Ganglionic eminence of the human fetal brain—new vistas
This review deals with recent findings concerning the complex functions of the ganglionic eminence (GE), which represents a conspicuous domain of the telencephalic proliferative zone and persists nearly throughout fetal life. The GE not only contains precursor neurons of the basal ganglia, it also contributes significantly to the population of interneurons in the cerebral cortex and to a population of thalamic neurons. The latter migrate through a distinct transient structure, the gangliothalamic body (GTB). The GE also represents an intermediate target for growing thalamic axons (on their way to the cerebral cortex) and cortical axons (on their way to the thalamus). In developmental neuropathology the GE plays an important role in prematurely born infants. The pathogenesis of GE bleedings is discussed with regard to the abundant expression of interleukin-6 (IL-6) receptors on GE cells. The consequences of such bleedings are discussed in view of cellular responses, such as the induction of leukemia inhibitory factor (LIF) expression in GE cells after hemorrhage. Anat Rec 267:191–195, 2002. © 2002 Wiley-Liss, Inc.
Proliferation of neuronal and most glial precursor cells takes place in the neuroepithelium lining the ventricles. Within the telencephalic proliferative zone a conspicuously thick domain protruding into the ventricular cavity is discernible nearly throughout fetal development. Thus, the telencephalic ganglionic eminence (GE) persists longer than other proliferative areas. Only by term has the GE nearly completely disappeared. As a consequence of the expansion of the cerebral hemisphere in a curved direction (to form the temporal lobe), the GE gains a C-shaped configuration. Sections of the middle third of the hemisphere pass twice through the GE. The superior part is found laterally in the floor of the central part of the lateral ventricle, with the caudate nucleus lying subjacent to it. The inferior part of the GE is seen in the roof of the inferior horn of the lateral ventricle, with the basolateral nuclei of the amygdala lying adjacent to it. In accordance with its C-shaped form, the superior and inferior parts of the GE are in continuity in sections of the occipital lobe (Kostovic, 1990; Ulfig et al., 2000c).
The GE gives rise to the precursor neurons of the striatum (putamen and caudate nucleus), the amygdala, and the basal nucleus of Meynert. During the second half of gestation the GE represents a major source of glial cells. This traditional view, as expressed in most textbooks on neuropathology, appears incomplete when novel findings on the GE (particularly those derived from the human fetal brain) are taken into consideration.
The first part of this review, therefore, focuses on which neuronal precursor cells are generated within the GE. The second part deals with another substantially different ontogenetic function of the GE. Its margin zones (i.e., the periphery of the GE) play a pivotal role in guiding outgrowing axons from the dorsal thalamus and the cerebral cortex, and vice versa. The GE represents an intermediate target for these axons. Finally, in the third chapter, the significance of the GE in developmental neuropathology is stressed. The GE is the most common site of intracranial hemorrhage (ICH). The latter is a frequent CNS complication in prematurely born infants.
NEURONAL POPULATIONS GENERATED IN THE GE
When slices of the developing cerebral cortex are cultured, the number of GABAergic interneurons is greatly reduced. When cortical slices are cultured with the GE attached, however, the number of GABAergic interneurons is significantly higher (Anderson et al., 1997). Moreover, mice lacking the homeodomain proteins DLX-1 and DLX-2, which are normally expressed in GE cells and regulate their differentiation, display a severe reduction in the number of cortical interneurons. These results and other data from the literature provide clear evidence that the majority of cortical (nonpyramidal) interneurons are generated in the GE. After leaving the GE these neuroblasts are found as tangentially migrating cells in the intermediate zone on their way to the cerebral cortex (Parnavelas, 2000). Axons of the intermediate zone may provide a substratum for this nonradial (i.e., tangential) migration. Axons of the developing corticofugal projection have been shown to contain the neural cell adhesion molecule TAG-1. Blocking of this molecule results in a distinct reduction of cortical interneurons. Thus, it is most probable that the migration of cortical interneurons from the GE is mediated by TAG-1 (Denaxa et al., 2001).
A subpopulation of the class of nonpyramidal interneurons expresses the calcium-binding protein calretinin (CR) (Andressen et al., 1993). Therefore, antibodies against CR can be used for detection of this cell type. In the human GE, particularly in its periphery, CR-immunoreactivity (ir) cells are found at midgestation. Moreover, CR-ir cells are seen in the intermediate zone. These cells, which appear as migratory neurons with a thicker leading and a thinner trailing process, are embedded in CR-ir fibers. The long axis of the bipolar cells runs parallel to the fibers (Ulfig, 2001b). The close association between tangentially oriented CR-ir cells and similarly-running CR-ir fibers supports the notion that these neuroblasts, most probably derived from the GE, migrate tangentially to reach the cortical anlage. The early expression of CR in migrating neurons may be related to the role of calcium in cell movement (Komuro and Rakic, 1996).
