Asymmetry of the Brain: Development and Implications.

Although the left and right hemispheres of our brains develop with a high degree of symmetry at both the anatomical and functional levels, it has become clear that subtle structural differences exist between the two sides and that each is dominant in processing specific cognitive tasks. As the result of evolutionary conservation or convergence, lateralization of the brain is found in both vertebrates and invertebrates, suggesting that it provides significant fitness for animal life. This widespread feature of hemispheric specialization has allowed the emergence of model systems to study its development and, in some cases, to link anatomical asymmetries to brain function and behavior. Here, we present some of what is known about brain asymmetry in humans and model organisms as well as what is known about the impact of environmental and genetic factors on brain asymmetry development. We specifically highlight the progress made in understanding the development of epithalamic asymmetries in zebrafish and how this model provides an exciting opportunity to address brain asymmetry at different levels of complexity.

The importance of population heterogeneities in detecting social learning as the foundation of animal cultural transmission.

High levels of within-population behavioural variation can have drastic demographic consequences, thus changing the evolutionary fate of populations. A major source of within-population heterogeneity is personality. Nonetheless, it is still relatively rarely accounted for in social learning studies that constitute the most basic process of cultural transmission. Here, we performed in female mosquitofish (Gambusia holbrooki) a social learning experiment in the context of mate choice, a situation called mate copying (MC), and for which there is strong evidence that it can lead to the emergence of persistent traditions of preferring a given male phenotype. When accounting for the global tendency of females to prefer larger males but ignoring differences in personality, we detected no evidence for MC. However, when accounting for the bold-shy dichotomy, we found that bold females did not show any evidence for MC, while shy females showed significant amounts of MC. This illustrates how the presence of variation in personality can hamper our capacity to detect MC. We conclude that MC may be more widespread than we thought because many studies ignored the presence of within-population heterogeneities.

Left/right asymmetric collective migration of parapineal cells is mediated by focal FGF signaling activity in leading cells.

The ability of cells to collectively interpret surrounding environmental signals underpins their capacity to coordinate their migration in various contexts, including embryonic development and cancer metastasis. One tractable model for studying collective migration is the parapineal, a left-sided group of neurons that arises from bilaterally positioned precursors that undergo a collective migration to the left side of the brain. In zebrafish, the migration of these cells requires Fgf8 and, in this study, we resolve how FGF signaling correlates with-and impacts the migratory dynamics of-the parapineal cell collective. The temporal and spatial dynamics of an FGF reporter transgene reveal that FGF signaling is activated in only few parapineal cells usually located at the leading edge of the parapineal during its migration. Overexpressing a constitutively active Fgf receptor compromises parapineal migration in wild-type embryos, while it partially restores both parapineal migration and mosaic expression of the FGF reporter transgene in fgf8-/- mutant embryos. Focal activation of FGF signaling in few parapineal cells is sufficient to promote the migration of the whole parapineal collective. Finally, we show that asymmetric Nodal signaling contributes to the restriction and leftwards bias of FGF pathway activation. Our data indicate that the first overt morphological asymmetry in the zebrafish brain is promoted by FGF pathway activation in cells that lead the collective migration of the parapineal to the left. This study shows that cell-state differences in FGF signaling in front versus rear cells is required to promote migration in a model of FGF-dependent collective migration.

Pitx2c ensures habenular asymmetry by restricting parapineal cell number.

Left-right (L/R) asymmetries in the brain are thought to underlie lateralised cognitive functions. Understanding how neuroanatomical asymmetries are established has been achieved through the study of the zebrafish epithalamus. Morphological symmetry in the epithalamus is broken by leftward migration of the parapineal, which is required for the subsequent elaboration of left habenular identity; the habenular nuclei flank the midline and show L/R asymmetries in marker expression and connectivity. The Nodal target pitx2c is expressed in the left epithalamus, but nothing is known about its role during the establishment of asymmetry in the brain. We show that abrogating Pitx2c function leads to the right habenula adopting aspects of left character, and to an increase in parapineal cell numbers. Parapineal ablation in Pitx2c loss of function results in right habenular isomerism, indicating that the parapineal is required for the left character detected in the right habenula in this context. Partial parapineal ablation in the absence of Pitx2c, however, reduces the number of parapineal cells to wild-type levels and restores habenular asymmetry. We provide evidence suggesting that antagonism between Nodal and Pitx2c activities sets an upper limit on parapineal cell numbers. We conclude that restricting parapineal cell number is crucial for the correct elaboration of epithalamic asymmetry.

