Zbtb16 mediates a switch between Fgf signalling regimes in the developing hindbrain.

Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the Zbtb16 transcription factor is required for this transition to happen in an orderly fashion. Zbtb16 expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Zbtb16, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in Zbtb16 mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.

Segmentation and patterning of the vertebrate hindbrain.

During early development, the hindbrain is sub-divided into rhombomeres that underlie the organisation of neurons and adjacent craniofacial tissues. A gene regulatory network of signals and transcription factors establish and pattern segments with a distinct anteroposterior identity. Initially, the borders of segmental gene expression are imprecise, but then become sharply defined, and specialised boundary cells form. In this Review, we summarise key aspects of the conserved regulatory cascade that underlies the formation of hindbrain segments. We describe how the pattern is sharpened and stabilised through the dynamic regulation of cell identity, acting in parallel with cell segregation. Finally, we discuss evidence that boundary cells have roles in local patterning, and act as a site of neurogenesis within the hindbrain.

A single cell transcriptome atlas of the developing zebrafish hindbrain.

Segmentation of the vertebrate hindbrain leads to the formation of rhombomeres, each with a distinct anteroposterior identity. Specialised boundary cells form at segment borders that act as a source or regulator of neuronal differentiation. In zebrafish, there is spatial patterning of neurogenesis in which non-neurogenic zones form at boundaries and segment centres, in part mediated by Fgf20 signalling. To further understand the control of neurogenesis, we have carried out single cell RNA sequencing of the zebrafish hindbrain at three different stages of patterning. Analyses of the data reveal known and novel markers of distinct hindbrain segments, of cell types along the dorsoventral axis, and of the transition of progenitors to neuronal differentiation. We find major shifts in the transcriptome of progenitors and of differentiating cells between the different stages analysed. Supervised clustering with markers of boundary cells and segment centres, together with RNA-seq analysis of Fgf-regulated genes, has revealed new candidate regulators of cell differentiation in the hindbrain. These data provide a valuable resource for functional investigations of the patterning of neurogenesis and the transition of progenitors to neuronal differentiation.

The Evolutionary History of Ephs and Ephrins: Toward Multicellular Organisms.

Eph receptor (Eph) and ephrin signaling regulate fundamental developmental processes through both forward and reverse signaling triggered upon cell-cell contact. In vertebrates, they are both classified into classes A and B, and some representatives have been identified in many metazoan groups, where their expression and functions have been well studied. We have extended previous phylogenetic analyses and examined the presence of Eph and ephrins in the tree of life to determine their origin and evolution. We have found that 1) premetazoan choanoflagellates may already have rudimental Eph/ephrin signaling as they have an Eph-/ephrin-like pair and homologs of downstream-signaling genes; 2) both forward- and reverse-downstream signaling might already occur in Porifera since sponges have most genes involved in these types of signaling; 3) the nonvertebrate metazoan Eph is a type-B receptor that can bind ephrins regardless of their membrane-anchoring structure, glycosylphosphatidylinositol, or transmembrane; 4) Eph/ephrin cross-class binding is specific to Gnathostomata; and 5) kinase-dead Eph receptors can be traced back to Gnathostomata. We conclude that Eph/ephrin signaling is of older origin than previously believed. We also examined the presence of protein domains associated with functional characteristics and the appearance and conservation of downstream-signaling pathways to understand the original and derived functions of Ephs and ephrins. We find that the evolutionary history of these gene families points to an ancestral function in cell-cell interactions that could contribute to the emergence of multicellularity and, in particular, to the required segregation of cell populations.

Role of forward and reverse signaling in Eph receptor and ephrin mediated cell segregation.

Eph receptor and ephrin signaling has a major role in segregating distinct cell populations to form sharp borders. Expression of interacting Ephs and ephrins typically occurs in complementary regions, such that polarised activation of both components occurs at the interface. Forward signaling through Eph receptors can drive cell segregation, but it is unclear whether reverse signaling through ephrins can also contribute. We have tested the role of reverse signaling, and of polarised versus non-polarised activation, in assays in which contact repulsion drives cell segregation and border sharpening. We find that polarised forward signaling drives stronger segregation than polarised reverse signaling. Nevertheless, reverse signaling contributes since bidirectional Eph and ephrin activation drives stronger segregation than unidirectional forward signaling alone. In contrast, non-polarised Eph activation drives little segregation. We propose that although polarised forward signaling is the principal driver of segregation, reverse signaling enables bidirectional repulsion which prevents mingling of each population into the other.

A feedback loop mediated by degradation of an inhibitor is required to initiate neuronal differentiation.

