Wnt genes define distinct boundaries in the developing human brain: implications for human forebrain patterning.

Understanding the factors that govern human forebrain regionalization along the dorsal-ventral and left-right (L-R) axes is likely to be relevant to a wide variety of neurodevelopmental and neuropsychiatric conditions. Recent work in lower vertebrates has identified several critical signaling molecules involved in embryonic patterning along these axes. Among these are the Wingless-Int (WNT) proteins, involved in the formation of dorsal central nervous system (CNS) structures, as well as in visceral L-R asymmetry. We examined the expression of WNT2b and WNT7b in the human brain, because these genes have highly distinctive expression patterns in the embryonic mouse forebrain. In the human fetal telencephalon, WNT2b expression appears to define the cortical hem, a dorsal signaling center previously characterized in mouse, which is also confirmed by BMP7 expression. In diencephalon, WNT2b expression is restricted to medial dorsal structures, including the developing pineal gland and habenular nucleus, both implicated in CNS L-R asymmetry in lower organisms. At 5 weeks gestation, WNT7b is expressed in cerebral cortical and diencephalic progenitor cells. As the cortical plate develops, WNT7b expression shifts, demarcating deep layer neurons of the neocortex and the hippocampal formation. Spatial and temporal expression patterns show startling similarity between human and mouse, suggesting that the developmental roles of these WNT genes may be highly conserved, despite the far greater size and complexity of the human forebrain.

Conserved expression of Hoxa1 in neurons at the ventral forebrain/midbrain boundary of vertebrates.

The previously described expression patterns of zebrafish and mouse Hoxa1 genes are seemingly very disparate, with mouse Hoxa1 expressed in the gastrula stage hindbrain and the orthologous zebrafish hoxa1a gene expressed in cell clusters within the ventral forebrain and midbrain. To investigate the evolution of Hox gene deployment within the vertebrate CNS, we have performed a comparative expression analysis of Hoxa1 orthologs in a range of vertebrate species, comprising representatives from the two major lineages of vertebrates (actinopterygians and sarcopterygians). We find that fore/midbrain expression of hoxa1a is conserved within the teleosts, as it is shared by the ostariophysan teleost zebrafish (Danio rerio) and the distantly related acanthopterygian teleost medaka (Oryzias latipes). Furthermore, we find that in addition to the described gastrula stage hindbrain expression of mouse Hoxa1, there is a previously unreported neurula stage expression domain, again located more anteriorly at the ventral fore/midbrain boundary. A two-phase expression profile in early hindbrain and later fore/midbrain is shared by the other tetrapod model organisms chick and Xenopus. We show that the anterior Hoxa1 expression domain is localized to the anterior terminus of the medial longitudinal fasciculus (MLF) in mouse, chick, and zebrafish. These findings suggest that anterior expression of Hoxa1 is a primitive characteristic that is shared by the two major vertebrate lineages. We conclude that Hox gene expression within the vertebrate CNS is not confined exclusively to the segmented hindbrain and spinal cord, but rather that a presumptive fore/midbrain expression domain arose early in vertebrate origins and has been conserved for at least 400 million years.

Neocortex patterning by the secreted signaling molecule FGF8.

A classic model proposes that the mammalian neocortex is divided into areas early in neurogenesis, but the molecular mechanisms that generate the area map have been elusive. Here we provide evidence that FGF8 regulates development of the map from a source in the anterior telencephalon. Using electroporation-mediated gene transfer in mouse embryos, we show that augmenting the endogenous anterior FGF8 signal shifts area boundaries posteriorly, reducing the signal shifts them anteriorly, and introducing a posterior source of FGF8 elicits partial area duplications, revealed by ectopic somatosensory barrel fields. These findings support a role for FGF signaling in specifying positional identity in the neocortex.

Patterning the mammalian cerebral cortex.

When and how is the area map of the cerebral cortex set up during development? Recent studies indicate that regional pattern emerges early in cortical neurogenesis, and that this pattern does not require cues from extrinsic innervation. Studies of mutant mice indicate a role for embryonic signaling centers and for specific transcription factors in regionalizing the cortex. Thus, it is increasingly probable that the cortex is partitioned using the same types of mechanisms--and in some cases, the same gene families--that are used in patterning other parts of the embryo. This emerging model is likely to be the basis for many future studies. However, new evidence also confirms the special nature of the cerebral cortex, in that cues from developing connections appear to modify and refine the final area map.

