Expanded expression of Sonic Hedgehog in Astyanax cavefish: multiple consequences on forebrain development and evolution

Ventral midline Sonic Hedgehog (Shh) signalling is crucial for growth and patterning of the embryonic forebrain. Here, we report how enhanced Shh midline signalling affects the evolution of telencephalic and diencephalic neuronal patterning in the blind cavefish Astyanax mexicanus, a teleost fish closely related to zebrafish. A comparison between cave- and surface-dwelling forms of Astyanax shows that cavefish display larger Shh expression in all anterior midline domains throughout development. This does not affect global forebrain regional patterning, but has several important consequences on specific regions and neuronal populations. First, we show expanded Nkx2.1a expression and higher levels of cell proliferation in the cavefish basal diencephalon and hypothalamus. Second, we uncover an Nkx2.1b-Lhx6-GABA-positive migratory pathway from the subpallium to the olfactory bulb, which is increased in size in cavefish. Finally, we observe heterochrony and enlarged Lhx7 expression in the cavefish basal forebrain. These specific increases in olfactory and hypothalamic forebrain components are Shh-dependent and therefore place the telencephalic midline organisers in a crucial position to modulate forebrain evolution through developmental events, and to generate diversity in forebrain neuronal patterning.     

Lhx2 mediates the activity of Six3 in zebrafish forebrain growth

The telencephalon shows the greatest degree of size variation in the vertebrate brain. Understanding the genetic cascade that regulates telencephalon growth is crucial to our understanding of how evolution of the normal human brain has supported such a variation in size. Here, we present a simple and quick approach to analyze this cascade that combines caged-mRNA technology and the use of antisense morpholino oligonucleotides in zebrafish embryos. Lhx2, a LIM-homeodomain protein, and Six3s (Six3b and Six3a), another homeodomain proteins, show very similar expression patterns early in forebrain development, and these are known to be involved in the growth of this part of the brain. The telencephalon of six3b and six3a double morphant (six3 morphant) embryos is markedly reduced in size due to impaired cellular proliferation. Head-specific overexpression of Lhx2 by photoactivation of a caged-lhx2 mRNA completely rescued this size reduction, whereas similar head-specific activation of Six3b could not rescue the knockdown effect of lhx2. In the forebrain of medaka embryos, Six3 facilitates cellular proliferation by sequestration of Geminin from Cdt1, a key component in the assembly of the prereplication complex. Our results suggest that Lhx2 may mediate an alternative or parallel pathway for control of cellular proliferation in the developing forebrain via Six3.     

Zebrafish forebrain and temporal conditioning

The rise of zebrafish as a neuroscience research model organism, in conjunction with recent progress in single-cell resolution whole-brain imaging of larval zebrafish, opens a new window of opportunity for research on interval timing. In this article, we review zebrafish neuroanatomy and neuromodulatory systems, with particular focus on identifying homologies between the zebrafish forebrain and the mammalian forebrain. The neuroanatomical and neurochemical basis of interval timing is summarized with emphasis on the potential of using zebrafish to reveal the neural circuits for interval timing. The behavioural repertoire of larval zebrafish is reviewed and we demonstrate that larval zebrafish are capable of expecting a stimulus at a precise time point with minimal training. In conclusion, we propose that interval timing research using zebrafish and whole-brain calcium imaging at single-cell resolution will contribute to our understanding of how timing and time perception originate in the vertebrate brain from the level of single cells to circuits.     

Molecular interactions coordinating the development of the forebrain and face

From an architectural point of view, the forebrain acts as a framework upon which the middle and upper face develops and grows. In addition to serving a structural role, we present evidence that the forebrain is a source of signals that shape the facial skeleton. In this study, we inhibited Sonic hedgehog (Shh) signaling from the neuroectoderm then examined the molecular changes and the skeletal alterations resulting from the treatment. One of the first changes we noted was that the dorsoventral polarity of the forebrain was disturbed, which manifested as a loss of Shh in the ventral telencephalon, a reduction in expression of the ventral markers Nkx2.1 and Dlx2, and a concomitant expansion of the dorsal marker Pax6. In addition to changes in the forebrain neuroectoderm, we observed altered gene expression patterns in the facial ectoderm. For example, Shh was not induced in the frontonasal ectoderm, and Ptc and Gli1 were reduced in both the ectoderm and adjacent mesenchyme. As a consequence, a signaling center in the frontonasal prominence was disrupted and the prominence failed to undergo proximodistal and mediolateral expansion. After 15 days of development, the upper beaks of the treated embryos were truncated, and the skeletal elements were located in more medial and proximal locations in relation to the skeletal elements of the lower jaw elements. These data indicate that a role of Shh in the forebrain is to regulate Shh expression in the face, and that together, these Shh domains mediate patterning within the frontonasal prominence and proximodistal outgrowth of the middle and upper face.     