In experimental studies, GE precursor interneurons destined for the cerebral cortex clearly avoid the striatum during their migration to the cortex (Marin et al., 2001). This avoidance is at least in part the result of chemorepulsive signals, namely the expression of class 3 semaphorin proteins in the striatum. To mediate the repulsive effect of class 3 semaphorins, the receptors neuropilin 1 and neuropilin 2 are required (Raper, 2000). Marin et al. (2001) demonstrated that neuropilin 1 and 2 are expressed in interneurons migrating towards the cerebral cortex, but never in cells entering the striatum. Taking these findings together, the following mechanism underlying the sorting of GE precursor neurons is conceivable: While migrating from the GE to the cortex, future cortical interneurons express neuropilin 1 and 2 and are thus repelled by striatal cell-expressing class 3 semaphorins. Future striatal neurons lacking neuropilin 1 or 2 may enter the striatum.
With regard to the GE as a source of the bulk of cortical interneurons, another experimental finding is worth mentioning. Fishell et al. (1993) demonstrated that cortical precursor cells migrate in varying directions in their proliferative zone. However, precursor cells do not cross the boundary between cortical and subcortical (i.e., GE) proliferative zones. Thus, the GE neurons cross the striato-cortical boundary only outside the proliferative zones.
Intense vimentin-immunoreactive radial glial fibers in the vicinity of the GE have been demonstrated in the second half of gestation (Ulfig et al., 1999). These radial fibers, which are known to provide scaffolding for migrating neurons, often run at right angles to each other. Such crossing of fibers is also seen in the intermediate zone. To date, tangential migration is generally believed to take place independently of radial glia (Pearlman et al., 1998). However, the findings summarized above provide evidence that GE precursor neurons migrating tangentially may be guided by correspondingly arranged radial glial fibers. However, it should be stressed that tangentially oriented vimentin-positive fibers have so far only been observed in the developing human brain.
The accumulation of crossing fibers next to the GE may thus be interpreted as follows: GE precursor neurons first migrate laterally (i.e., radially), and then they change direction and migrate tangentially. Both migratory pathways may be formed by radial glial fibers (Ulfig, 2000b).
Rakic and Sidman (1969) first described tangential migration in the fetal human brain. They demonsrated that many neurons of the thalamic pulvinar arise in the telencephalic GE and migrate to the diencephalon during gestational months 5–8.
Various nuclei of the dorsal thalamus have been shown to receive neurons from the GE (Letinic and Kostovic, 1997). The precursor cells have to cross the telencephalo-diencephalic boundary and then migrate through a distinct transient structure, referred to as the gangliothalamic body (GTB). The GTB represents a thin band of migratory neurons lying upon the superior thalamic nuclei (in the lateral third or lateral half). The lateral half of the GTB merges with the medial edge of the GE at the telencephalic-diencephalic sulcus (i.e., the terminal groove).
The aforementioned findings and interpretations were recently substantiated by investigations performed on cultured human brain slices (Letinic and Rakic, 2001). Those studies provided direct evidence that neurons expressing GABA migrate from the GE to the dorsal thalamus and settle mainly with in the pulvinar and the mediodorsal nucleus. These tangentially migrating neurons appear to be independent of glial fibers, relying instead on contacts with other neurons. This homotypic-neurophilic guidance, which closely resembles the chain migration of neurons to the olfactory bulb in rodents, depends on the presence of polysialylated neuronal cell adhesion molecule (PSA-NCAM). The latter has also been demonstrated by Letinic and Rakic (2001), who also showed that the dorsal thalamus attracts GE cells.
The GTB can be regarded as a conduit through which neuronal precursors migrate into the dorsal thalamus. In the human fetal brain the GTB can reliably be demonstrated in microtubule-associated protein (MAP) 1b immunopreparations (Ulfig et al., 2000b). The GTB contains bipolar MAP1b-ir cells, whose long axes run parallel with the ependymal surface. These cells form a thin band lying upon the superior thalamic nuclei.
The GTB has not yet been delineated in the nonhuman primate brain, and GE cells migrating towards the dorsal thalamus have not been shown in the macaque monkey or in any other mammalian species investigated so far (Rakic, 1990; Hatten, 1999).
On the whole, the GTB represents a migratory pathway that probably is unique to the human brain, and significantly contributes to an expansion of associative thalamic nuclei being involved in higher cognitive functions (Letinic and Rakic, 2001; Rao and Wu, 2001).
