Nervous System Regionalization Entails Axial Allocation before Neural Differentiation.

Neural induction in vertebrates generates a CNS that extends the rostral-caudal length of the body. The prevailing view is that neural cells are initially induced with anterior (forebrain) identity; caudalizing signals then convert a proportion to posterior fates (spinal cord). To test this model, we used chromatin accessibility to define how cells adopt region-specific neural fates. Together with genetic and biochemical perturbations, this identified a developmental time window in which genome-wide chromatin-remodeling events preconfigure epiblast cells for neural induction. Contrary to the established model, this revealed that cells commit to a regional identity before acquiring neural identity. This "primary regionalization" allocates cells to anterior or posterior regions of the nervous system, explaining how cranial and spinal neurons are generated at appropriate axial positions. These findings prompt a revision to models of neural induction and support the proposed dual evolutionary origin of the vertebrate CNS.

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

Steering cell migration by alternating blebs and actin-rich protrusions.

BACKGROUND: High directional persistence is often assumed to enhance the efficiency of chemotactic migration. Yet, cells in vivo usually display meandering trajectories with relatively low directional persistence, and the control and function of directional persistence during cell migration in three-dimensional environments are poorly understood. RESULTS: Here, we use mesendoderm progenitors migrating during zebrafish gastrulation as a model system to investigate the control of directional persistence during migration in vivo. We show that progenitor cells alternate persistent run phases with tumble phases that result in cell reorientation. Runs are characterized by the formation of directed actin-rich protrusions and tumbles by enhanced blebbing. Increasing the proportion of actin-rich protrusions or blebs leads to longer or shorter run phases, respectively. Importantly, both reducing and increasing run phases result in larger spatial dispersion of the cells, indicative of reduced migration precision. A physical model quantitatively recapitulating the migratory behavior of mesendoderm progenitors indicates that the ratio of tumbling to run times, and thus the specific degree of directional persistence of migration, are critical for optimizing migration precision. CONCLUSIONS: Together, our experiments and model provide mechanistic insight into the control of migration directionality for cells moving in three-dimensional environments that combine different protrusion types, whereby the proportion of blebs to actin-rich protrusions determines the directional persistence and precision of movement by regulating the ratio of tumbling to run times.

Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis.

During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. In this study, we show that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx3 mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis.

A polarised population of dynamic microtubules mediates homeostatic length control in animal cells.

Because physical form and function are intimately linked, mechanisms that maintain cell shape and size within strict limits are likely to be important for a wide variety of biological processes. However, while intrinsic controls have been found to contribute to the relatively well-defined shape of bacteria and yeast cells, the extent to which individual cells from a multicellular animal control their plastic form remains unclear. Here, using micropatterned lines to limit cell extension to one dimension, we show that cells spread to a characteristic steady-state length that is independent of cell size, pattern width, and cortical actin. Instead, homeostatic length control on lines depends on a population of dynamic microtubules that lead during cell extension, and that are aligned along the long cell axis as the result of interactions of microtubule plus ends with the lateral cell cortex. Similarly, during the development of the zebrafish neural tube, elongated neuroepithelial cells maintain a relatively well-defined length that is independent of cell size but dependent upon oriented microtubules. A simple, quantitative model of cellular extension driven by microtubules recapitulates cell elongation on lines, the steady-state distribution of microtubules, and cell length homeostasis, and predicts the effects of microtubule inhibitors on cell length. Together this experimental and theoretical analysis suggests that microtubule dynamics impose unexpected limits on cell geometry that enable cells to regulate their length. Since cells are the building blocks and architects of tissue morphogenesis, such intrinsically defined limits may be important for development and homeostasis in multicellular organisms.

Involvement of Lgl and Mahjong/VprBP in cell competition.

During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby "loser cells" undergo apoptosis are not clearly understood. Lgl (lethal giant larvae) is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj(-/-)) do not have obvious patterning defects during embryonic or larval development. However, mahj(-/-) cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl(-/-) cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK) is increased in mahj(-/-) or lgl(-/-) mutant cells, and expression of Puckered (Puc), an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl(-/-) cells strongly suppresses JNK activation and blocks apoptosis of lgl(-/-) cells in the wild-type wing disc epithelium. These data indicate that Mahjong interacts with Lgl biochemically and genetically and that Mahjong and Lgl function in the same pathway to regulate cellular competitiveness. As far as we are aware, this is the first report that cell competition can occur in a mammalian cell culture system.

Developmental cell fate choice in neural tube progenitors employs two distinct cis-regulatory strategies.

