Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon

Midway between the anterior neural border and the midbrain-hindbrain boundary, two well-known local signalling centres in the early developing brain, is a further transverse boundary with putative signalling properties -- the zona limitans intrathalamica (ZLI). Here, we describe formation of the ZLI in zebrafish in relation to expression of sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), and to development of the forebrain regions that flank the ZLI: the prethalamus and thalamus. We find that enhanced Hh signalling increases the size of prethalamic and thalamic gene expression domains, whereas lack of Hh signalling leads to absence of these domains. In addition, we show that shh and twhh display both unique and redundant functions during diencephalic patterning. Genetic ablation of the basal plate shows that Hh expression in the ZLI alone is sufficient for diencephalic differentiation. Furthermore, acquisition of correct prethalamic and thalamic gene expression is dependent on direct Hh signalling. We conclude that proper maturation of the diencephalon requires ZLI-derived Hh signalling.     

Thalamic development induced by Shh in the chick embryo

Patterning of the early neural tube is achieved in part by the inductive signals, which arise from neuroepithelial signaling centers. The zona limitans intrathalamica (ZLI) is a neuroepithelial domain in the alar plate of the diencephalon which separates the prethalamus from the thalamus. The ZLI has recently been considered to be a possible secondary organizer, effecting its inductions via sonic hedgehog (Shh), a signaling molecule which drives morphogenetic information for the thalamus. Using experimental embryological techniques involving the generation of chimeric embryos, we show that the formation of the ZLI in the diencephalic alar plate is due to an interaction between the prechordal and epichordal plate neuroepithelia. We also provide evidence that Shh expression in the ZLI underlies the morphogenetic activity of this putative diencephalic organizer. Ectopic Shh led to the auto-induction of its own gene expression in host cells, as well as to the expression of other genes involved in diencephalic regionalization and histogenesis. Analysis of long-term surviving embryos after Shh ectopic expression demonstrated that Shh was able to induce thalamic structures and local overgrowth. Overall, these results indicate that Shh expressed in the ZLI plays an important role in diencephalic growth and in the development of the thalamus.     

Patterning the zebrafish diencephalon by the conserved zinc-finger protein Fezl

The forebrain constitutes the most anterior part of the central nervous system, and is functionally crucial and structurally conserved in all vertebrates. It includes the dorsally positioned telencephalon and eyes, the ventrally positioned hypothalamus, and the more caudally located diencephalon [from rostral to caudal: the prethalamus, the zona limitans intrathalamica (ZLI), the thalamus and the pretectum]. Although antagonizing Wnt proteins are known to establish the identity of the telencephalon and eyes, it is unclear how various subdivisions are established within the diencephalon--a complex integration center and relay station of the vertebrate brain. The conserved forebrain-specific zinc-finger-containing protein Fezl plays a crucial role in regulating neuronal differentiation in the vertebrate forebrain. Here, we report a new and essential role of zebrafish Fezl in establishing regional subdivisions within the diencephalon. First, reduced activity of fezl results in a deficit of the prethalamus and a corresponding expansion of the ZLI. Second, Gal4-UAS-mediated fezl overexpression in late gastrula is capable of expanding the prethalamus telencephalon and hypothalamus at the expense of the ZLI and other fore- and/or mid-brain regions. Such altered brain regionalization is preceded by the early downregulation of wnt expression in the prospective diencephalon. Finally, fezl overexpression is able to restore the anterior forebrain and downregulate wnt expression in Headless- and/or Tcf3 (also known as Tcf7l1a)-deficient embryos. Our findings reveal that Fezl is crucial for establishing regional subdivisions within the diencephalon and may also play a role in the development of the telencephalon and hypothalamus.     

Determining the fate of Shh-expressing cells in the diencephalon using a BAC transgenic reporter

The thalamus and prethalamus consist of multiple distinct nuclei and their boundary is demarcated by the zona limitans intrathalamica (ZLI). The development of the primordial thalamus and prethalamus proceed within the caudal diencephalon. Shh has been shown to be essential for diencephalic patterning and regionalization. To understand the role of Shh in the specification of distinct thalamic and prethalamic nuclei, we developed a lineage marker for diencephalic cells expressing Shh by using bacterial artificial chromosome (BAC) transgenesis. A genomic fragment containing ～210 kb of the mouse Shh locus was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. This transgenic BAC reporter faithfully mimicked the pattern of endogenous Shh expression in the caudal diencephalon, including the ZLI. Fate mapping analysis at multiple developmental stages showed that descendents of Shh-expressing progenitor cells derived from ZLI contribute to a population of cells in the ventral lateral geniculate nucleus.     