Detailed field pattern is intrinsic to the embryonic mouse hippocampus early in neurogenesis.

There is accumulating evidence that the mammalian cerebral cortex is regionally specified early in neurogenesis. However, the degree and scale of the regional pattern that is intrinsic to different parts of the cortical primordium remains unclear. Here, we show that detailed patterning-the accurate positioning of several areas or fields-is intrinsic to the part of the primordium that generates the hippocampus. A caudomedial portion of the cortical primordium, the site from which the hippocampus arises, was isolated from potential extrinsic patterning cues by maintaining it in explant culture. Explants were prepared at embryonic day (E) 12.5, which is early in hippocampal neurogenesis in the mouse and 3 d before individual fields are seen by differential gene expression. Allowed to develop for 3 d in vitro, E12.5 explants upregulate field-specific patterns of gene expression with striking temporal and spatial accuracy. Possible sources of patterning signals intrinsic to the explants were evaluated by removing the cortical hem or presumptive extrahippocampal cortex from the explants. To expose cells to different local positional cues, explant fragments were grafted into ectopic positions in a larger explant. None of these manipulations altered the development of patterned, field-specific gene expression. Finally, explants harvested at E10.5 also upregulate field-specific gene expression, although less robustly. Some hippocampal patterning information is therefore intrinsic to the caudomedial cortical primordium at the time that the first hippocampal neurons are born at E10.5. By E12.5, hippocampal field patterning appears to be well established and resistant to the manipulation of several potential intrinsic cues.

LIM-homeodomain gene Lhx2 regulates the formation of the cortical hem.

We are interested in the early mechanisms that initiate regional patterning in the dorsal telencephalon, which gives rise to cerebral cortex. Members of the LIM-homeodomain (LIM-HD) family of transcription factors are implicated in patterning and cell fate specification in several systems including the mammalian forebrain. Mice in which Lhx2 is disrupted were reported to have reduced telencephalic development, and the hippocampal primordium appeared to be missing, by morphological observation. We hypothesized that this may be due to a defect in the cortical hem, a Wnt- and Bmp-rich putative signaling center in the medial telencephalon, a source of regulatory signals for hippocampal development. We asked if the expression of any known hem-specific signaling molecule is deficient in Lhx2-/- mice. Our results reveal, unexpectedly, that at embryonic day (E)12.5, what appears to be some spared 'lateral' cortex is instead an expanded cortical hem. Normally restricted to the extreme medial edge of the telencephalon, the hem covers almost the entire dorsal telencephalon in the Lhx2-/- mice. This indicates a role for Lhx2 in the regulation of the extent of the cortical hem. In spite of an expanded, mislocated hem in the Lhx2-/- telencephalon, a potential source of ectopic dorsalizing cues, no hippocampal differentiation is detected in tissue adjacent to the mutant hem, nor does the overall dorsoventral patterning appear perturbed. We propose that Lhx2 is involved at a crucial early step in patterning the telencephalon, where the neuroepithelium is first divided into presumptive cortical tissue, and the cortical hem. The defect in the Lhx2-/- telencephalon appears to be at this step.

Emx2 is required for growth of the hippocampus but not for hippocampal field specification.

The vertebrate Emx genes are expressed in a nested pattern in early embryonic cerebral cortex, such that a medial strip of cortex expresses Emx2 but not Emx1. This pattern suggests that Emx genes could play a role in specifying different areas or fields of the cortex along the mediolateral axis. Such a role has been supported by the observation that in mice lacking functional Emx2 the hippocampus is shrunken and the most medial field of the cortex, the hippocampal dentate gyrus, appears by cytoarchitecture to be missing (Pellegrini et al., 1996; Yoshida et al., 1997). Use of region-specific molecular markers shows, however, that hippocampal fields are specified and correctly positioned in the Emx2 mutant. In particular, a dentate cell population is generated, although it fails to form a morphological gyrus. This failure may be part of a more widespread medial cortical defect in the mutant. Examination of cortical cell proliferation and differentiation indicates a disruption of the maturation of the medial cortex in the absence of Emx2. Thus, Emx2 is required for normal growth and maturation of the hippocampus but not for the specification of cells to particular hippocampal field identities.

Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toes(J).

