Retinaldehyde dehydrogenase 2 (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic forebrain development by controlling FGF and sonic hedgehog signaling

Although retinoic acid (RA) has been implicated as one of the diffusible signals regulating forebrain development, patterning of the forebrain has not been analyzed in detail in knockout mouse mutants deficient in embryonic RA synthesis. We show that the retinaldehyde dehydrogenase 2 (RALDH2) enzyme is responsible for RA synthesis in the mouse craniofacial region and forebrain between the 8- and 15-somite stages. Raldh2-/- knockout embryos exhibit defective morphogenesis of various forebrain derivatives, including the ventral diencephalon, the optic and telencephalic vesicles. These defects are preceded by regionally decreased cell proliferation in the neuroepithelium, correlating with abnormally low D-cyclin gene expression. Increases in cell death also contribute to the morphological deficiencies at later stages. Molecular analyses reveal abnormally low levels of FGF signaling in the craniofacial region, and impaired sonic hedgehog signaling in the ventral diencephalon. Expression levels of several regulators of diencephalic, telencephalic and optic development therefore cannot be maintained. These results unveil crucial roles of RA during early mouse forebrain development, which may involve the regulation of the expansion of neural progenitor cells through a crosstalk with FGF and sonic hedgehog signaling pathways.     

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.     

A subset of clones in the chick telencephalon arranged in rostrocaudal arrays

Background Different areas of the vertebrate central nervous system appear to follow different rules during development for determining the position of sibling cells. For example, in the chick hindbrain, clones are frequently confined to a single functional unit that derives from a single rhombomere. In contrast, clones in the mammalian cerebral cortex often cross functional boundaries because of the extensive migration of sibling cells in orthogonal directions. We have investigated whether the pattern of clonal distribution in the chick telencephalon is similar to that of the hindbrain or to the more functionally analogous mammalian cerebral cortex. Progenitor cells in the chick telencephalon were marked using a retroviral library encoding alkaline phosphatase and over 10⁵ distinct molecular tags. Patterns of dispersion were detected using alkaline phosphatase histochemistry, followed by the recovery and sequencing of the molecular tag. We also analyzed the phenotypes of cells that occurred within the clones.Results A subset of progenitors gave rise to clones that were found in rostrocaudal arrays resembling tubes. Arrays were restricted in the mediolateral and dorsoventral planes but could span up to 4 mm in the rostrocaudal direction. They were found throughout the telencephalon and a single clone often spanned more than one telencephalic nucleus. Rostrocaudal clones comprised 60% of clones containing five or more cells and contained many different types of neurons, astrocytes, oligodendrocytes, or various combinations of these cell types.Conclusions Telencephalic progenitors are multipotent, producing progeny that become distinct cell types. Clonally related cells can migrate rostrocaudally within domains that are restrained in the mediolateral and dorsoventral directions. A subset of rostrocaudal clones resemble those seen in the mammalian cerebral cortex, with respect to the crossing of functional boundaries, but all rostrocaudal clones differ from the cerebral cortical clones in the pattern of spread of sibling cells, with the rostrocaudal clones being more constrained in the mediolateral and dorsoventral directions. A role for lineage in the patterning of the chick forebrain is supported by these observations. In addition, these data suggest a role for cues within the telencephalic marginal zone that serve to guide clones in their rostrocaudal migration.     

Shh and Gli3 regulate formation of the telencephalic-diencephalic junction and suppress an isthmus-like signaling source in the forebrain

