH) signal reception, its conduction through the primary cilium and activation of Gli transcription factors (TF). How HH signalling induces cell-type-specific responses through Gli TF in stem cells (SCs) and their cilia-dependence remain open questions. Here, we use conditional mouse mutants to genetically dissect the roles of Gli and cilia HH mediators in the skin epithelium. Upon Gli2 depletion, hair follicle (HF) morphogenesis is delayed whereas sebaceous gland (SG) formation is enhanced, suggesting a dual role for Gli2 during appendage formation. By controlling proliferation of SG SCs, Gli2 impacts on the number and size of individual SG lobes. Whereas ablation of Gli3 shows no detectable phenotypes, HF cell fate is blocked in Gli2/3 double knockouts. In addition, loss of cilia phenocopies the loss of Gli2 but not the Gli2/3 mutants. Our study reveals that Gli3 exerts activator and cilia-independent functions in the absence of Gli2. Bullet pointsO_LIEpidermal Gli2 and Gli3 together mediate Hedgehog signalling and are essential for proper appendage formation C_LIO_LIGli3 activator function compensates for the loss of Gli2 during HF morphogenesis C_LIO_LIGli2 controls HF stem cell sub-compartments and SG formation C_LIO_LICilia mediate epidermal Gli2 but not Gli3 signalling in the skin epithelium C_LI

cell biology↗

bioRxiv2023

Generation and characterization of a Dkk4-Cre knock-in mouse line

Ectodermal appendages in mammals, such as teeth, mammary glands, sweat glands and hair follicles, are generated during embryogenesis through a series of mesenchymal-epithelial interactions. Canonical Wnt signaling and its inhibitors are implicated in the early steps of ectodermal appendage development and patterning. To study the activation dynamics of the Wnt target and inhibitor Dickkopf4 (Dkk4) in ectodermal appendages, we used CRSIPR/Cas9 to generate a Dkk4-Cre knock-in mouse (Mus musculus) line, where the Cre recombinase cDNA replaces the expression of endogenous Dkk4. Using Cre reporters, the Dkk4-Cre activity was evident at the prospective sites of ectodermal appendages, overlapping with the Dkk4 mRNA expression. Unexpectedly, a predominantly mesenchymal cell population in the embryo posterior also showed Dkk4-Cre activity. Lineage-tracing suggested that these cells are likely derived from a few Dkk4-Cre-expressing cells in the epiblast at early gastrulation. Finally, our analyses of Dkk4-Cre-expressing cells in developing hair follicle epithelial placodes revealed intra- and inter-placodal cellular heterogeneity, supporting emerging data on the positional and transcriptional variability in placodes. Collectively, we propose the new Dkk4-Cre knock-in mouse line as a suitable model to study Wnt and DKK4 inhibitor dynamics in early mouse development and ectodermal appendage morphogenesis.

developmental biology↗

bioRxiv2022

Mouse SAS-6 is required for centriole formation in embryos and integrity in embryonic stem cells

Cell division fidelity is crucial for stem cell propagation and the maintenance of pluripotency. Centrosomes, which organize the mitotic spindle microtubules to ensure proper bipolar cell division, have a core of a pair of centrioles that duplicate once per cell cycle. At the onset of centriole biogenesis, SAS-6 forms a cartwheel structure, which is the precursor for the forming procentrioles. SAS-6 is essential for centriole formation in human cell lines and other organisms. However, the functions of SAS-6 in mouse stem cells remain to be elucidated. Here, we report that Sas-6-null mouse embryos lack centrioles, activate the mitotic surveillance cell death pathway and arrest at mid-gestation. In contrast, SAS-6 is not strictly required for centriole formation in mouse embryonic stem cells (mESCs) in vitro, but is still important to regulate centriole length, symmetry and ability to template cilia. Remarkably, centrioles appeared after just one day of culture of Sas-6-null blastocysts, from which mESCs are normally derived. Finally, the number of cells with centrosomes is drastically decreased upon the exit from a pluripotent state. Collectively, our data suggest a differential requirement for mouse SAS-6 in centriole formation or integrity depending on the cellular context, and highlight the robustness of mESCs in using SAS-6-independent centriole-duplication pathways.