Roles of organizer factors and BMP antagonism in mammalian forebrain establishment

A critical question in mammalian development is how the forebrain is established. In amphibians, bone morphogenetic protein (BMP) antagonism emanating from the gastrula organizer is key. Roles of BMP antagonism and the organizer in mammals remain unclear. Anterior visceral endoderm (AVE) promotes early mouse head development, but its function is controversial. Here, we explore the timing and regulation of forebrain establishment in the mouse. Forebrain specification requires tissue interaction through the late streak stage of gastrulation. Foxa2(-/-) embryos lack both the organizer and its BMP antagonists, yet about 25% show weak forebrain gene expression. A similar percentage shows ectopic AVE gene expression distally. The distal VE may thus be a source of forebrain promoting signals in these embryos. In wild-type ectoderm explants, AVE promoted forebrain specification, while anterior mesendoderm provided maintenance signals. Embryological and molecular data suggest that the AVE is a source of active BMP antagonism in vivo. In prespecification ectoderm explants, exogenous BMP antagonists triggered forebrain gene expression and inhibited posterior gene expression. Conversely, BMP inhibited forebrain gene expression, an effect that could be antagonized by anterior mesendoderm, and promoted expression of some posterior genes. These results lead to a model in which BMP antagonism supplied by exogenous tissues promotes forebrain establishment and maintenance in the murine ectoderm.     

Role of retinoic acid during forebrain development begins late when Raldh3 generates retinoic acid in the ventral subventricular zone

Retinoic acid (RA) synthesized by Raldh3 in the frontonasal surface ectoderm of chick embryos has been suggested to function in early forebrain patterning by regulating Fgf8, Shh, and Meis2 expression. Similar expression of Raldh3 exists in E8.75 mouse embryos, but Raldh2 is also expressed in the optic vesicle at this stage suggesting that both genes may play a role in early forebrain patterning. Furthermore, Raldh3 is expressed later in the forebrain itself (lateral ganglionic eminence; LGE) starting at E12.5, suggesting a later role in forebrain neurogenesis. Here we have analyzed mouse embryos carrying single or double null mutations in Raldh2 and Raldh3 for defects in forebrain development. Raldh2(-/-);Raldh3(-/-) embryos completely lacked RA signaling activity in the early forebrain, but exhibited relatively normal expression of Fgf8, Shh, and Meis2 in the forebrain. Thus, we find no clear requirement for RA in controlling expression of these important forebrain patterning genes, but Raldh3 expression in the frontonasal surface ectoderm was found to be needed for normal Fgf8 expression in the olfactory pit. Our studies revealed that later expression of Raldh3 in the subventricular zone of the LGE is required for RA signaling activity in the ventral forebrain. Importantly, expression of dopamine receptor D2 in E18.5 Raldh3(-/-) embryos was essentially eliminated in the developing nucleus accumbens, a tissue lying close to the source of RA provided by Raldh3. Our results suggest that the role of RA during forebrain development begins late when Raldh3 expression initiates in the ventral subventricular zone.     

Tetraploidy in mice, embryonic cell number, and the grain of the developmental map.

Tetraploid mice prepared by electrofusion develop for up to 14 days in utero. The embryos are essentially normal save that the forebrain and its associated tissues fail to develop properly. Here, we report measurements of cell counts in tissues and volume measurements of tetraploid and control embryos together with observations on the morphology of tetraploid embryos. The results show that the tetraploid embryos are about 85% normal size, but have only a little under half the number of cells of control embryos, with their nuclei being about twice the size of those of diploid cells. Close examination of sectioned material, in contrast, showed that tetraploid morphology and morphogenesis were indistinguishable from those of controls, except in forebrain-associated material. This conclusion gives some insight into an important developmental question, how fine can the developmental map be for normal cellular differentiation to proceed? As tetraploids have only about half the expected number of cells, the ability of these embryos to develop normally in all regions except the forebrain and its derivatives argues that pattern formation mechanisms can cope with the abnormally small number of cells in all regions except the forebrain. The results as a whole argue for size regulation in mammalian embryos being achieved by assaying absolute size rather than counting cell numbers.     