Neuronal differentiation is regulated by proneural genes that promote neurogenesis and inhibitory mechanisms that maintain progenitors. This raises the question of how the up-regulation of proneural genes required to initiate neurogenesis occurs in the presence of such inhibition. We carried out loss and gain of gene function, an interaction screen for binding partners, and biochemical analyses to uncover the regulation, developmental role, and mechanism of action of a ubiquitination adaptor protein, Btbd6a (BTB domain containing 6a). We find that the proneural gene neurog1 up-regulates btbd6a, which in turn is required for up-regulation of neurog1. Btbd6a is an adaptor for the Cul3 ubiquitin ligase complex, and we find that it binds to the transcriptional repressor Plzf (promyelocytic leukemia zinc finger). Btbd6a promotes the relocation of Plzf from nucleus to cytoplasm and targets Plzf for ubiquitination and degradation. plzfa is expressed widely in the neural epithelium; when overexpressed, it inhibits neurogenesis, and this inhibition is reversed by btbd6a. The antagonism of endogenous plzfa by btbd6a is required for neurogenesis, since the block in neuronal differentiation caused by btbd6a knockdown is alleviated by plzfa knockdown. These findings reveal a feedback loop mediated by degradation of an inhibitor that is essential for progenitors to undergo the transition to neuronal differentiation.

Mechanisms of boundary formation by Eph receptor and ephrin signaling.

The formation of sharp borders, across which cell intermingling is restricted, has a crucial role in the establishment and maintenance of organized tissues. Signaling of Eph receptors and ephrins underlies formation of a number of boundaries between and within tissues during vertebrate development. Eph-ephrin signaling can regulate several types of cell response-adhesion, repulsion and tension-that can in principle underlie the segregation of cells and formation of sharp borders. Recent studies have implicated each of these cell responses as having important roles at different boundaries: repulsion at the mesoderm-ectoderm border, decreased adhesion at the notochord-presomitic mesoderm border, and tension at boundaries within the hindbrain and forebrain. These distinct responses to Eph receptor and ephrin activation may in part be due to the adhesive properties of the tissue.

An inducible transgene expression system for zebrafish and chick.

We have generated an inducible system to control the timing of transgene expression in zebrafish and chick. An estrogen receptor variant (ERT2) fused to the GAL4 transcriptional activator rapidly and robustly activates transcription within 3 hours of treatment with the drug 4-hydroxy-tamoxifen (4-OHT) in tissue culture and transgenic zebrafish. We have generated a broadly expressed inducible ERT2-GAL4 zebrafish line using the ubiquitin (ubi) enhancer. In addition, use of ERT2-GAL4 in conjunction with tissue-specific enhancers enables the control of transgene expression in both space and time. This spatial restriction and the ability to sustain forced expression are important advantages over the currently used heat-shock promoters. Moreover, in contrast to currently available TET and LexA systems, which require separate constructs with their own unique recognition sequences, ERT2-GAL4 is compatible with the growing stock of UAS lines being generated in the community. We also applied the same inducible system to the chick embryo and find that it is fully functional, suggesting that this strategy is generally applicable.

A Hox gene controls lateral line cell migration by regulating chemokine receptor expression downstream of Wnt signaling.

The posterior lateral line primordium in zebrafish provides an amenable model to study mechanisms of collective cell migration. The directed migration of the cell cluster along the path of Sdf1a chemokine requires two receptors, Cxcr4b and Cxcr7b, which are expressed in the leading and trailing part of the primordium, respectively. The polarized expression of receptors is regulated by Wnt signaling, but downstream players mediating this control remain to be found. Here, we show that the Hox homeobox gene Hoxb8a is a critical component that acts downstream of the Wnt pathway to coordinate the expression of both chemokine receptors. We find that Hoxb8a is expressed in the leading part of the primordium and is required for the correct speed and extent of migration. Hoxb8a expression is dependent upon Wnt activity and needed both for cxcr4b expression and to repress and thus restrict cxcr7b expression to the trailing zone of the primordium. In the absence of Wnt activity, overexpressed Hoxb8a is able to repress cxcr7b but not up-regulate cxcr4b expression. Together with results from expressing dominant activator and repressor constructs, these findings suggest that Hoxb8a is induced by and cooperates with Wnt signaling to up-regulate cxcr4b, and acts through multiple mechanisms to repress cxcr7b expression.

Chemokine and Fgf signalling act as opposing guidance cues in formation of the lateral line primordium.