Otx1 and Otx2 activities are required for the normal development of the mouse inner ear

The Otx1 and Otx2 genes are two murine orthologues of the Orthodenticle (Otd) gene in Drosophila. In the developing mouse embryo, both Otx genes are expressed in the rostral head region and in certain sense organs such as the inner ear. Previous studies have shown that mice lacking Otx1 display abnormal patterning of the brain, whereas embryos lacking Otx2 develop without heads. In this study, we examined, at different developmental stages, the inner ears of mice lacking both Otx1 and Otx2 genes. In wild-type inner ears, Otx1, but not Otx2, was expressed in the lateral canal and ampulla, as well as part of the utricle. Ventral to the mid-level of the presumptive utricle, Otx1 and Otx2 were co-expressed, in regions such as the saccule and cochlea. Paint-filled membranous labyrinths of Otx1-/- mutants showed an absence of the lateral semicircular canal, lateral ampulla, utriculosaccular duct and cochleosaccular duct, and a poorly defined hook (the proximal part) of the cochlea. Defects in the shape of the saccule and cochlea were variable in Otx1-/- mice and were much more severe in an Otx1-/-;Otx2(+/)- background. Histological and in situ hybridization experiments of both Otx1-/- and Otx1-/-;Otx2(+/)- mutants revealed that the lateral crista was absent. In addition, the maculae of the utricle and saccule were partially fused. In mutant mice in which both copies of the Otx1 gene were replaced with a human Otx2 cDNA (hOtx2(1)/ hOtx2(1)), most of the defects associated with Otx1-/- mutants were rescued. However, within the inner ear, hOtx2 expression failed to rescue the lateral canal and ampulla phenotypes, and only variable rescues were observed in regions where both Otx1 and Otx2 are normally expressed. These results suggest that both Otx genes play important and differing roles in the morphogenesis of the mouse inner ear and the development of its sensory organs.     

The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment—the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.     

Inductive signal and tissue responsiveness defining the tectum and the cerebellum

The mes/metencephalic boundary (isthmus) has an organizing activity for mesencephalon and metencephalon. The candidate signaling molecule is Fgf8 whose mRNA is localized in the region where the cerebellum differentiates. Responding to this signal, the cerebellum differentiates in the metencephalon and the tectum differentiates in the mesencephalon. Based on the assumption that strong Fgf8 signal induces the cerebellum and that the Fgf8b signal is stronger than that of Fgf8a, we carried out experiments to misexpress Fgf8b and Fgf8a in chick embryos. Fgf8a did not affect the expression pattern of Otx2, Gbx2 or Irx2. En2 expression was upregulated in the mesencephalon and in the diencephalon by Fgf8a. Consequently, Fgf8a misexpression resulted in the transformation of the presumptive diencephalon to the fate of the mesencephalon. In contrast, Fgf8b repressed Otx2 expression, but upregulated Gbx2 and Irx2 expression in the mesencephalon. As a result, Fgf8b completely changed the fate of the mesencephalic alar plate to cerebellum. Quantitative analysis showed that Fgf8b signal is 100 times stronger than Fgf8a signal. Co-transfection of Fgf8b with Otx2 indicates that Otx2 is a key molecule in mesencephalic generation. We have shown by RT-PCR that both Fgf8a and Fgf8b are expressed, Fgf8b expression prevailing in the isthmic region. The results all support our working hypothesis that the strong Fgf8 signal induces the neural tissue around the isthmus to differentiate into the cerebellum.     

A lineage specific enhancer drives Otx2 expression in teleost organizer tissues

In mouse Otx2 plays essential roles in anterior-posterior axis formation and head development in anterior visceral endoderm and anterior mesendoderm. The Otx2 expression in these sites is regulated by VE and CM enhancers at the 5' proximal to the translation start site, and we proposed that these enhancers would have been established in ancestral sarcoptergians after divergence from actinopterigians for the use of Otx2 as the head organizer gene (Kurokawa et al., 2010). This would make doubtful an earlier proposal of ours that a 1.1 kb fragment located at +14.4 to +15.5 kb 3' (3'En) of fugu Otx2a gene harbors enhancers phylogenetically and functionally homologous to mouse VE and CM enhancers (Kimura-Yoshida et al., 2007). In the present study, we demonstrate that fugu Otx2a is not expressed in the dorsal margin of blastoderm, shield and early anterior mesendoderm, and that the fugu Otx2a 3'En do not exhibit activities at these sites of fugu embryos. We conclude that the fugu Otx2a 3'En does not harbor an organizer enhancer, but encodes an enhancer for the expression in later anterior mesendodermal tissues. Instead, in fugu embryos Otx2b is expressed in the dorsal margin of blastoderm at blastula stage and shield at 50% epiboly, and this expression is directed by an enhancer, 5'En, located at -1000 to -800 bp, which is uniquely conserved among teleost Otx2b orthologues.     

