Cre recombinase microinjection for single-cell tracing and localised gene targeting.

Tracing and manipulating cells in embryos are essential to understand development. Lipophilic dye microinjections, viral transfection and iontophoresis have been key to map the origin of the progenitor cells that form the different organs in the post-implantation mouse embryo. These techniques require advanced manipulation skills and only iontophoresis, a demanding approach of limited efficiency, has been used for single-cell labelling. Here, we perform lineage tracing and local gene ablation using cell-permeant Cre recombinase (TAT-Cre) microinjection. First, we map the fate of undifferentiated progenitors to the different heart chambers. Then, we achieve single-cell recombination by titrating the dose of TAT-Cre, which allows clonal analysis of nascent mesoderm progenitors. Finally, injecting TAT-Cre to Mycnflox/flox embryos in the primitive heart tube revealed that Mycn plays a cell-autonomous role in maintaining cardiomyocyte proliferation. This tool will help researchers identify the cell progenitors and gene networks involved in organ development, helping to understand the origin of congenital defects.

Dissecting the Complexity of Early Heart Progenitor Cells.

Early heart development depends on the coordinated participation of heterogeneous cell sources. As pioneer work from Adriana C. Gittenberger-de Groot demonstrated, characterizing these distinct cell sources helps us to understand congenital heart defects. Despite decades of research on the segregation of lineages that form the primitive heart tube, we are far from understanding its full complexity. Currently, single-cell approaches are providing an unprecedented level of detail on cellular heterogeneity, offering new opportunities to decipher its functional role. In this review, we will focus on three key aspects of early heart morphogenesis: First, the segregation of myocardial and endocardial lineages, which yields an early lineage diversification in cardiac development; second, the signaling cues driving differentiation in these progenitor cells; and third, the transcriptional heterogeneity of cardiomyocyte progenitors of the primitive heart tube. Finally, we discuss how single-cell transcriptomics and epigenomics, together with live imaging and functional analyses, will likely transform the way we delve into the complexity of cardiac development and its links with congenital defects.

In primary airway epithelial cells, the unjamming transition is distinct from the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.

A gene regulatory network to control EMT programs in development and disease.

The Epithelial to Mesenchymal Transition (EMT) regulates cell plasticity during embryonic development and in disease. It is dynamically orchestrated by transcription factors (EMT-TFs), including Snail, Zeb, Twist and Prrx, all activated by TGF-ß among other signals. Here we find that Snail1 and Prrx1, which respectively associate with gain or loss of stem-like properties and with bad or good prognosis in cancer patients, are expressed in complementary patterns during vertebrate development and in cancer. We show that this complementarity is established through a feedback loop in which Snail1 directly represses Prrx1, and Prrx1, through direct activation of the miR-15 family, attenuates the expression of Snail1. We also describe how this gene regulatory network can establish a hierarchical temporal expression of Snail1 and Prrx1 during EMT and validate its existence in vitro and in vivo, providing a mechanism to switch and select different EMT programs with important implications in development and disease.

MicroRNAs Establish the Right-Handed Dominance of the Heart Laterality Pathway in Vertebrates.

Despite their external bilateral symmetry, vertebrates have internal left/right (L/R) asymmetries required for optimal organ function. BMP-induced epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) triggers L/R asymmetric cell movements toward the midline, higher from the right, which are crucial for heart laterality in vertebrates. However, how the L/R asymmetric levels of EMT factors are achieved is not known. Here, we show that the posterior-to-anterior Nodal wave upregulates several microRNAs (miRNAs) to transiently attenuate the levels of EMT factors (Prrx1a and Snail1) on the left LPM in a Pitx2-independent manner in the fish and mouse. These data clarify the role of Nodal in heart laterality and explain how Nodal and BMP exert their respective dominance on the left and right sides through the mutual inhibition of their respective targets, ensuring the proper balance of L/R information required for heart laterality and morphogenesis.

A right-handed signalling pathway drives heart looping in vertebrates.