No evidence for mate copying in Danio rerio.

The zebrafish Danio rerio is an important model organism, but little is known about its mating preferences and how these are influenced by personality traits like boldness. In this study, we tested two strains of zebrafish and addressed whether females used social information to build a mating preference, a behavior called mate copying, and whether this social learning was affected by boldness. Thus, we provided positive social information for small males to test whether female zebrafish changed their mate preference after observing a pair of a small and a large male with a demonstrator female next to the small one. After that, we tested the observer female in a test maze to evaluate boldness. We found no significant evidence for mate copying as females did not change their preference for the small male after witnessing the large male alone and the small male interacting with another female and chose consistently larger males in a control without opportunity to copy. Whether the female was defined as shy or bold had no effect on mate copying. We conclude that mate copying is probably inexistent or only relatively weak in this species.

Cell-to-cell heterogeneity in Sox2 and Bra expression guides progenitor motility and destiny.

Although cell-to-cell heterogeneity in gene and protein expression within cell populations has been widely documented, we know little about its biological functions. By studying progenitors of the posterior region of bird embryos, we found that expression levels of transcription factors Sox2 and Bra, respectively involved in neural tube (NT) and mesoderm specification, display a high degree of cell-to-cell heterogeneity. By combining forced expression and downregulation approaches with time-lapse imaging, we demonstrate that Sox2-to-Bra ratio guides progenitor's motility and their ability to stay in or exit the progenitor zone to integrate neural or mesodermal tissues. Indeed, high Bra levels confer high motility that pushes cells to join the paraxial mesoderm, while high levels of Sox2 tend to inhibit cell movement forcing cells to integrate the NT. Mathematical modeling captures the importance of cell motility regulation in this process and further suggests that randomness in Sox2/Bra cell-to-cell distribution favors cell rearrangements and tissue shape conservation.

The THAP domain: a novel protein motif with similarity to the DNA-binding domain of P element transposase.

We have identified a novel evolutionarily conserved protein motif - designated the THAP domain - that defines a new family of cellular factors. We have found that the THAP domain presents striking similarities with the site-specific DNA-binding domain (DBD) of Drosophila P element transposase, including a similar size, N-terminal location, and conservation of the residues that define the THAP motif, such as the C2CH signature (Cys-Xaa(2-4)-Cys-Xaa(35-50)-Cys-Xaa(2)-His). Our results suggest that the THAP domain is a novel example of a DBD that is shared between cellular proteins and transposases from mobile genomic parasites.

Notch signaling restricts FGF pathway activation in parapineal cells to promote their collective migration.

Coordinated migration of cell collectives is important during embryonic development and relies on cells integrating multiple mechanical and chemical cues. Recently, we described that focal activation of the FGF pathway promotes the migration of the parapineal in the zebrafish epithalamus. How FGF activity is restricted to leading cells in this system is, however, unclear. Here, we address the role of Notch signaling in modulating FGF activity within the parapineal. While Notch loss-of-function results in an increased number of parapineal cells activating the FGF pathway, global activation of Notch signaling decreases it; both contexts result in defects in parapineal migration and specification. Decreasing or increasing FGF signaling in a Notch loss-of-function context respectively rescues or aggravates parapineal migration defects without affecting parapineal cells specification. We propose that Notch signaling controls the migration of the parapineal through its capacity to restrict FGF pathway activation to a few leading cells.

Divergence in regulation of the PEA3 family of ETS transcription factors.

