From morphogen to morphogenesis and back.

A long-term aim of the life sciences is to understand how organismal shape is encoded by the genome. An important challenge is to identify mechanistic links between the genes that control cell-fate decisions and the cellular machines that generate shape, therefore closing the gap between genotype and phenotype. The logic and mechanisms that integrate these different levels of shape control are beginning to be described, and recently discovered mechanisms of cross-talk and feedback are beginning to explain the remarkable robustness of organ assembly. The 'full-circle' understanding of morphogenesis that is emerging, besides solving a key puzzle in biology, provides a mechanistic framework for future approaches to tissue engineering.

A conserved role for Snail as a potentiator of active transcription.

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila ∼25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. Here we demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, we identified 106 genes that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of "Snail-activated" genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, we show that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which we demonstrate is essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development.

Conditional deletion of histone deacetylase 1 in T cells leads to enhanced airway inflammation and increased Th2 cytokine production.

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4(+) T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.

Nlcam modulates midline convergence during anterior neural plate morphogenesis.

During development, different cell types must undergo distinct morphogenetic programs so that tissues develop the right dimensions in the appropriate place. In early eye morphogenesis, retinal progenitor cells (RPCs) move first towards the midline, before turning around to migrate out into the evaginating optic vesicles. Neighbouring forebrain cells, however, converge rapidly and then remain at the midline. These differential behaviours are regulated by the transcription factor Rx3. Here, we identify a downstream target of Rx3, the Ig-domain protein Nlcam, and characterise its role in regulating cell migration during the initial phase of optic vesicle morphogenesis. Through sophisticated live imaging and comprehensive cell tracking experiments in zebrafish, we show that ectopic expression of Nlcam in RPCs, as is observed in Rx3 mutants, causes enhanced convergence of these cells. Expression levels of Nlcam therefore regulate the migratory properties of RPCs. Our results provide evidence that the two phases of optic vesicle morphogenesis: slowed convergence and outward-directed migration, are under different genetic control. We propose that Nlcam forms part of the guidance machinery directing rapid midline migration of forebrain precursors, where it is normally expressed, and that its ectopic expression upon loss of Rx3 imparts these migratory characteristics upon RPCs.

In vivo time-lapse imaging in medaka--n-heptanol blocks contractile rhythmical movements.

Medaka is an ideal model system for developmental studies as it combines the advantages of powerful genetics and classical embryology. Due to the accessibility, transparency and fast development, embryogenesis and morphogenesis can be followed in vivo. Microscopic time-lapse imaging, however, requires the immobilization of the object to be observed. In medaka rhythmical contractile movements of the blastoderm during early development hampered time-lapse studies, as they cause the embryo to rotate vividly. Here we show that the contractile movements can be reduced by continuous treatment with the gap-junction uncoupling agent n-heptanol up to the 12-somite stage (stage 23) without interfering with development. This allows for the first time to perform high-resolution time-lapse studies in medaka.

Mutations affecting retina development in Medaka.

In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation.

Role for Traf4 in polarizing adherens junctions as a prerequisite for efficient cell shape changes.

Apical constriction of epithelial cells is a widely used morphogenetic mechanism. In the Drosophila embryo, the apical constrictions that internalize the mesoderm are controlled by the transcription factor Twist and require intact adherens junctions and a contractile acto-myosin network. We find that adherens junctions in constricting mesodermal cells undergo extensive remodeling. A Twist target gene encoding a member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family, Traf4, is involved in this process. While TRAFs are best known for their functions in inflammatory responses, Traf4 appears to have a different role, and its mechanism of action is poorly understood. We show that Traf4 is required for efficient apical constriction during ventral furrow formation and for proper localization of Armadillo to the apical position in constricting cells. Traf4 and Armadillo interact with each other physically and functionally. Traf4 acts in a TNF receptor- and Jun N-terminal protein kinase (JNK)-independent manner to fine-tune the assembly of adherens junctions in the invaginating mesodermal cells.

The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation.