Little is known about the mechanisms that control the development of regional identity in the mammalian telencephalon. The Gli family of transcription factor genes is involved in the regulation of pattern at many sites in the embryo and is expressed in the embryonic mouse telencephalon. We have analyzed telencephalic patterning in the extra-toes (J) (Xt(J)) mouse mutant, which carries a deletion in the Gli family member Gli3. We report that dorsoventral patterning of the telencephalon is dramatically disrupted in the Xt(J) mutant. Specific dorsal telencephalic cell types and gene expression patterns are lost in homozygous Xt(J) mutants, and features of ventral telencephalic identity develop ectopically in the dorsal telencephalon. This partial ventralization of the dorsal telencephalon does not appear to be induced by an expansion of Sonic hedgehog expression in the telencephalon, but may be due to a loss of Bmp and Wnt gene expression in a putative dorsal telencephalic signaling center, the cortical hem. Our findings suggest that in dorsal telencephalon Gli3 is needed to repress ventral telencephalic identity.

Patterning events and specification signals in the developing hippocampus.

The mouse hippocampus is an attractive model system in which to study patterning of a cortical structure. Ongoing studies indicate that hippocampal areas or fields are specified many days before birth -- possibly involving signals from within the cortical mantle. Although the hippocampal CA fields are distinguished by cytoarchitecture only after birth, molecular differences between fields appear by late gestation. Moreover, these embryonic fields are already specified to develop additional features that characterize the mature fields. The basic division of the hippocampus into fields may be specified still earlier. Thus, if medial cortical neuroepithelium is isolated in vitro early in hippocampal neurogenesis, it can autonomously generate features of a patterned hippocampus. In vivo, the spatial progression of initial field differentiation suggests that signals regulating growth and patterning could arise from sources close to the hippocampal poles. Observations of mouse mutants indicate that the cortical hem, an embryonic structure close to one pole of the hippocampus, is a source of such regulatory signals.

The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice.

In the developing vertebrate CNS, members of the Wnt gene family are characteristically expressed at signaling centers that pattern adjacent parts of the neural tube. To identify candidate signaling centers in the telencephalon, we isolated Wnt gene fragments from cDNA derived from embryonic mouse telencephalon. In situ hybridization experiments demonstrate that one of the isolated Wnt genes, Wnt7a, is broadly expressed in the embryonic telencephalon. By contrast, three others, Wnt3a, 5a and a novel mouse Wnt gene, Wnt2b, are expressed only at the medial edge of the telencephalon, defining the hem of the cerebral cortex. The Wnt-rich cortical hem is a transient, neuron-containing, neuroepithelial structure that forms a boundary between the hippocampus and the telencephalic choroid plexus epithelium (CPe) throughout their embryonic development. Indicating a close developmental relationship between the cortical hem and the CPe, Wnt gene expression is upregulated in the cortical hem both before and just as the CPe begins to form, and persists until birth. In addition, although the cortical hem does not show features of differentiated CPe, such as expression of transthyretin mRNA, the CPe and cortical hem are linked by shared expression of members of the Bmp and Msx gene families. In the extra-toesJ (XtJ) mouse mutant, telencephalic CPe fails to develop. We show that Wnt gene expression is deficient at the cortical hem in XtJ/XtJ mice, but that the expression of other telencephalic developmental control genes, including Wnt7a, is maintained. The XtJ mutant carries a deletion in Gli3, a vertebrate homolog of the Drosophila gene cubitus interruptus (ci), which encodes a transcriptional regulator of the Drosophila Wnt gene, wingless. Our observations indicate that Gli3 participates in Wnt gene regulation in the vertebrate telencephalon, and suggest that the loss of telencephalic choroid plexus in XtJ mice is due to defects in the cortical hem that include Wnt gene misregulation.

Early specification and autonomous development of cortical fields in the mouse hippocampus.