Here, we report the cloning of a cDNA encoding zebrafish ER81, a member of the PEA3 family of Ets transcription factors. Strikingly, the spatial and temporal expression of er81 is significantly different from its Xenopus orthologue, XER81, whose expression is more reminiscent of the FGF dependant zebrafish PEA3 family members. In keeping with this observation, while pea3, erm and XER81 require FGF activity for their expression, er81 does not require FGF signalling. Our results suggest that, since the vertebrate specific expansion of the PEA3 subfamily of Ets transcription factors, the regulation of PEA3 genes has been independently modified during the evolution of different vertebrate lineages.

Habenular Neurogenesis in Zebrafish Is Regulated by a Hedgehog, Pax6 Proneural Gene Cascade.

The habenulae are highly conserved nuclei in the dorsal diencephalon that connect the forebrain to the midbrain and hindbrain. These nuclei have been implicated in a broad variety of behaviours in humans, primates, rodents and zebrafish. Despite this, the molecular mechanisms that control the genesis and differentiation of neural progenitors in the habenulae remain relatively unknown. We have previously shown that, in zebrafish, the timing of habenular neurogenesis is left-right asymmetric and that in the absence of Nodal signalling this asymmetry is lost. Here, we show that habenular neurogenesis requires the homeobox transcription factor Pax6a and the redundant action of two proneural bHLH factors, Neurog1 and Neurod4. We present evidence that Hedgehog signalling is required for the expression of pax6a, which is in turn necessary for the expression of neurog1 and neurod4. Finally, we demonstrate by pharmacological inhibition that Hedgehog signalling is required continuously during habenular neurogenesis and by cell transplantation experiments that pathway activation is required cell autonomously. Our data sheds light on the mechanism underlying habenular development that may provide insights into how Nodal signalling imposes asymmetry on the timing of habenular neurogenesis.

THAP1 is a nuclear proapoptotic factor that links prostate-apoptosis-response-4 (Par-4) to PML nuclear bodies.

Promyelocytic leukemia (PML) nuclear bodies (PML NBs) are discrete subnuclear domains organized by the promyelocytic leukemia protein PML, a tumor suppressor essential for multiple apoptotic pathways. We have recently described a novel family of cellular factors, the THAP proteins, characterized by the presence at their amino-terminus of an evolutionary conserved putative DNA-binding motif, designated THAP domain. Here, we report that THAP1 is a novel nuclear proapoptotic factor associated with PML NBs, which potentiates both serum withdrawal- and TNF alpha-induced apoptosis, and interacts with prostate-apoptosis-response-4 (Par-4), a well characterized proapoptotic factor, previously linked to prostate cancer and neurodegenerative diseases. We show that endogenous Par-4 colocalizes with ectopic THAP1 within PML NBs in primary endothelial cells and fibroblasts. In addition, we found that Par-4 is a component of PML NBs in blood vessels, a major site of PML expression in vivo. Finally, we investigated the role of the THAP domain in THAP1 activities and found that this putative DNA-binding domain is not required for Par-4 binding and localization within PML NBs, but is essential for THAP1 proapoptotic activity. Together, our results provide an unexpected link between a nuclear factor of the THAP family, the proapoptotic protein Par-4 and PML nuclear bodies.

Tcf7l2 is required for left-right asymmetric differentiation of habenular neurons.

BACKGROUND: Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown. RESULTS: In a forward genetic screen for mutations that result in loss of habenular asymmetry, we identified two mutant alleles of tcf7l2, a gene that encodes a transcriptional regulator of Wnt signaling. In tcf7l2 mutants, most neurons on both sides differentiate with dHbl identity. Consequently, the habenulae develop symmetrically, with both sides adopting a pronounced leftward character. Tcf7l2 acts cell automously in nascent equipotential neurons, and on the right side, it promotes dHbm and suppresses dHbl differentiation. On the left, the parapineal prevents this Tcf7l2-dependent process, thereby promoting dHbl differentiation. CONCLUSIONS: Tcf7l2 is essential for lateralized fate selection by habenular neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.

Breaking symmetry: the zebrafish as a model for understanding left-right asymmetry in the developing brain.