Most animals show external bilateral symmetry, which hinders the observation of multiple internal left-right (L/R) asymmetries that are fundamental to organ packaging and function. In vertebrates, left identity is mediated by the left-specific Nodal-Pitx2 axis that is repressed on the right-hand side by the epithelial-mesenchymal transition (EMT) inducer Snail1 (refs 3, 4). Despite some existing evidence, it remains unclear whether an equivalent instructive pathway provides right-hand-specific information to the embryo. Here we show that, in zebrafish, BMP mediates the L/R asymmetric activation of another EMT inducer, Prrx1a, in the lateral plate mesoderm with higher levels on the right. Prrx1a drives L/R differential cell movements towards the midline, leading to a leftward displacement of the cardiac posterior pole through an actomyosin-dependent mechanism. Downregulation of Prrx1a prevents heart looping and leads to mesocardia. Two parallel and mutually repressed pathways, respectively driven by Nodal and BMP on the left and right lateral plate mesoderm, converge on the asymmetric activation of the transcription factors Pitx2 and Prrx1, which integrate left and right information to govern heart morphogenesis. This mechanism is conserved in the chicken embryo, and in the mouse SNAIL1 acts in a similar manner to Prrx1a in zebrafish and PRRX1 in the chick. Thus, a differential L/R EMT produces asymmetric cell movements and forces, more prominent from the right, that drive heart laterality in vertebrates.

Snail2 and Zeb2 repress P-cadherin to define embryonic territories in the chick embryo.

Snail and Zeb transcription factors induce epithelial-to-mesenchymal transition (EMT) in embryonic and adult tissues by direct repression of E-cadherin transcription. The repression of E-cadherin transcription by the EMT inducers Snail1 and Zeb2 plays a fundamental role in defining embryonic territories in the mouse, as E-cadherin needs to be downregulated in the primitive streak and in the epiblast, concomitant with the formation of mesendodermal precursors and the neural plate, respectively. Here, we show that in the chick embryo, E-cadherin is weakly expressed in the epiblast at pre-primitive streak stages where it is substituted for by P-cadherin We also show that Snail2 and Zeb2 repress P-cadherin transcription in the primitive streak and the neural plate, respectively. This indicates that E- and P-cadherin expression patterns evolved differently between chick and mouse. As such, the Snail1/E-cadherin axis described in the early mouse embryo corresponds to Snail2/P-cadherin in the chick, but both Snail factors and Zeb2 fulfil a similar role in chick and mouse in directly repressing ectodermal cadherin genes to contribute to the delamination of mesendodermal precursors at gastrulation and the proper specification of the neural ectoderm during neural induction.

Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1.

The epithelial-mesenchymal transition (EMT) is required in the embryo for the formation of tissues for which cells originate far from their final destination. Carcinoma cells hijack this program for tumor dissemination. The relevance of the EMT in cancer is still debated because it is unclear how these migratory cells colonize distant tissues to form macrometastases. We show that the homeobox factor Prrx1 is an EMT inducer conferring migratory and invasive properties. The loss of Prrx1 is required for cancer cells to metastasize in vivo, which revert to the epithelial phenotype concomitant with the acquisition of stem cell properties. Thus, unlike the classical EMT transcription factors, Prrx1 uncouples EMT and stemness, and is a biomarker associated with patient survival and lack of metastasis.

Mutual exclusion of transcription factors and cell behaviour in the definition of vertebrate embryonic territories.

Early embryonic territories are transient entities under permanent remodelling to form newly derived cell populations that will eventually give rise to the adult tissues and organs. A vast effort has been devoted to identifying the determinants and mechanisms that define embryonic territories. Indeed, studies in the vertebrate embryo from the morula stage to the segregation of the main embryonic layers-ectoderm, mesoderm and endoderm-have highlighted the importance of the mutual exclusion/repression between pairs of transcription factors, in coordination with the control exerted over cell division, adhesion and motility.

Reciprocal repression between Sox3 and snail transcription factors defines embryonic territories at gastrulation.