Studies of the specification of distinct areas in the developing cerebral cortex have until now focused mainly on neocortex. We demonstrate that the hippocampus, an archicortical structure, offers an elegant, alternative system in which to explore cortical area specification. Individual hippocampal areas, called CA fields, display striking molecular differences in maturity. We use these distinct patterns of gene expression as markers of CA field identity, and show that the two major hippocampal fields, CA1 and CA3, are specified early in hippocampal development, during the period of neurogenesis. Two field-specific markers display consistent patterns of expression from the embryo to the adult. Presumptive CA1 and CA3 fields (Pca1, Pca3) can therefore be identified between embryonic days 14.5 and 15.5 in the mouse, a week before the fields are morphologically distinct. No other individual cortical areas have been detected by gene expression as early in development. Indeed, other features that distinguish between the CA fields appear after birth, indicating that mature CA field identity is acquired over at least 3 weeks. To determine if Pca1 and Pca3 are already specified to acquire mature CA field identities, the embryonic fields were isolated from further potential specification cues by maintaining them in slice culture. CA field development proceeds in slices of the entire embryonic hippocampus. More strikingly, slices restricted to Pca1 or Pca3 alone also develop appropriate mature features of CA1 or CA3. Pca1 and Pca3 are therefore able to develop complex characteristics of mature CA field identity autonomously, that is, without contact or innervation from other fields or other parts of the brain. Because Pca1 and Pca3 can be identified before major afferents grow into the hippocampus, innervation may also be unnecessary for the initial division of the hippocampus into separate fields. Providing a clue to the source of the true specifying signals, the earliest field markers appear first at the poles of the hippocampus, then progress inwards. General hippocampal development does not follow this pronounced pattern. We suggest that the sources of signals that specify hippocampal field identity lie close to the hippocampal poles, and that the signals operate first on cells at the poles, then move inwards.

Multiple restricted lineages in the embryonic rat cerebral cortex.

We have labelled precursor cells in the embryonic rat cerebral cortex using BAG, a retroviral vector that expresses beta-galactosidase. We had previously reported that labelled precursor cells generate clusters of labelled cells that could be classified into four types by their morphological appearance and anatomical distribution (Price and Thurlow, 1988). In this study, we have used immunohistochemistry and intracellular dye labelling to identify the cell types that make up these clusters. We discovered that clusters are almost always composed of a single cell type. In addition to clusters composed entirely of neurones, we found four different types of glial cell clusters. In the grey matter, glial clusters are composed either of protoplasmic astrocytes, or of cells that have an astrocyte morphology, but no glial filaments. In the white matter, clusters are composed of either fibrous astrocytes or oligodendocytes. Our results indicate that each of these different cortical cell types is generated from a separate population of precursor cells.

Neuronal precursor cells in the rat hippocampal formation contribute to more than one cytoarchitectonic area.

We have tested the hypothesis that cell lineage restriction boundaries define the borders between cytoarchitectonic areas in the cerebral cortex. Clonally related cells were identified using a retroviral marking technique, and the dispersion of neuronal clones was examined with respect to the transitions between cortical areas. We chose to study the hippocampal formation because we found that clones of hippocampal neurons, unlike those in neocortex, are compact and readily identifiable in the adult and that transitions between areas in the hippocampus are sharp relative to the spread of a typical clone. We conclude, contrary to the hypothesis, that clones of neurons transgress the boundaries between areas in the hippocampal formation, that border-crossing clones are observed as frequently as would be expected if clones spread freely over the hippocampus with no constraint imposed by area borders, and that different types of pyramidal neurons, characteristic of different areas, may appear to a single clone. different areas, may appear in a single clone.

Inhibition of protein kinase C prevents phorbol ester- but not muscarine-induced depolarizations in the rat superior cervical ganglion.

The role of protein kinase C (PKC) activation in mediating muscarinic depolarization was assessed in the rat superior cervical ganglion. Staurosporine, an inhibitor of PKC, abolished a depolarization elicited by the direct PKC activator beta-phorbol 12,13-dibutyrate, but had little effect on the response to muscarine. Thus, activation of PKC may not be an obligatory transduction step between muscarinic receptor stimulation and depolarization.

9-Amino-1,2,3,4-tetrahydroacridine (THA) blocks agonist-induced potassium conductance in rat hippocampal neurones.

The actions of 9-amino-1,2,3,4-tetrahydroacridine (THA) were studied on rat CA1 pyramidal neurones under voltage-clamp in transverse slices of hippocampus maintained in vitro. As previously reported, THA reduced the resting conductance of cells; THA also suppressed inward rectification activated by hyperpolarization by up to 75% (The dose of THA which reduced the response by 50% (IC50) was 300 microM). More sensitive to the action of THA was the outward K+ current activated in CA1 neurones by 5-HT, adenosine and baclofen. This was completely blocked by THA (IC50 = 28 microM). The cooperativity of this latter action of THA with its well-known anticholinesterase activity is discussed in relation to the therapeutic effects of THA in treating Alzheimer's disease.

Neural associations of the substantia innominata in the rat: afferent connections.