It is interesting to note that oligodendrocyte precursors also leave the GE and follow tangential migration routes to reach various white matter areas of the forebrain (Spassky et al., 1998; Ulfig et al., 2002).
GE—AN INTERMEDIATE TARGET FOR OUTGROWING AXONS
Outgrowing axons may use several types of guidance cues to find their way to their final target. In the development of some pathways, fibers make characteristic sharp changes of directions as they reach a specific site (Bate, 1976). Cells of such a particular circumscribed point exert a guiding role and may be regarded as an intermediate target, which orchestrates the pathfinding of a developing projection through sequential target recognition (Bentley and Keshishian, 1982).
Recently, the GE has been shown to be an intermediate target for corticofugal and thalamocortical fibers. On the way to their target, both thalamic and cortical axons appear to pause within the mantle region of the GE (Metin and Godement, 1996). This part of the GE contains postmitotic neurons, which are characterized by a complex morphology.
The GE cells building up the mantle region may be directed towards this region by a chemoattractant, or they may migrate in random directions and reach the mantle region by coincidence.
In the human fetal brain, antibodies directed against SNAP-25 may be used for the demonstration of corticofugal axons reaching the GE. Synaptosomal-associated protein of 25kDa (SNAP-25) belongs to the soluble N-methylmaleimide-sensitive factor attachment protein receptor (SNARE), which is required for docking and fusion of synaptic vesicles in exocytosis (Söllner et al., 1993; Gerst, 1999). In the mature brain, SNAP-25 is transported to presynaptic terminals by fast axonal transport; therefore, it is mainly detected in terminals. During development, however, SNAP-25 accumulates in axons and therefore can be demonstrated in SNAP-25 immunopreparations. SNAP-25-immunoreactive fibers are found during weeks 19–24 of gestation—for instance, within the internal capsule. At the ventricular edge they display curvature and seem to be directed towards the lateral margin of the GE. Intense punctate immunoreactivity is present in this region. In double labelings, CR-ir nerve cells are observed to be surrounded by SNAP-25-ir puncta. Thus, fibers of the intermediate zone, probably originating from subplate (SP) neurons, make a turn to reach the lateral margin of the GE (Ulfig et al., 2000d; Ulfig and Chan, 2002).
A likely candidate for inducing the orientation of growing axons towards the GE has been demonstrated to be netrin-1, a chemoattractant that is released by cells of the GE (Metin et al., 1997). An increasing gradient of netrin-1 is likely to attract growing axons towards the lateral margin of the GE. The latter thus constitutes an intermediate target where growing axons appear to pause.
The medial margin of the GE most probably also functions as an intermediate target for growing axons. This functional role of the medial margin can be demonstrated when antibodies are applied against MAP1b. MAP1b, which is the most abundant MAP in the developing brain, is involved (along with other proteins) as an intracellular regulator of process outgrowth. At midgestation, MAP1b-ir fiber bundles are seen in the dorsal thalamus. They merge with a band-like structure that shows intense punctate immunolabeling (indicative of fiber termination) and represents the medial marginal zone of the GE. These results indicate that fibers seemingly originating in the dorsal thalamus terminate in the medial marginal zone of the GE (Ulfig et al., 2000b). Thus, fibers destined for the cortex appear to pause when they reach this portion of the GE, which may act as a cellular guidepost.
Recent experimental data has shown that the chemoattractant netrin-1 is required for the proper development of thalamocortical axons. Dorsal thalamic axons are attracted by netrin-1. This effect is mediated by two receptors, DCC and neogenin, which are expressed by dorsal thalamic neurons (Braisted et al., 2000).
On the whole, the medial and lateral marginal zones of the GE are involved in transient neuronal circuitries that are essential for the establishment of mature projections between the thalamus and the cerebral cortex. It has been postulated that cortical (or thalamic) axons may be waiting at the intermediate target (i.e., the GE) for interactions with reciprocal thalamic (or cortical) axons (the “handshake hypothesis” (Molnar, 1998)).
Thalamocortical and corticofugal fibers both interact with another intermediate target after leaving the GE: the subplate (for thalamocortical axons) and the perireticular nucleus (for corticofugal axons) (Ulfig et al., 2000c).
GE cells have been shown to project to certain parts of the cortex-anlage during early fetal development. In the human fetal brain this early efferent projection from the GE can be visualized using anti-synaptogyrin. This synapse-related protein is among the most abundant synaptic vesicle components. During early development it accumulates in somata and axons (Ulfig et al., 2000a).
