Michael Brand

The midbrain--hindbrain boundary organizer.

Cell fate in the cephalic neural primordium is controlled by an organizer located at the midbrain-hindbrain boundary. Studies in chick, mouse and zebrafish converge to show that mutually repressive interactions between homeodomain transcription factors of the Otx and Gbx class position this organizer in the neural primordium. Once positioned, independent signaling pathways converge in their activity to drive organizer function. Fibroblast growth factors secreted from the organizer are necessary for, and sufficient to mimic, organizer activity in patterning the midbrain and anterior hindbrain, and are tightly controlled by feedback inhibition.

Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules

It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

The Endosomal Protein Appl1 Mediates Akt Substrate Specificity and Cell Survival in Vertebrate Development

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain

Post-embryonic neurogenesis is a fundamental feature of the vertebrate brain. However, the level of adult neurogenesis decreases significantly with phylogeny. In the first part of this review, a comparative analysis of adult neurogenesis and its putative roles in vertebrates are discussed. Adult neurogenesis in mammals is restricted to two telencephalic constitutively active zones. On the contrary, non-mammalian vertebrates display a considerable amount of adult neurogenesis in many brain regions. The phylogenetic differences in adult neurogenesis are poorly understood. However, a common feature of vertebrates (fish, amphibians and reptiles) that display a widespread adult neurogenesis is the substantial post-embryonic brain growth in contrast to birds and mammals. It is probable that the adult neurogenesis in fish, frogs and reptiles is related to the coordinated growth of sensory systems and corresponding sensory brain regions. Likewise, neurons are substantially added to the olfactory bulb in smell-oriented mammals in contrast to more visually oriented primates and songbirds, where much fewer neurons are added to the olfactory bulb. The second part of this review focuses on the differences in brain plasticity and regeneration in vertebrates. Interestingly, several recent studies show that neurogenesis is suppressed in the adult mammalian brain. In mammals, neurogenesis can be induced in the constitutively neurogenic brain regions as well as ectopically in response to injury, disease or experimental manipulations. Furthermore, multipotent progenitor cells can be isolated and differentiated in vitro from several otherwise silent regions of the mammalian brain. This indicates that the potential to recruit or generate neurons in non-neurogenic brain areas is not completely lost in mammals. The level of adult neurogenesis in vertebrates correlates with the capacity to regenerate injury, for example fish and amphibians exhibit the most widespread adult neurogenesis and also the greatest capacity to regenerate central nervous system injuries. Studying these phenomena in non-mammalian vertebrates may greatly increase our understanding of the mechanisms underlying regeneration and adult neurogenesis. Understanding mechanisms that regulate endogenous proliferation and neurogenic permissiveness in the adult brain is of great significance in therapeutical approaches for brain injury and disease.

Acute Inflammation Initiates the Regenerative Response in the Adult Zebrafish Brain

The Good Side of Inflammation The zebrafish brain is much more adept than the human brain at recovering after traumatic injury. Kyritsis et al. (p. 1353, published online 8 November; see the Perspective by Stella) investigated the cellular events that support regeneration in the zebrafish brain. Although inflammation is part of the response in both settings, the zebrafish brain goes on to initiate proliferation of replacement neurons. By inciting inflammation without neuronal damage, radial glial cells could be pushed into neurogenesis. An inflammatory response to traumatic injury promotes neurogenesis and repair in the zebrafish brain. The zebrafish regenerates its brain after injury and hence is a useful model organism to study the mechanisms enabling regenerative neurogenesis, which is poorly manifested in mammals. Yet the signaling mechanisms initiating such a regenerative response in fish are unknown. Using cerebroventricular microinjection of immunogenic particles and immunosuppression assays, we showed that inflammation is required and sufficient for enhancing the proliferation of neural progenitors and subsequent neurogenesis by activating injury-induced molecular programs that can be observed after traumatic brain injury. We also identified cysteinyl leukotriene signaling as an essential component of inflammation in the regenerative process of the adult zebrafish brain. Thus, our results demonstrate that in zebrafish, in contrast to mammals, inflammation is a positive regulator of neuronal regeneration in the central nervous system.

Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development.

