SAS-6 (SASS6) is essential for centriole formation in human cells and other organisms but its functions in the mouse are unclear. Here, we report that
-mutant mouse embryos lack centrioles, activate ...the mitotic surveillance cell death pathway, and arrest at mid-gestation. In contrast, SAS-6 is not required for centriole formation in mouse embryonic stem cells (mESCs), but is essential to maintain centriole architecture. Of note, centrioles appeared after just one day of culture of
-mutant blastocysts, from which mESCs are derived. Conversely, the number of cells with centrosomes is drastically decreased upon the exit from a mESC pluripotent state. At the mechanistic level, the activity of the master kinase in centriole formation, PLK4, associated with increased centriolar and centrosomal protein levels, endow mESCs with the robustness in using a SAS-6-independent centriole-biogenesis pathway. Collectively, our data suggest a differential requirement for mouse SAS-6 in centriole formation or integrity depending on PLK4 activity and centrosome composition.
Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize ...centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4 -null embryos lack centrosomes but survive until midgestation. As expected, Sas4 ⁻/⁻ mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4 ⁻/⁻ embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4 ⁻/⁻ p53 ⁻/⁻ double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4 ⁻/⁻ mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo.
Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. 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 characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we 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 the interactions between PEs and their 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. 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 within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo.
Abstract
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 leads 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 alter their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggest that the first and major phase of epidermal development is boosted by high proliferation rates in both basal and suprabasally-committed keratinocytes 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.
Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the ...functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illuminates an unexpected function for polycomb proteins in facilitating neural induction by endowing major anterior neural loci with a permissive regulatory topology.
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•Poised enhancers are necessary for the induction of major anterior neural genes•Poised enhancers physically contact their target genes in embryonic stem cells•The interaction between poised enhancers and their target genes is PRC2 dependent•PRC2 provides major anterior neural loci with a permissive regulatory topology
Cruz-Molina et al. demonstrate that pluripotent-associated poised enhancers are necessary for the induction of anterior neural genes. Poised enhancers physically interact with their target genes in embryonic stem cells in a PRC2-dependent manner. Thus, polycomb proteins might facilitate neural induction by providing anterior neural loci with a permissive regulatory topology.
While the transcriptional network of human embryonic stem cells (hESCs) has been extensively studied, relatively little is known about how post-transcriptional modulations determine hESC function. ...RNA-binding proteins play central roles in RNA regulation, including translation and turnover. Here we show that the RNA-binding protein CSDE1 (cold shock domain containing E1) is highly expressed in hESCs to maintain their undifferentiated state and prevent default neural fate. Notably, loss of CSDE1 accelerates neural differentiation and potentiates neurogenesis. Conversely, ectopic expression of CSDE1 impairs neural differentiation. We find that CSDE1 post-transcriptionally modulates core components of multiple regulatory nodes of hESC identity, neuroectoderm commitment and neurogenesis. Among these key pro-neural/neuronal factors, CSDE1 binds fatty acid binding protein 7 (FABP7) and vimentin (VIM) mRNAs, as well as transcripts involved in neuron projection development regulating their stability and translation. Thus, our results uncover CSDE1 as a central post-transcriptional regulator of hESC identity and neurogenesis.
Summary
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 cellular 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.
A key initial event in hair follicle morphogenesis is the localised thickening of the skin epithelium to form a placode, partitioning future hair follicle epithelium from interfollicular epidermis. ...Although many developmental signalling pathways are implicated in follicle morphogenesis, the role of epidermal growth factor (EGF) and keratinocyte growth factor (KGF, also known as FGF7) receptors are not defined. EGF receptor (EGFR) ligands have previously been shown to inhibit developing hair follicles; however, the underlying mechanisms have not been characterised. Here we show that receptors for EGF and KGF undergo marked downregulation in hair follicle placodes from multiple body sites, whereas the expression of endogenous ligands persist throughout hair follicle initiation. Using embryonic skin organ culture, we show that when skin from the sites of primary pelage and whisker follicle development is exposed to increased levels of two ectopic EGFR ligands (HBEGF and amphiregulin) and the FGFR2(IIIb) receptor ligand KGF, follicle formation is inhibited in a time- and dose-dependent manner. We then used downstream molecular markers and microarray profiling to provide evidence that, in response to KGF and EGF signalling, epidermal differentiation is promoted at the expense of hair follicle fate. We propose that hair follicle initiation in placodes requires downregulation of the two pathways in question, both of which are crucial for the ongoing development of the interfollicular epidermis. We have also uncovered a previously unrecognised role for KGF signalling in the formation of hair follicles in the mouse.
Ectodermal appendage morphogenesis requires continuous epithelial–mesenchymal cross-talk during development. Canonical Wnt signaling has been shown to be pivotal during this process and its ...inhibition leads to the absence of any morphological or molecular signs of appendage formation, including hair follicles (HFs). In the mouse, primary HFs arise
in utero starting just before E14.5, when the first morphological signs of a placode are discernible. In this study, our goal was to identify novel factors expressed during primary HF morphogenesis. We performed transcriptional profiling of the developing epidermis at 12 h intervals between E12.5 and E15.5. One of the significantly differentially expressed genes was the Wnt inhibitor
Dickkopf 4,
Dkk4. We show that Dkk4 mRNA increases sharply in the dorso-lateral epidermis around E14 and then decreases until E15.5. Using whole mount
in situ hybridization, we show that
Dkk4 mRNA is localized to the pre-placodes at sites of presumptive epithelial–mesenchymal interactions during appendage morphogenesis, including the dental lamina, mammary gland, eccrine gland, and primary and secondary HFs.
In silico analysis, reporter gene assays as well as
in vitro transfections of LEF1 and β-catenin show that
Dkk4 is a potential downstream target of canonical Wnt signaling. In addition, we demonstrate a direct physical interaction between LEF1/β-catenin complex and the
Dkk4 promoter using ChIP. We propose that Dkk4 acts in a negative feedback loop to attenuate canonical Wnt signaling, and may facilitate a switch to the non-canonical Wnt planar cell polarity (PCP) pathway that is involved in cell movements during morphogenesis.
Centrosomes are dominant microtubule organizing centers in animal cells with a pair of centrioles at their core. They template cilia during interphase and help organize the mitotic spindle for a more ...efficient cell division. Here, we review the roles of centrosomes in the early developing mouse and during organ formation. Mammalian cells respond to centrosome loss‐of‐function by activating the mitotic surveillance pathway, a timing mechanism that, when a defined mitotic duration is exceeded, leads to p53‐dependent cell death in the descendants. Mouse embryos without centrioles are highly susceptible to this pathway and undergo embryonic arrest at mid‐gestation. The complete loss of the centriolar core results in earlier and more severe phenotypes than that of other centrosomal proteins. Finally, different developing tissues possess varying thresholds and mount graded responses to the loss of centrioles that go beyond the germ layer of origin.
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