developmental biology↗

bioRxiv2021

Translational specialization in pluripotency by RBPMS poises future lineage-decisions

The blueprints for developing organs are preset at the early stages of embryogenesis. Transcriptional and epigenetic mechanisms are proposed to preset developmental trajectories. However, we reveal that the competence for future cardiac fate of human embryonic stem cells (hESCs) is preset in pluripotency by a specialized mRNA translation circuit controlled by RBPMS. RBPMS is recruited to active ribosomes in hESCs to control the translation of essential factors needed for cardiac commitment program, including WNT signaling. Consequently, RBPMS loss specifically and severely impedes cardiac mesoderm specification leading to patterning and morphogenesis defects in human cardiac organoids. Mechanistically, RBPMS specializes mRNA translation, selectively via 3UTR binding and globally by promoting translation initiation. Accordingly, RBPMS loss causes translation initiation defects highlighted by aberrant retention of the EIF3 complex and depletion of EIF5A from mRNAs, thereby abrogating ribosome recruitment. We reveal how future fate trajectories are preprogrammed during embryogenesis by specialized mRNA translation. Teaser: Cardiac fate competence is preprogrammed in pluripotency by specialized mRNA translation of factors initiating cardiogenesis

developmental biology↗

bioRxiv2021

The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

Poised enhancers (PEs) represent a limited and genetically distinct set of distal regulatory elements that control the induction of developmental genes in a hierarchical and non-redundant manner. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally identified and subsequently characterized using embryonic stem cells (ESC) as an in vitro differentiation system, it is currently unknown whether PEs are functionally conserved in vivo. Here, we generate and mine various types of genomic data to show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that, in mouse pluripotent cells, the interactions between PEs and their bivalent target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. proximity to CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation, preferentially within specific vertebrate clades, with only a small subset being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting evolutionary conserved PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential and non-redundant roles during the induction of major developmental genes in vivo.

molecular biology↗

bioRxiv2020

Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development

Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas-4 null mice leads to embryonic arrest around E9. Centriole loss in Sas-4-/- embryos causes prolonged mitosis and p53-dependent cell death. Studies in vitro discovered a similar USP28-, 53BP1-, and p53-dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas-4-/- embryos. The data indicates that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.

developmental biology↗

bioRxiv2020

Epidermal stratification is uncoupled from centrosome-dependent cell division orientation of the basal progenitors

The development of complex stratified epithelial barriers in mammals is initiated from single-layered epithelia. How stratification is initiated and fueled are still open questions. Previous studies on skin epidermal stratification suggested a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors. Here, we use centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetically ablating centrosomes from the developing epidermis led to the activation of the p53-, 53BP1- and USP28-dependent mitotic surveillance pathway causing a thinner epidermis and hair follicle arrest. The centrosome/p53-double mutant keratinocyte progenitors significantly altered their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggested that the first and major phase of epidermal stratification is boosted by high proliferation rates of the basal and suprabasal transit-amplifying cells as well as cell delamination, whereas the second phase maybe uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

developmental biology↗

bioRxiv2019

Centrioles control the capacity, but not the specificity, of cytotoxic T cell killing

Immunological synapse formation between cytotoxic T lymphocytes (CTLs) and the target cells they aim to destroy is accompanied by reorientation of the CTL centrosome to a position beneath the synaptic membrane. Centrosome polarization is thought to enhance the potency and specificity of killing by driving lytic granule fusion at the synapse and thereby the release of perforin and granzymes toward the target cell. To test this model, we employed a genetic strategy to delete centrioles, the core structural components of the centrosome. Centriole deletion altered microtubule architecture, as expected, but surprisingly had no effect on lytic granule polarization and directional secretion. Nevertheless, CTLs lacking centrioles did display substantially reduced killing potential, which was associated with defects in both lytic granule biogenesis and synaptic actin remodeling. These results reveal an unexpected role for the intact centrosome in controlling the capacity, but not the specificity, of cytotoxic killing.

immunology↗