How does left-right asymmetry develop in the brain and how does the resultant asymmetric circuitry impact on brain function and lateralized behaviors? By enabling scientists to address these questions at the levels of genes, neurons, circuitry and behavior,the zebrafish model system provides a route to resolve the complexity of brain lateralization. In this review, we present the progress made towards characterizing the nature of the gene networks and the sequence of morphogenetic events involved in the asymmetric development of zebrafish epithalamus. In an attempt to integrate the recent extensive knowledge into a working model and to identify the future challenges,we discuss how insights gained at a cellular/developmental level can be linked to the data obtained at a molecular/genetic level. Finally, we present some evolutionary thoughts and discuss how significant discoveries made in zebrafish should provide entry points to better understand the evolutionary origins of brain lateralization.

Chemotrap-1: an engineered soluble receptor that blocks chemokine-induced migration of metastatic cancer cells in vivo.

Cancer and dendritic cells recognize and migrate toward chemokines secreted from lymphatics and use this mechanism to invade the lymphatic system, and cancer cells metastasize through it. The lymphatic-secreted chemokine ligand CCL21 has been identified as a key regulatory molecule in the switch to a metastatic phenotype in melanoma and breast cancer cells. However, it is not known whether CCL21 inhibition is a potential therapeutic strategy for inhibition of metastasis. Here, we describe an engineered CCL21-soluble inhibitor, Chemotrap-1, which inhibits migration of metastatic melanoma cells in vivo. Two-hybrid, pull-down, and coimmunoprecipitation assays allowed us to identify a naturally occurring human zinc finger protein with CCL21 chemokine-binding properties. Further analyses revealed a short peptide (∼70 amino acids), with a predicted coiled-coil structure, which is sufficient for association with CCL21. This CCL21 chemokine-binding peptide was then fused to the Fc region of human IgG1 to generate Chemotrap-1, a human chemokine-binding Fc fusion protein. Surface plasmon resonance and chemotaxis assays showed that Chemotrap-1 binds CCL21 and inhibits CCL21-induced migration of melanoma cells in vitro with subnanomolar affinity. In addition, Chemotrap-1 blocked migration of melanoma cells toward lymphatic endothelial cells in vitro and in vivo. Finally, Chemotrap-1 strongly reduced lymphatic invasion, tracking, and metastasis of CCR7-expressing melanoma cells in vivo. Together, these results show that CCL21 chemokine inhibition by Chemotrap-1 is a potential therapeutic strategy for metastasis and provide further support for the hypothesis that lymphatic-mediated metastasis is a chemokine-dependent process.

Nodal signalling imposes left-right asymmetry upon neurogenesis in the habenular nuclei.

The habenulae are evolutionarily conserved bilateral nuclei in the epithalamus that relay input from the forebrain to the ventral midbrain. In zebrafish, the habenulae display left-right (L/R) asymmetries in gene expression and axonal projections. The elaboration of habenular asymmetries requires the presence of a second asymmetric structure, the parapineal, the laterality of which is biased by unilateral Nodal signalling. Here we show that neurons are present earlier in the left habenula than in the right, but, in contrast to other habenular asymmetry phenotypes, this asymmetry in neurogenesis is not dependent on the parapineal. Embryos in which the L/R asymmetry in Nodal signalling is abolished display symmetric neurogenesis, revealing a requirement for this pathway in asymmetrically biasing neurogenesis. Our results provide evidence of a direct requirement for unilateral Nodal activity in establishing an asymmetry per se, rather than solely in biasing its laterality.

An Fgf8-dependent bistable cell migratory event establishes CNS asymmetry.

Neuroanatomical and functional asymmetries are universal features of the vertebrate CNS, but how asymmetry is generated is unknown. Here we show that zebrafish fgf8 mutants do not elaborate forebrain asymmetries, demonstrated by the failure of the parapineal nucleus to migrate from its initial midline position to the left side of the brain. Local provision of Fgf8 restores the asymmetric migration of parapineal cells, usually to the left, irrespective of the location of the Fgf8 source. This laterality bias is due to left-sided Nodal signaling and when the bias in Nodal signaling is removed, parapineal cells migrate toward the source of Fgf8 protein. This study presents a mechanism for breaking neuroanatomical symmetry through Fgf8-dependent regulation of bistable left- or right-sided migration of the parapineal. The combined action of Fgf and Nodal signals ensures the establishment of neuroanatomical asymmetries with consistent laterality.