Differential transcriptional control as the major molecular event in generating Otx1-/- and Otx2-/- divergent phenotypes

Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, show a limited amino acid sequence divergence. Their embryonic expression patterns overlap in spatial and temporal profiles with two major exceptions: until 8 days post coitum (d.p.c. ) only Otx2 is expressed in gastrulating embryos, and from 11 d.p.c. onwards only Otx1 is transcribed within the dorsal telencephalon. Otx1 null mice exhibit spontaneous epileptic seizures and multiple abnormalities affecting primarily the dorsal telencephalic cortex and components of the acoustic and visual sense organs. Otx2 null mice show heavy gastrulation abnormalities and lack the rostral neuroectoderm corresponding to the forebrain, midbrain and rostral hindbrain. In order to define whether these contrasting phenotypes reflect differences in expression pattern or coding sequence of Otx1 and Otx2 genes, we replaced Otx1 with a human Otx2 (hOtx2) full-coding cDNA. Interestingly, homozygous mutant mice (hOtx2(1)/hOtx2(1)) fully rescued epilepsy and corticogenesis abnormalities and showed a significant improvement of mesencephalon, cerebellum, eye and lachrymal gland defects. In contrast, the lateral semicircular canal of the inner ear was never recovered, strongly supporting an Otx1-specific requirement for the specification of this structure. These data indicate an extended functional homology between OTX1 and OTX2 proteins and provide evidence that, with the exception of the inner ear, in Otx1 and Otx2 null mice contrasting phenotypes stem from differences in expression patterns rather than in amino acid sequences.     

Shh-dependent formation of the ZLI is opposed by signals from the dorsal diencephalon

The zona limitans intrathalamica (ZLI) is located at the border between the prospective ventral thalamus and dorsal thalamus, and functions as a diencephalic signaling center. Little is known about the mechanism controlling ZLI formation. Using a combination of fate-mapping studies and in vitro assays, I show that the differentiation of the ZLI from progenitor cells in the alar plate is initiated by a Shh-dependent signal from the basal plate. The subsequent dorsal progression of ZLI differentiation requires ongoing Shh signaling, and is constrained by inhibitory factors derived from the dorsal diencephalon. These studies demonstrate that self-organizing signals from the basal plate regulate the formation of a potential patterning center in the ZLI in an orthogonal orientation in the alar plate, and thus create the potential for coordinated thalamic patterning in two dimensions.     

Otx2 and Otx1 protect diencephalon and mesencephalon from caudalization into metencephalon during early brain regionalization

Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2^{ΔFM1ΔFM2/ΔFM1ΔFM2}) and examined the TKO (Otx1^−/−Otx2^{ΔFM1ΔFM2/ΔFM1ΔFM2}) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers.     

Emx2 directs the development of diencephalon in cooperation with Otx2

The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2(-/-)Otx2(+/-) double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2(+/Emx2)). Emx2(-/-)Otx2(+/-) double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2(+/Emx2) knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.     

Otx1 and Otx2 in the development and evolution of the mammalian brain.

In the last decade, a number of genes related to the induction, specification and regionalization of the brain were isolated and their functional properties currently are being dissected. Among these, Otx1 and Otx2 play a pivotal role in several processes of brain morphogenesis. Findings from several groups now confirm the importance of Otx2 in the early specification of neuroectoderm destined to become fore-midbrain, the existence of an Otx gene dosage-dependent mechanism in patterning the developing brain, and the involvement of Otx1 in corticogenesis. Some of these properties appear particularly fascinating when considered in evolutionary terms and highlight the central role of Otx genes in the establishment of the genetic program defining the complexity of a vertebrate brain. This review deals with the major aspects related to the roles played by Otx1 and Otx2 in the development and evolution of the mammalian brain.     

The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice

The prevailing model to explain the formation of topographic projections in the nervous system stipulates that this process is governed by information located within the projecting and targeted structures. In mammals, different thalamic nuclei establish highly ordered projections with specific neocortical domains and the mechanisms controlling the initial topography of these projections remain to be characterized. To address this issue, we examined Ebf1(-/-) embryos in which a subset of thalamic axons does not reach the neocortex. We show that the projections that do form between thalamic nuclei and neocortical domains have a shifted topography, in the absence of regionalization defects in the thalamus or neocortex. This shift is first detected inside the basal ganglia, a structure on the path of thalamic axons, and which develops abnormally in Ebf1(-/-) embryos. A similar shift in the topography of thalamocortical axons inside the basal ganglia and neocortex was observed in Dlx1/2(-/-) embryos, which also have an abnormal basal ganglia development. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projections neurons. Thus, our study shows that: (1) different thalamic nuclei do not establish projections independently of each other; (2) a shift in thalamocortical topography can occur in the absence of major regionalization defects in the dorsal thalamus and neocortex; and (3) the basal ganglia may contain decision points for thalamic axons' pathfinding and topographic organization. These observations suggest that the topography of thalamocortical projections is not strictly determined by cues located within the neocortex and may be regulated by the relative positioning of thalamic axons inside the basal ganglia.