The directional migration of many cell populations occurs as a coherent group. An amenable model is provided by the posterior lateral line in zebrafish, which is formed by a cohesive primordium that migrates from head to tail and deposits future neuromasts at intervals. We found that prior to the onset of migration, the compact state of the primordium is not fully established, as isolated cells with lateral line identity are present caudal to the main primordium. These isolated cells are retained in position such that they fuse with the migrating primordium as it advances, and later contribute to the leading zone and terminal neuromasts. We found that the isolated lateral line cells are positioned by two antagonistic cues: Fgf signalling attracts them towards the primordium, which counteracts Sdf1α/Cxcr4b-mediated caudal attraction. These findings reveal a novel chemotactic role for Fgf signalling in which it enables the coalescence of the lateral line primordium from an initial fuzzy pattern into a compact group of migrating cells.

Signalling from hindbrain boundaries regulates neuronal clustering that patterns neurogenesis.

During central nervous system development, neural progenitors are patterned to form discrete neurogenic and non-neurogenic zones. In the zebrafish hindbrain, neurogenesis is organised by Fgf20a emanating from neurons located at each segment centre that inhibits neuronal differentiation in adjacent progenitors. Here, we have identified a molecular mechanism that clusters fgf20a-expressing neurons in segment centres and uncovered a requirement for this positioning in the regulation of neurogenesis. Disruption of hindbrain boundary cell formation alters the organisation of fgf20a-expressing neurons, consistent with a role of chemorepulsion from boundaries. The semaphorins Sema3fb and Sema3gb, which are expressed by boundary cells, and their receptor Nrp2a are required for clustering of fgf20a-expressing neurons at segment centres. The dispersal of fgf20a-expressing neurons that occurs following the disruption of boundaries or of Sema3fb/Sema3gb signalling leads to reduced FGF target gene expression in progenitors and an increased number of differentiating neurons. Sema3 signalling from boundaries thus links hindbrain segmentation to the positioning of fgf20a-expressing neurons that regulates neurogenesis.

EphB-ephrin-B2 interactions are required for thymus migration during organogenesis.

Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.

Morpholino artifacts provide pitfalls and reveal a novel role for pro-apoptotic genes in hindbrain boundary development.

Morpholino antisense oligonucleotides (MOs) are widely used as a tool to achieve loss of gene function, but many have off-target effects mediated by activation of Tp53 and associated apoptosis. Here, we re-examine our previous MO-based loss-of-function studies that had suggested that Wnt1 expressed at hindbrain boundaries in zebrafish promotes neurogenesis and inhibits boundary marker gene expression in the adjacent para-boundary regions. We find that Tp53 is highly activated and apoptosis is frequently induced by the MOs used in these studies. Co-knockdown of Tp53 rescues the decrease in proneural and neuronal marker expression, which is thus an off-target effect of MOs. While loss of gene expression can be attributed to cell loss through apoptotic cell death, surprisingly we find that the ectopic expression of hindbrain boundary markers is also dependent on Tp53 activity and its downstream apoptotic effectors. We examine whether this non-specific activation of hindbrain boundary gene expression provides insight into the endogenous mechanisms underlying boundary cell specification. We find that the pro-apoptotic Bcl genes puma and bax-a are required for hindbrain boundary marker expression, and that gain of function of the Bcl-caspase pathway leads to ectopic boundary marker expression. These data reveal a non-apoptotic role for pro-apoptotic genes in the regulation of gene expression at hindbrain boundaries. In light of these findings, we discuss the precautions needed in performing morpholino knockdowns and in interpreting the data derived from their use.

Clonal behaviour of myogenic precursor cells throughout the vertebrate lifespan.

To address questions of stem cell diversity during skeletal myogenesis, a Brainbow-like genetic cell lineage tracing method, dubbed Musclebow2, was derived by enhancer trapping in zebrafish. It is shown that, after initial formation of the primary myotome, at least 15 muscle precursor cells (mpcs) seed each somite, where they proliferate but contribute little to muscle growth prior to hatching. Thereafter, dermomyotome-derived mpc clones rapidly expand while some progeny undergo terminal differentiation, leading to stochastic clonal drift within the mpc pool. No evidence of cell-lineage-based clonal fate diversity was obtained. Neither fibre nor mpc death was observed in uninjured animals. Individual marked muscle fibres persist across much of the lifespan indicating low rates of nuclear turnover. In adulthood, early-marked mpc clones label stable blocks of tissue comprising a significant fraction of either epaxial or hypaxial somite. Fusion of cells from separate early-marked clones occurs in regions of clone overlap. Wounds are regenerated from several local mpcs; no evidence for specialised stem mpcs was obtained. In conclusion, our data indicate that most mpcs in muscle tissue contribute to local growth and repair and suggest that cellular turnover is low in the absence of trauma.

Interplay of Eph-Ephrin Signalling and Cadherin Function in Cell Segregation and Boundary Formation.