Validating in utero electroporation for the rapid analysis of gene regulatory elements in the murine telencephalon.

With the ultimate goal of understanding how genetic modules have evolved in the telencephalon, we set out to modernize the functional analysis of cross-species cis-regulatory elements in mouse. In utero electroporation is rapidly replacing transgenesis as the method of choice for gain- and loss-of-function studies in the murine telencephalon, but the application of this technique to the analysis of transcriptional regulation has yet to be fully explored and exploited. To empirically define the developmental stages required to target specific populations of neurons in the dorsal telencephalon, or pallium, which gives rise to the neocortex in mouse, we performed a temporal and spatial analysis of the migratory properties of electroporated versus birth-dated cells. Next, we compared the activities of two known Ngn2 enhancers via transgenesis and in utero electroporation, demonstrating that the latter technique more faithfully reports the endogenous telencephalic expression pattern observed in an Ngn2lacZ knock-in line. Finally, we used this approach to test the telencephalic activities of a series of deletion constructs comprised of the zebrafish ER81 upstream regulatory region, allowing us to identify a previously uncharacterized enhancer that displays cross-species activity in the murine piriform cortex and lateral neocortex, yet not in more medial domains of the forebrain. Taken together, our data supports the contention that in utero technology can be exploited to rapidly examine the architecture and evolution of pallial-specific cis-regulatory elements.

Mechano-sensory organ regeneration in adults: the zebrafish lateral line as a model.

In this report, we present a study of regeneration of the lateral line, a collection of mechano-sensory organ, in the adult zebrafish caudal fin. As all neuromasts are innervated by axon fibers, neuronal regeneration is a key issue in the regenerating process. We first show that support cells from the last neuromast adjacent to the amputation plane divide and migrate to colonize the blastema in order to reform the missing part of the lateral line. We then show that nerve re-growth takes place later than neuromast progenitor cell migration. We also provide evidence that new growth cones form at the amputation plane and subsequently follow the migrating placode-like structure to re-innervate regenerated neuromasts as they differentiate. Altogether, our observations indicate that caudal lateral line regeneration is not a mere recapitulation of the ontogenic process.

The THAP domain of THAP1 is a large C2CH module with zinc-dependent sequence-specific DNA-binding activity.

We have recently described an evolutionarily conserved protein motif, designated the THAP domain, which defines a previously uncharacterized family of cellular factors (THAP proteins). The THAP domain exhibits similarities to the site-specific DNA-binding domain of Drosophila P element transposase, including a putative metal-coordinating C2CH signature (CX(2-4)CX(35-53)CX(2)H). In this article, we report a comprehensive list of approximately 100 distinct THAP proteins in model animal organisms, including human nuclear proapoptotic factors THAP1 and DAP4/THAP0, transcriptional repressor THAP7, zebrafish orthologue of cell cycle regulator E2F6, and Caenorhabditis elegans chromatin-associated protein HIM-17 and cell-cycle regulators LIN-36 and LIN-15B. In addition, we demonstrate the biochemical function of the THAP domain as a zinc-dependent sequence-specific DNA-binding domain belonging to the zinc-finger superfamily. In vitro binding-site selection allowed us to identify an 11-nucleotide consensus DNA-binding sequence specifically recognized by the THAP domain of human THAP1. Mutations of single nucleotide positions in this sequence abrogated THAP-domain binding. Experiments with the zinc chelator 1,10-o-phenanthroline revealed that the THAP domain is a zinc-dependent DNA-binding domain. Site-directed mutagenesis of single cysteine or histidine residues supported a role for the C2CH motif in zinc coordination and DNA-binding activity. The four other conserved residues (P, W, F, and P), which define the THAP consensus sequence, were also found to be required for DNA binding. Together with previous genetic data obtained in C. elegans, our results suggest that cellular THAP proteins may function as zinc-dependent sequence-specific DNA-binding factors with roles in proliferation, apoptosis, cell cycle, chromosome segregation, chromatin modification, and transcriptional regulation.

Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules.

Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.