Histone deacetylases (HDACs) are chromatin-modifying enzymes that are involved in the regulation of proliferation, differentiation and development. HDAC inhibitors induce cell cycle arrest, differentiation, or apoptosis in tumor cells and are therefore promising antitumor agents. Numerous genes were found to be deregulated upon HDAC inhibitor treatment; however, the relevant target enzymes are still unidentified. HDAC1 is required for mouse development and unrestricted proliferation of embryonic stem cells. We show here that HDAC1 reversibly regulates cellular proliferation and represses the cyclin-dependent kinase inhibitor p21 in embryonic stem cells. Disruption of the p21 gene rescues the proliferation phenotype of HDAC1(-/-) embryonic stem cells but not the embryonic lethality of HDAC1(-/-) mice. In the absence of HDAC1, mouse embryonic fibroblasts scarcely undergo spontaneous immortalization and display increased p21 expression. Chromatin immunoprecipitation assays demonstrate a direct regulation of the p21 gene by HDAC1 in mouse embryonic fibroblasts. Transformation with simian virus 40 large T antigen or ablation of p21 restores normal immortalization of primary HDAC1(-/-) fibroblasts. Our data demonstrate that repression of the p21 gene is crucial for HDAC1-mediated control of proliferation and immortalization. HDAC1 might therefore be one of the relevant targets for HDAC inhibitors as anticancer drugs.

ojoplano-mediated basal constriction is essential for optic cup morphogenesis.

Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.

Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution.

Neurosecretory control centers form part of the forebrain in many animal phyla, including vertebrates, insects, and annelids. The evolutionary origin of these centers is largely unknown. To identify conserved, and thus phylogenetically ancient, components of neurosecretory brain centers, we characterize and compare neurons that express the prohormone vasotocin (vasopressin/oxytocin)-neurophysin in the developing forebrain of the annelid Platynereis dumerilii and of the zebrafish. These neurons express the same tissue-restricted microRNA, miR-7, and conserved, cell-type-specific combinations of transcription factors (nk2.1, rx, and otp) that specify their identity, as evidenced by the specific requirement of zebrafish rx3 for vasotocin-neurophysin expression. MiR-7 also labels another shared population of neurons containing RFamides. Since the vasotocinergic and RFamidergic neurons appear to be directly sensory in annelid and fish, we propose that cell types with dual sensory-neurosecretory properties were the starting point for the evolution of neurosecretory brain centers in Bilateria.

Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct.

Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish.

Individual cell migration serves as the driving force for optic vesicle evagination.

The cellular mechanisms underlying organ formation are largely unknown. We visualized early vertebrate eye morphogenesis at single-cell resolution by in vivo imaging in medaka (Oryzias latipes). Before optic vesicle evagination, retinal progenitor cells (RPCs) modulate their convergence in a fate-specific manner. Presumptive forebrain cells converge toward the midline, whereas medial RPCs remain stationary, predetermining the site of evagination. Subsequent optic vesicle evagination is driven by the active migration of individual RPCs. The analysis of mutants demonstrated that the retina-specific transcription factor rx3 determines the convergence and migration behaviors of RPCs. Hence, the migration of individual cells mediates essential steps of organ morphogenesis.

Medial floor plate formation in zebrafish consists of two phases and requires trunk-derived Midkine-a.

The medial floor plate (MFP) organizes the specification of neurons and outgrowth of axons in the ventral spinal cord of vertebrates. We show that the growth factor Midkine-a, expressed in the paraxial mesoderm, is required for formation of the MFP in zebrafish. Our epistatic analyses demonstrate that development of MFP comprises two independent sequential phases. Following initial MFP induction in the gastrula organizer, Midkine-a regulates allocation of MFP cells during subsequent development. Thus in zebrafish, trunk-derived signals are required for complete MFP formation from a common pool of organizer-derived midline precursor cells.

Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression.

Histone deacetylases (HDACs) modulate chromatin structure and transcription, but little is known about their function in mammalian development. HDAC1 was implicated previously in the repression of genes required for cell proliferation and differentiation. Here we show that targeted disruption of both HDAC1 alleles results in embryonic lethality before E10.5 due to severe proliferation defects and retardation in development. HDAC1-deficient embryonic stem cells show reduced proliferation rates, which correlate with decreased cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1). Similarly, expression of p21 and p27 is up-regulated in HDAC1-null embryos. In addition, loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 and concomitant changes in other histone modifications. The expression of HDAC2 and HDAC3 is induced in HDAC1-deficient cells, but cannot compensate for loss of the enzyme, suggesting a unique function for HDAC1. Our study provides the first evidence that a histone deacetylase is essential for unrestricted cell proliferation by repressing the expression of selective cell cycle inhibitors.