In many developing tissues, the patterns of gene expression that assign cell fate are organized by graded secreted signals. Cis-regulatory elements (CREs) interpret these signals to control gene expression, but how this is accomplished remains poorly understood. In the neural tube, a gradient of the morphogen sonic hedgehog (Shh) patterns neural progenitors. We identify two distinct ways in which CREs translate graded Shh into differential gene expression in mouse neural progenitors. In most progenitors, a common set of CREs control gene activity by integrating cell-type-specific inputs. By contrast, the most ventral progenitors use a unique set of CREs, established by the pioneer factor FOXA2. This parallels the role of FOXA2 in endoderm, where FOXA2 binds some of the same sites. Together, the data identify distinct cis-regulatory strategies for the interpretation of morphogen signaling and raise the possibility of an evolutionarily conserved role for FOXA2 across tissues.

Correction: Ivanovitch et al. Growth and Morphogenesis during Early Heart Development in Amniotes. J. Cardiovasc. Dev. Dis. 2017, 4, 20.

In the published publication [...].

Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis.

During vertebrate heart development, two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here, we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development.

Growth and Morphogenesis during Early Heart Development in Amniotes.

In this review, we will focus on the growth and morphogenesis of the developing heart, an aspect of cardiovascular development to which Antoon Moorman and colleagues have extensively contributed. Over the last decades, genetic studies and characterization of regionally regulated gene programs have provided abundant novel insights into heart development essential to understand the basis of congenital heart disease. Heart morphogenesis, however, is inherently a complex and dynamic three-dimensional process and we are far from understanding its cellular basis. Here, we discuss recent advances in studying heart morphogenesis and regionalization under the light of the pioneering work of Moorman and colleagues, which allowed the reinterpretation of regional gene expression patterns under a new morphogenetic framework. Two aspects of early heart formation will be discussed in particular: (1) the initial formation of the heart tube and (2) the formation of the cardiac chambers by the ballooning process. Finally, we emphasize that in addition to analyses based on fixed samples, new approaches including clonal analysis, single-cell sequencing, live-imaging and quantitative analysis of the data generated will likely lead to novel insights in understanding early heart tube regionalization and morphogenesis in the near future.

A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics.

How left-right patterning drives asymmetric morphogenesis is unclear. Here, we have quantified shape changes during mouse heart looping, from 3D reconstructions by HREM. In combination with cell labelling and computer simulations, we propose a novel model of heart looping. Buckling, when the cardiac tube grows between fixed poles, is modulated by the progressive breakdown of the dorsal mesocardium. We have identified sequential left-right asymmetries at the poles, which bias the buckling in opposite directions, thus leading to a helical shape. Our predictive model is useful to explore the parameter space generating shape variations. The role of the dorsal mesocardium was validated in Shh-/- mutants, which recapitulate heart shape changes expected from a persistent dorsal mesocardium. Our computer and quantitative tools provide novel insight into the mechanism of heart looping and the contribution of different factors, beyond the simple description of looping direction. This is relevant to congenital heart defects.

Precocious acquisition of neuroepithelial character in the eye field underlies the onset of eye morphogenesis.

Using high-resolution live imaging in zebrafish, we show that presumptive eye cells acquire apicobasal polarity and adopt neuroepithelial character prior to other regions of the neural plate. Neuroepithelial organization is first apparent at the margin of the eye field, whereas cells at its core have mesenchymal morphology. These core cells subsequently intercalate between the marginal cells contributing to the bilateral expansion of the optic vesicles. During later evagination, optic vesicle cells shorten, drawing their apical surfaces laterally relative to the basal lamina, resulting in further laterally directed evagination. The early neuroepithelial organization of the eye field requires Laminin1, and ectopic Laminin1 can redirect the apicobasal orientation of eye field cells. Furthermore, disrupting cell polarity through combined abrogation of the polarity protein Pard6γb and Laminin1 severely compromises optic vesicle evagination. Our studies elucidate the cellular events underlying early eye morphogenesis and provide a framework for understanding epithelialization and complex tissue formation.

Live-imaging of single stem cells within their niche reveals that a U3snoRNP component segregates asymmetrically and is required for self-renewal in Drosophila.

Stem cells generate self-renewing and differentiating progeny over many rounds of asymmetric divisions. How stem cell growth rate and size are maintained over time remains unknown. We isolated mutations in a Drosophila melanogaster gene, wicked (wcd), which induce premature differentiation of germline stem cells (GSCs). Wcd is a member of the U3 snoRNP complex required for pre-ribosomal RNA maturation. This general function of Wcd contrasts with its specific requirement for GSC self-renewal. However, live imaging of GSCs within their niche revealed a pool of Wcd-forming particles that segregate asymmetrically into the GSCs on mitosis, independently of the Dpp signal sent by the niche. A fraction of Wcd also segregated asymmetrically in dividing larval neural stem cells (NSCs). In the absence of Wcd, NSCs became smaller and produced fewer neurons. Our results show that regulation of ribosome synthesis is a crucial parameter for stem cell maintenance and function.