In human holoprosencephaly (HPE), the forebrain does not separate fully into two hemispheres. Further, the border between the telencephalon and diencephalon, the telencephalic/diencephalic junction (TDJ), is often indistinct, and the ventricular system can be blocked at the third ventricle, creating a forebrain ‘holosphere’. Mice deficient in Sonic Hedgehog (Shh) have previously been described to show HPE and associated cyclopia. Here we report that the third ventricle is blocked in Shh null mutants, similar to human HPE, and that characteristic telencephalic and diencephalic signaling centers, the cortical hem and zona limitans intrathalamica (ZLI), are merged, obliterating the TDJ. The resulting forebrain holosphere comprises Foxg1-positive telencephalic- and Foxg1-negative diencephalic territories. Loss of one functional copy of Gli3 in Shh nulls rescues ventricular collapse and substantially restores the TDJ. Characteristic regional gene expression patterns are rescued on the telencephalic side of the TDJ but not in the diencephalon.Further analysis of compound Shh;Gli3 mutants revealed an unexpected type of signaling center deregulation. In Shh;Gli3 mutants, adjacent rings of Fgf8 and Wnt3a expression are induced in the diencephalon at the ZLI, reminiscent of the Fgf8/Wnt1-expressing isthmic organizer. Neither Shh nor Gli3 single mutants show this forebrain double ring of Fgf/Wnt expression; thus both Shh and Gli3 are independently required to suppress it. Adjacent tissue is not respecified to a midbrain/hindbrain fate, but shows overgrowth, consistent with ectopic mitogen expression.Our observations indicate that the separation of the telencephalon and diencephalon depends on interactions between Shh and Gli3, and, moreover, demonstrate that both Shh and Gli3 suppress a potential Fgf/Wnt signaling source in the forebrain. That optional signaling centers are actively repressed in normal development is a striking new insight into the processes of vertebrate brain development.     

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.     

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.     

The flat-top gene is required for the expansion and regionalization of the telencephalic primordium

The telencephalic vesicles form in the mouse embryo by the expansion of precursor regions in the anterior neural tube. Once the vesicles have formed, discrete dorsal and ventral territories can be recognized that later give rise to cortical and subcortical structures, respectively. To investigate the mechanisms that regulate the expansion and regionalization of the telencephalon, we have carried out a screen to identify recessive mutations that disrupt these events. We isolated a mouse mutant in which an early and critical step in development of the telencephalic vesicles is disrupted. Telencephalic primordia are present in flat-top embryos but they fail to progress to form the telencephalic vesicles. An increased rate of proliferation in the forebrain neurectoderm that accompanies telencephalic expansion in wild-type embryos fails to occur in flat-top embryos. Regionalization events that would normally take place during expansion of the primordia also fail to occur. Thus the phenotype of the flat-top mouse reveals that outgrowth of the telencephalic vesicles and their regionalization are coupled processes.     

Aromatase and 5β-reductase activity in cultures of developing zebra finch brain: An investigation of sex and regional differences

Estrogen treatment of hatchling female zebra finches causes the masculine development of singing behavior and of the telencephalic brain regions involved in the control of song. However, early estrogen treatment of males also blocks masculine development of copulatory behavior, presumably controlled by diencephalic regions. In an effort to determine whether the differences in estrogen action are related to sex and regional differences in androgen metabolism (estrogen synthesis or androgen inactivation), we measured aromatase and 5β-reductase activity in dissociated-cell cultures made separately from the telencephalon, diencephalon, and also cerebellum of hatching zebra finches under a variety of conditions. Cultures from all three brain regions express high levels of aromatase and 5β-reductase activity. Comparisons between telencephalic and diencephalic cultures of the activity and kinetics of aromatase suggest that the telencephalic cultures convert androgen to estrogen more efficiently than diencephalic cultures, which might be important in the differential action of estrogen in the two brain regions. However, the activity of neither aromatase nor 5β-reductase was significantly different between the sexes in either telencephalic or diencephalic cultures. Thus, comparisons between the sexes do not support the idea that differences in posthatching aromatase or 5β-reductase activity account for the pattern of sexual differentiation of the song and copulatory systems. © 1995 John Wiley & Sons, Inc.     

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.     