Several molecules of the Fibroblast growth factor family have been implicated in the development of the vertebrate brain, but the effectors of these molecules remain largely unknown. Here we study Erm and Pea3, two ETS domain transcription factors, and show that their expression correlates closely with the domains of fgf8 and fgf3 expression. In situ hybridization analysis in wild-type and acerebellar (ace) mutant embryos defective for fgf8 demonstrates a requirement of Fgf8 for normal expression levels of erm and pea3 transcripts in and close to various domains of Fgf8 action, including the prospective midbrain-hindbrain region, the somites, the neural crest, the forebrain, and developing eyes. Morpholino-oligomer-assisted gene knock-down experiments targeted against fgf8 and fgf3 suggest that Fgf3 and Fgf8 are co-regulators of these genes in the early forebrain anlage. Furthermore, inhibition of Fgf signaling by overexpression of sprouty4 or application of the Fgf inhibitor SU5402 leads to a loss of all erm and pea3 expression domains. Conversely, ectopically provided fgf3 mRNA or implanted beads coated with Fgf8 elicit ectopic transcription of erm and pea3. Both activation and loss of transcripts can be observed within short time frames. We conclude that both the transcriptional onset and maintenance of these factors are tightly coupled to Fgf signaling and propose that erm and pea3 transcription is a direct readout of cells to Fgf levels. Given the knowledge that has accumulated on the posttranslational control of ETS domain factors and their combinatorial interactions with other transcription factors, we suggest that the close coupling of erm and pea3 transcription to Fgf signaling might serve to integrate Fgf signaling with other signals to establish refined patterns in embryonic development.

Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.

Severe traumatic injury to the adult mammalian CNS leads to life-long loss of function. By contrast, several non-mammalian vertebrate species, including adult zebrafish, have a remarkable ability to regenerate injured organs, including the CNS. However, the cellular and molecular mechanisms that enable or prevent CNS regeneration are largely unknown. To study brain regeneration mechanisms in adult zebrafish, we developed a traumatic lesion assay, analyzed cellular reactions to injury and show that adult zebrafish can efficiently regenerate brain lesions and lack permanent glial scarring. Using Cre-loxP-based genetic lineage-tracing, we demonstrate that her4.1-positive ventricular radial glia progenitor cells react to injury, proliferate and generate neuroblasts that migrate to the lesion site. The newly generated neurons survive for more than 3 months, are decorated with synaptic contacts and express mature neuronal markers. Thus, regeneration after traumatic lesion of the adult zebrafish brain occurs efficiently from radial glia-type stem/progenitor cells.

Bone Regenerates via Dedifferentiation of Osteoblasts in the Zebrafish Fin

While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.

Boundary formation and maintenance in tissue development

The formation and maintenance of boundaries between neighbouring groups of embryonic cells is vital for development because groups of cells with distinct functions must often be kept physically separated. Furthermore, because cells at the boundary often take on important signalling functions by acting as organizing centres, boundary shape and integrity can also control the outcome of many downstream patterning events. Recent experimental findings and theoretical descriptions have shed new light on classic questions about boundaries. In particular, in the past couple of years the role of forces acting in epithelial tissues to maintain boundaries has emerged as a new principle in understanding how early pattern is made into permanent anatomy.

Adult neurogenesis and brain regeneration in zebrafish

Adult neurogenesis is a widespread trait of vertebrates; however, the degree of this ability and the underlying activity of the adult neural stem cells differ vastly among species. In contrast to mammals that have limited neurogenesis in their adult brains, zebrafish can constitutively produce new neurons along the whole rostrocaudal brain axis throughout its life. This feature of adult zebrafish brain relies on the presence of stem/progenitor cells that continuously proliferate, and the permissive environment of zebrafish brain for neurogenesis. Zebrafish has also an extensive regenerative capacity, which manifests itself in responding to central nervous system injuries by producing new neurons to replenish the lost ones. This ability makes zebrafish a useful model organism for understanding the stem cell activity in the brain, and the molecular programs required for central nervous system regeneration. In this review, we will discuss the current knowledge on the stem cell niches, the characteristics of the stem/progenitor cells, how they are regulated and their involvement in the regeneration response of the adult zebrafish brain. We will also emphasize the open questions that may help guide the future research.