A dynamic fate map of the forebrain shows how vertebrate eyes form and explains two causes of cyclopia

Mechanisms for shaping and folding sheets of cells during development are poorly understood. An example is the complex reorganisation of the forebrain neural plate during neurulation, which must fold a sheet into a tube while evaginating two eyes from a single contiguous domain within the neural plate. We, for the first time, track these cell rearrangements to show that forebrain morphogenesis differs significantly from prior hypotheses. We postulate a new model for forebrain neurulation and demonstrate how mutations affecting two signalling pathways can generate cyclopic phenotypes by disrupting normal cell movements or introducing new erroneous behaviours.     

Bilateral feedback projections to the forebrain in the premotor network for singing in zebra finches

A discrete neural circuit mediates the production of learned vocalizations in oscine songbirds. Although this circuit includes some bilateral pathways at midbrain and medullary levels, the forebrain components of the song control network are not directly connected across the midline. There have been no previous reports of bilateral projections from medullary and midbrain vocal control nuclei back to the forebrain song system, but the existence of such bilateral corollary discharge pathways was strongly suggested by the recent observation that unilateral stimulation of a forebrain song nucleus during singing leads to a rapid readjustment of premotor activity in the contralateral forebrain. In the present study, we used neuroanatomical tracers to demonstrate bilateral projections from (a) the rostral ventrolateral medulla (RVL), which may control respiratory aspects of vocalization, to nucleus uvaeformis (Uva), and (b) the dorsomedial intercollicular nucleus (DM), a midbrain vocal control region, to Uva. Both RVL and DM receive descending projections from the forebrain song nucleus robustus archistriatalis, and Uva projects directly to the forebrain song nuclei interfacialis and high vocal center. We suggest that the bilateral feedback projections from DM and RVL to Uva function to coordinate the two hemispheres during singing in adult songbirds and to convey internal feedback of premotor signals to the forebrain in young birds that are learning to sing. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 27–40, 1998     

Temporally- and spatially regulated generation of distinct descendants by sonic hedgehog-expressing progenitors in the forebrain

The generation of distinct neural subtypes depends on the activities of cell-extrinsic and -intrinsic factors during the development of the vertebrate CNS. Previous studies have provided a molecular basis for how neural progenitors are patterned and generate distinct descendants that are spatially and temporally regulated by inductive signals secreted by polarized sources. However, it still remains unknown how the generation of neural descendants by progenitors located at polarized sources of inductive signals is controlled. Sonic hedgehog (Shh), which is expressed at the ventral midline in the forebrain, has been shown to play a critical role for the patterning and specification of distinct neural subtypes in the forebrain. Here, we analyzed the identities and distributions of Shh-descendants generated at discrete time points in the forebrain by using a ShhcreER(T2) mouse driver line in which a tamoxifen-inducible Cre cassette was inserted into the Shh locus together with a Z/EG mouse reporter line. Our results showed that Shh-expressing neural progenitors generated neuronal and glial descendants distributed throughout the telencephalon and diencephalon in a temporally distinct manner. Furthermore, our results showed that Shh-progenitors are located at two spatially distinct sub-domains that can be characterized by their temporally distinct patterns of Shh expression. These results suggest that temporally- and spatially controlled mechanisms that specify neural subtypes operate in the Shh-expressing progenitor domain, and raise the possibility that the distinct temporal gradient of Shh activity might be responsible for the generation of distinct neural subtypes in the telencephalon.     

Lack of the murine homeobox gene Hesx1 leads to a posterior transformation of the anterior forebrain

The homeobox gene Hesx1 is an essential repressor that is required within the anterior neural plate for normal forebrain development in mouse and humans. Combining genetic cell labelling and marker analyses, we demonstrate that the absence of Hesx1 leads to a posterior transformation of the anterior forebrain (AFB) during mouse development. Our data suggest that the mechanism underlying this transformation is the ectopic activation of Wnt/beta-catenin signalling within the Hesx1 expression domain in the AFB. When ectopically expressed in the developing mouse embryo, Hesx1 alone cannot alter the normal fate of posterior neural tissue. However, conditional expression of Hesx1 within the AFB can rescue the forebrain defects observed in the Hesx1 mutants. The results presented here provide new insights into the function of Hesx1 in forebrain formation.     

Signals from the edges: The cortical hem and antihem in telencephalic development

The early cortical primordium develops from a sheet of neuroepithelium that is flanked by distinct signaling centers. Of these, the hem and the antihem are positioned as longitudinal stripes, running rostro-caudally along the medial and lateral faces, respectively, of each telencepahlic hemisphere. In this review we examine the similarities and differences in how these two signaling centers arise, their roles in patterning adjacent tissues, and the cells and structures they contribute to. Since both the hem and the antihem have been identified across many vertebrate phyla, they appear to be part of an evolutionary conserved set of mechanisms that play fundamental roles in forebrain development.