Spatial domains in the developing forebrain: developmental regulation of a restricted cell surface protein

We have isolated a monoclonal antibody, mAb 52G9, that recognizes a 55-kDa cell surface protein restricted to the early embryonic rat forebrain and to placode-derived structures. In the central nervous system (CNS), 52G9 immunoreactivity appears at Embryonic Day 11 (E11) in the rostral-most area of the telencephalon. It then spreads to the neuroepithelium of the telencephalon and basal diencephalon. Most strikingly, it appears at E14 in a distinct zone at the caudal end of the ventral diencephalic neuroepithelium. This area is sharply defined by strong 52G9 immunoreactivity bounded by unlabeled neuroepithelium. The pattern revealed by 52G9 is the first biochemical demonstration of spatial domains in the forebrain at a time prior to neuronal differentiation. By E18, 52G9 immunoreactivity has progressively disappeared from the forebrain; the glomerular layer of the olfactory bulb is the only 52G9-positive area in the CNS. The olfactory, otic, and hypophyseal placodes, which can be identified as early as E10, are also 52G9 positive as are their derivatives, the sensory epithelial of the nasal passage and inner ear, and also Rathke's pouch. The distribution and regulation of the 52G9 protein suggests that this novel cell surface molecule may be involved in the formation of spatial domains in the developing forebrain.     

A novel role for retinoids in patterning the avian forebrain during presomite stages

Retinoids, and in particular retinoic acid (RA), are known to induce posterior fates in neural tissue. However, alterations in retinoid signalling dramatically affect anterior development. Previous reports have demonstrated a late role for retinoids in patterning craniofacial and forebrain structures, but an earlier role in anterior patterning is not well understood. We show that enzymes involved in synthesizing retinoids are expressed in the avian hypoblast and in tissues directly involved in head patterning, such as anterior definitive endoderm and prechordal mesendoderm. We found that in the vitamin A-deficient (VAD) quail model, which lacks biologically active RA from the first stages of development, anterior endodermal markers such as Bmp2, Bmp7, Hex and the Wnt antagonist crescent are affected during early gastrulation. Furthermore, prechordal mesendodermal and prospective ventral telencephalic markers are expanded posteriorly, Shh expression in the axial mesoderm is reduced, and Bmp2 and Bmp7 are abnormally expressed in the ventral midline of the neural tube. At early somite stages, VAD embryos have increased cell death in ventral neuroectoderm and foregut endoderm, but normal cranial neural crest production, whereas at later stages extensive apoptosis occurs in head mesenchyme and ventral neuroectoderm. As a result, VAD embryos end up with a single and reduced telencephalic vesicle and an abnormally patterned diencephalon. Therefore, we propose that retinoids have a dual role in patterning the anterior forebrain during development. During early gastrulation, RA acts in anterior endodermal cells to modulate the anteroposterior (AP) positional identity of prechordal mesendodermal inductive signals to the overlying neuroectoderm. Later on, at neural pore closure, RA is required for patterning of the mesenchyme of the frontonasal process and the forebrain by modulating signalling molecules involved in craniofacial morphogenesis.     

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.     

Six3 cooperates with Hedgehog signaling to specify ventral telencephalon by promoting early expression of Foxg1a and repressing Wnt signaling

Six3 exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly (HPE), a forebrain malformation that results, at least in part, from abnormal telencephalon development. However, the roles of Six3 in telencephalon patterning and differentiation are not well understood. To address the role of Six3 in telencephalon development, we analyzed zebrafish embryos deficient in two out of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. We found that telencephalon forms in six3b;six7-deficient embryos; however, ventral telencephalic domains are smaller and dorsal domains are larger. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog (Hh) signaling in telencephalon development. Unlike in mice, we observe that Hh signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hh signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hh-independent role for Six3 in telencephalon patterning. We further find that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of the Wnt/β-catenin pathway.     