In developing amniote embryos, the first epithelial-to-mesenchymal transition (EMT) occurs at gastrulation, when a subset of epiblast cells moves to the primitive streak and undergoes EMT to internalize and generate the mesoderm and the endoderm. We show that in the chick embryo this decision to internalize is mediated by reciprocal transcriptional repression of Snail2 and Sox3 factors. We also show that the relationship between Sox3 and Snail is conserved in the mouse embryo and in human cancer cells. In the embryo, Snail-expressing cells ingress at the primitive streak, whereas Sox3-positive cells, which are unable to ingress, ensure the formation of ectodermal derivatives. Thus, the subdivision of the early embryo into the two main territories, ectodermal and mesendodermal, is regulated by changes in cell behavior mediated by the antagonistic relationship between Sox3 and Snail transcription factors.

Attenuation of Notch signalling by the Down-syndrome-associated kinase DYRK1A.

Notch signalling is used throughout the animal kingdom to spatially and temporally regulate cell fate, proliferation and differentiation. Its importance is reflected in the dramatic effects produced on both development and health by small variations in the strength of the Notch signal. The Down-syndrome-associated kinase DYRK1A is coexpressed with Notch in various tissues during embryonic development. Here we show that DYRK1A moves to the nuclear transcription compartment where it interacts with the intracellular domain of Notch promoting its phosphorylation in the ankyrin domain and reducing its capacity to sustain transcription. DYRK1A attenuates Notch signalling in neural cells both in culture and in vivo, constituting a novel mechanism capable of modulating different developmental processes that can also contribute to the alterations observed during brain development in animal models of Down syndrome.

Snail genes at the crossroads of symmetric and asymmetric processes in the developing mesoderm.

Retinoic acid (RA) signalling ensures that vertebrate mesoderm segmentation is bilaterally synchronized, and corrects transient interferences from asymmetric left-right (L-R) signals involved in organ lateralization. Snail genes participate in both these processes and, although they are expressed symmetrically in the presomitic mesoderm (PSM), Snail1 transcripts are asymmetrically distributed in the L-R lateral mesoderm. We show that the alteration of the symmetric Snail expression in the PSM induces asynchronous somite formation. Furthermore, in the absence of RA signalling, normal asymmetric Snail1 expression in the lateral mesoderm is extended to the PSM, desynchronizing somitogenesis. Thus, Snail1 is the first cue corrected by RA in the PSM to ensure synchronized bilateral segmentation.

Snail blocks the cell cycle and confers resistance to cell death.

The Snail zinc-finger transcription factors trigger epithelial-mesenchymal transitions (EMTs), endowing epithelial cells with migratory and invasive properties during both embryonic development and tumor progression. During EMT, Snail provokes the loss of epithelial markers, as well as changes in cell shape and the expression of mesenchymal markers. Here, we show that in addition to inducing dramatic phenotypic alterations, Snail attenuates the cell cycle and confers resistance to cell death induced by the withdrawal of survival factors and by pro-apoptotic signals. Hence, Snail favors changes in cell shape versus cell division, indicating that with respect to oncogenesis, although a deregulation/increase in proliferation is crucial for tumor formation and growth, this may not be so for tumor malignization. Finally, the resistance to cell death conferred by Snail provides a selective advantage to embryonic cells to migrate and colonize distant territories, and to malignant cells to separate from the primary tumor, invade, and form metastasis.

Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell-fates in Xenopus.

We analysed the role of Notch signalling during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway we found that Notch expands the floor plate domain of sonic hedgehog and pintallavis and represses the notochordal markers chordin and brachyury, with a concomitant reduction of the notochord size. We propose that within a population of the early organiser with equivalent potential to develop either as notochord or floor plate, Notch activation favours floor plate development at the expense of the notochord, preferentially before mid gastrula. We present evidence that sonic hedgehog down-regulates chordin, suggesting that secreted Sonic hedgehog may be involved or reinforcing the cell-fate switch executed by Notch. We also show that Notch signalling requires Presenilin to modulate this switch.