The segregation of distinct cell populations to form sharp boundaries is crucial for stabilising tissue organisation, for example during hindbrain segmentation in craniofacial development. Two types of mechanisms have been found to underlie cell segregation: differential adhesion mediated by cadherins, and Eph receptor and ephrin signalling at the heterotypic interface which regulates cell adhesion, cortical tension and repulsion. An interplay occurs between these mechanisms since cadherins have been found to contribute to Eph-ephrin-mediated cell segregation. This may reflect that Eph receptor activation acts through multiple pathways to decrease cadherin-mediated adhesion which can drive cell segregation. However, Eph receptors mainly drive cell segregation through increased heterotypic tension or repulsion. Cadherins contribute to cell segregation by antagonising homotypic tension within each cell population. This suppression of homotypic tension increases the difference with heterotypic tension triggered by Eph receptor activation, and it is this differential tension that drives cell segregation and border sharpening.

Inhibition of BMPs by follistatin is required for FGF3 expression and segmental patterning of the hindbrain.

A network of molecular interactions is required in the developing vertebrate hindbrain for the formation and anterior-posterior patterning of the rhombomeres. FGF signaling is required in this network to upregulate the expression of the Krox20 and Kreisler segmentation genes, but little is known of how FGF gene expression is regulated in the hindbrain. We show that the dynamic expression of FGF3 in chick hindbrain segments and boundaries is similar to that of the BMP antagonist, follistatin. Consistent with a regulatory relationship between BMP signaling and FGF3 expression, we find that an increase in BMP activity due to blocking of follistatin translation by morpholino antisense oligonucleotides or overexpression of BMP results in strong inhibition of FGF3 expression. Conversely, addition of follistatin leads to an increase in the level of FGF3 expression. Furthermore, the segmental inhibition of BMP activity by follistatin is required for the expression of Krox20, Hoxb1 and EphA4 in the hindbrain. In addition, we show that the maintenance of FGF3 gene expression requires FGF activity, suggestive of an autoregulatory loop. These results reveal an antagonistic relationship between BMP activity and FGF3 expression that is required for correct segmental gene expression in the chick hindbrain, in which follistatin enables FGF3 expression by inhibiting BMP activity.

Boundary cells regulate a switch in the expression of FGF3 in hindbrain rhombomeres.

BACKGROUND: During formation of the vertebrate central nervous system, the hindbrain is organized into segmental units, called rhombomeres (r). These cell-lineage restricted segments are separated by a subpopulation of cells known as boundary cells. Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells. However, little is known regarding their functions and the mechanisms that regulate their formation. RESULTS: Hindbrain boundary cells express several signaling molecules, such as FGF3, which at earlier developmental stages is transiently expressed in specific rhombomeres. We show that chick embryos that lack boundary cells due to overexpression of truncated EphA4 receptor in the hindbrain have continued segmental expression of FGF3 at stages when it is normally restricted to hindbrain boundaries. Furthermore, surgical ablation of the boundary between r3 and r4, or blocking of the contact of r4 with boundary cells, results in sustained FGF3 expression in this segment. CONCLUSION: These findings suggest that boundary cells are required for the downregulation of segmental FGF3, presumably mediated by a soluble factor(s) that emanates from boundaries. We propose that this new function of boundary cells enables a switch in gene expression that may be required for stage-specific functions of FGF3 in the developing hindbrain.

Diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly.

Eph receptor tyrosine kinases and ephrins have key roles in regulation of the migration and adhesion of cells required to form and stabilize patterns of cell organization during development. Activation of Eph receptors or ephrins can lead either to cell repulsion or to cell adhesion and invasion, and recent work has found that cells can switch between these distinct responses. This review will discuss biochemical mechanisms and developmental roles of the diverse cell responses controlled by Eph receptors and ephrins.

Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain.

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. We have investigated a potential role of Notch signaling and find that in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center.

Actomyosin regulation by Eph receptor signaling couples boundary cell formation to border sharpness.

The segregation of cells with distinct regional identity underlies formation of a sharp border, which in some tissues serves to organise a boundary signaling centre. It is unclear whether or how border sharpness is coordinated with induction of boundary-specific gene expression. We show that forward signaling of EphA4 is required for border sharpening and induction of boundary cells in the zebrafish hindbrain, which we find both require kinase-dependent signaling, with a lesser input of PDZ domain-dependent signaling. We find that boundary-specific gene expression is regulated by myosin II phosphorylation, which increases actomyosin contraction downstream of EphA4 signaling. Myosin phosphorylation leads to nuclear translocation of Taz, which together with Tead1a is required for boundary marker expression. Since actomyosin contraction maintains sharp borders, there is direct coupling of border sharpness to boundary cell induction that ensures correct organisation of signaling centres.