Conditional cell ablation via diphtheria toxin reveals distinct requirements for the basal plate in the regional identity of diencephalic subpopulations

The mammalian diencephalon is the caudal derivative of the embryonic forebrain. Early events in diencephalic regionalization include its subdivision along the dorsoventral and anteroposterior axes. The prosomeric model by Puelles and Rubenstein (1993) suggests that the alar plate of the posterior diencephalon is partitioned into three different prosomeres (designated p1–p3), which develop into the pretectum, thalamus, and prethalamus, respectively. Here, we report the developmental consequences of genetic ablation of cell populations from the diencephalic basal plate. The strategy for conditionally regulated cell ablation is based on the targeted expression of the diphtheria toxin gene (DTA) to the diencephalic basal plate via tamoxifen‐ induced, Cre‐mediated recombination of the ROSA^DTA allele. We show that activation of DTA leads to specific cell loss in the basal plate of the posterior diencephalon, and disrupted early regionalization of distinct alar territories. In the basal plate‐deficient embryos, the p1 alar plate exhibited reduced expression of subtype‐specific markers in the pretectum, whereas p2 alar plate failed to further subdivide into two discrete thalamic subpopulations. We also show that these defects lead to abnormal nuclear organization at later developmental stages. Our data have implications for increased understanding of the interactive roles between discrete diencephalic compartments. genesis 53:356–365, 2015. © 2015 Wiley Periodicals, Inc.     

Chemoattractant axon guidance cues regulate de novo axon trajectories in the embryonic forebrain of zebrafish

The development of axon tracts in the early vertebrate brain is controlled by combinations of soluble, membrane-bound and extracellular matrix molecules. How these multiple and sometimes conflicting guidance cues are integrated in order to establish stereotypical pathways remains to be determined. We show here that when interactions between the chemoattractive signal Netrin1a and its receptor Dcc are suppressed using a loss-of-function approach, a novel axon trajectory emerges in the dorsal diencephalon. Axons arising from a subpopulation of telencephalic neurons failed to project rostrally into the anterior commissure in the absence of either Netrin1a or Dcc. Instead these axons inappropriately exited the telencephalon and ectopically coursed caudally into virgin neuroepithelium. This response was highly specific since loss-of-function of Netrin1b, a paralogue of Netrin1a, generated a distinct phenotype in the rostral brain. These results show that a subpopulation of telencephalic neurons, when freed from long-range chemoattraction mediated by Netrin1a-Dcc interactions, follow alternative instructive cues that lead to creation of an ectopic axon bundle in the diencephalon. This work provides insight into how integration of multiple guidance signals defines the initial scaffold of axon tracts in the embryonic vertebrate forebrain.     

Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors

The olfactory bulb, neocortex and archicortex arise from a common pool of progenitors in the dorsal telencephalon. We studied the consequences of supplying excess Notch1 signal in vivo on the cellular and regional destinies of telencephalic precursors using bicistronic replication defective retroviruses. After ventricular injections mid-neurogenesis (E14.5), activated Notch1 retrovirus markedly inhibited the generation of neurons from telencephalic precursors, delayed the emergence of cells from the subventricular zone (SVZ), and produced an augmentation of glial progeny in the neo- and archicortex. However, activated Notch1 had a distinct effect on the progenitors of the olfactory bulb, markedly reducing the numbers of cells of any type that migrated there. To elucidate the mechanism of the cell fate changes elicited by Notch1 signals in the cortical regions, short- and long-term cultures of E14.5 telencephalic progenitors were examined. These studies reveal that activated Notch1 elicits a cessation of proliferation that coincides with an inhibition of the generation of neurons. Later, during gliogenesis, activated Notch1 triggers a rapid cellular proliferation with a significant increase in the generation of cells expressing GFAP. To examine the generation of cells destined for the olfactory bulb, we used stereotaxic injections into the early postnatal anterior subventricular zone (SVZa). We observed that precursors of the olfactory bulb responded to Notch signals by remaining quiescent and failing to give rise to differentiated progeny of any type, unlike cortical precursor cells, which generated glia instead of neurons. These data show that forebrain precursors vary in their response to Notch signals according to spatial and temporal cues, and that Notch signals influence the composition of forebrain regions by modulating the rate of proliferation of neural precursor cells.