The afferent connections of the substantia innominata (SI) in the rat were determined employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), in combination with histochemical procedures to characterize the neuropil of the SI and identify cholinergic cells. Both neurochemical and connectional data establish that the SI is organized into a dorsal and a ventral division. Each of these divisions is strongly affiliated with a different region of the amygdala, and, together with its amygdalar affiliate, forms part of one of two largely distinct constellations of interconnected forebrain and brainstem cell groups. The dorsal SI receives selective innervation from the lateral part of the bed nucleus of the stria terminalis, the central and basolateral nuclei of the amygdala, the fundus of the striatum, distinctive perifornical and caudolateral zones of the lateral hypothalamus, and caudal brainstem structures including the dorsal raphe nucleus, parabrachial nucleus, and nucleus of the solitary tract. Projections preferentially directed to the ventral SI arise from the medial part of the bed nucleus of the stria terminalis, the rostral two-thirds of the medial nucleus of the amygdala, a large region of the rat amygdala that lies ventral to the central nucleus, the medial preoptic area, anterior hypothalamus, medialmost lateral hypothalamus, and the ventromedial hypothalamus. Both SI divisions appear to receive afferents from the dorsomedial and posterior hypothalamus, supramammillary region, ventral tegmental area, and the peripeduncular area of the midbrain. Projections to the SI whose selectivity was not determined originate from medial prefrontal, insular, perirhinal, and entorhinal cortex and from midline thalamic nuclei. Findings from both PHA-L and WGA-HRP experiments additionally indicate that cell groups preferentially innervating a single SI division maintain numerous projections to one another, thus forming a tightly linked assembly of structures. In the rat, cholinergic neurons that are scattered throughout the SI and in parts of the globus pallidus make up a cell population equivalent to the primate basal nucleus of Meynert (Mesulam et al.: Neuroscience 10:1185-1201, '83). PHA-L-filled axons, labelled from lectin deposits in the dorsal raphe nucleus, peripeduncular area, ventral tegmental area, or caudomedial hypothalamus were occasionally seen to approach individual cholinergic neurons int he SI, and to contact the surface of such cells with axonal varicosities (putative synaptic boutons.(ABSTRACT TRUNCATED AT 400 WORDS)

Efferent connections of the substantia innominata in the rat.

The efferent connections of the substantia innominata (SI) were investigated employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The projections of the SI largely reciprocate the afferent connections described by Grove (J. Comp. Neurol. 277:315-346, '88) and thus further distinguish a dorsal and a ventral division in the SI. Efferents from both the dorsal and ventral divisions of the SI descend as far caudal as the ventral tegmental area, substantia nigra, and peripeduncular area, but projections to pontine and medullary structures appear to originate mainly from the dorsal SI. Within the amygdala and hypothalamus, which receive widespread innervation from the SI, the dorsal SI projects preferentially to the lateral part of the bed nucleus of the stria terminalis; the lateral, basolateral, and central nuclei of the amygdala; the lateral preoptic area; paraventricular nucleus of the hypothalamus; and certain parts of the lateral hypothalamus, prominently including the perifornical and caudolateral zones described previously. The ventral SI projects more heavily to the medial part of the bed nucleus of the stria terminalis; the anterior amygdaloid area; a ventromedial amygdaloid region that includes but is not limited to the medial nucleus; the lateral and medial preoptic areas; and the anterior hypothalamus. Modest projections reach the lateral hypothalamus, with at least a slight preference for the medial part of the region, and the ventromedial and arcuate hypothalamic nuclei. Both SI divisions appear to innervate the dorsomedial and posterior hypothalamus and the supramammillary region. In the thalamus, the subparafascicular, gustatory, and midline nuclei receive a light innervation from the SI, which projects more densely to the medial part of the mediodorsal nucleus and the reticular nucleus. Cortical efferents from at least the midrostrocaudal part of the SI are distributed primarily in piriform, infralimbic, prelimbic, anterior cingulate, granular and agranular insular, perirhinal, and entorhinal cortices as well as in the main and accessory olfactory bulbs. The cells of origin for many projections arising from the SI were identified as cholinergic or noncholinergic by combining the retrograde transport of WGA-HRP with histochemical and immunohistochemical procedures to demonstrate acetylcholinesterase activity or choline acetyltransferase immunoreactivity. Most of the descending efferents of the SI appear to arise primarily or exclusively from noncholinergic cells.(ABSTRACT TRUNCATED AT 400 WORDS)