The early projection from the GE may provide a scaffold that interacts with outgrowing corticofugal fibers. Thus, cortical axons may navigate their way towards the GE along a pathway formed by pioneering axons of GE cells (Metin and Godement, 1996; Molnar, 1998). In this manner cortical axons may reach the GE as their intermediate target.
On the whole, the GE represents a neurochemically and functionally heterogeneous structure.
GE IN DEVELOPMENTAL NEUROPATHOLOGY
Hemorrhage of the GE (ICH) is a major central nervous system complication of preterm delivery. ICH can result in severe handicap in the infant, impairing motor and cognitive development (Volpe, 1996). In particular, the mechanisms underlying cognitive deficits are poorly understood to date.
In general, brain injury induces various responses, such as changes in trophic factor expression. Very recently it was shown that leukemia inhibitory factor (LIF), which belongs to the family of neuropoetic cytokines, is expressed in GE cells after bleedings (Taga and Kishimoto, 1997; Ulfig, 2001a). A moderate number of LIF-ir cells were seen in the vicinity of the bleedings, whereas no LIF-ir structure was observed in the control group. This is the only cellular response after bleeding described so far. LIF expression may be a result of circuit disruption or direct mechanical injury, either of which may lead to glial activation (Banner et al., 1997). LIF may act as a stimulator of astrocyte differentiation (Nishiyama et al., 1993). As the GE is a major source of oligodendrocytes for various brain areas, their number could be reduced due to an astrocyte-inducing activity exerted by LIF (Birling and Price, 1998). The occurrence of, or increase in LIF expression in the GE may also interfere with normal developmental processes taking place within the GE. Experimental studies have demonstrated neurotrophic and neurotoxic effects, as well as a reduction in neuronal death after lesions (Murphy et al., 1991; Cheema et al., 1994). It is so far not clear what kind of action is exerted on neuronal precursor cells of the GE via LIF.
Intrauterine infection has been well established as a leading cause of preterm labor (Watts et al., 1992). The infection is associated with increased concentrations of cytokines in the amniotic fluid. Moreover, the concentrations of interleukin-6 (IL-6) (Weeks et al., 1997), a multifunctional inflammatory cytokine, in the umbilical cord plasma have been shown to be significantly elevated in cases of ICH. Between weeks 22 and 28 of gestation, IL-6 receptor is highly expressed in the GE (Ulfig and Friese, 1999). Thus, it may be assumed that IL-6 activates GE cells, which are known to be a major source of the plasminogen activator (Gilles et al., 1971). This protease is involved in the remodeling and final involution of the GE. Enhanced activity of the plasminogen activator can induce an increase in fibrinolytic activity. Thus, any fibrin developing in the GE can be lysed, and the occurrence of profuse bleeding after initial capillary rupture can be explained (Volpe, 1996).
It is generally believed that the prognosis of ICH is dependent on the size of the bleeding. However, a strict correlation has not been detected. Taking into consideration the data on the complex organization of the GE, it may be assumed that the exact location of the bleeding may largely influence the developmental outcome of ICH. If the margin of the GE is involved in a small lesion, consequences may be severe, as this part of the GE is involved in the establishment of connections.
Periventricular leukomalacia (PVL) commonly results in circumscribed foci in the white matter in close proximity to the lateral ventricle. Thus, damage of neurons migrating from the GE towards the cerebral cortex may occur. Cognitive or mental deficits often observed in PVL may be attributed to such lesions of migrating neurons (i.e., prospective interneurons of the cerebral cortex). Another cytokine has been proposed to play a contributory role in the pathogenesis of PVL: tumor necrosis factor (TNF) (Leviton, 1993; Ulfig, 2000a). TNF is also secreted as a result of infection, and it may mediate preterm delivery (via prostaglandins) and PVL. In PVL, production of TNF by microglia is regarded as a response to endotoxin, which is associated with infection. TNF may promote destruction of myelin-forming oligodendrocytes. In line with this assumption, a paucity of myelin is observed in PVL foci. However, it should be stressed that the involvement of TNF in PVL is only one hypothesis regarding the genesis of this white matter injury.
Bleedings in the GE may also extend into the GTB, which contains migrating neurons destined for the dorsal thalamus. This aspect has so far been totally overlooked in developmental neuropathology.
Evidence has been provided that the GE has a distinctly higher potential to generate myelin-forming oligodendrocytes than other parts of the proliferative zone (Birling and Price, 1998). The sequential maturation of oligodendrocytes is influenced by growth factors, such as ciliary neurotrophic factor (CNTF) (Louis et al., 1993; Mayer et al., 1994). In a recent study CNTF was shown to be abundantly expressed in GE cells during months 5–7 of gestation (Ulfig, 2001b). Thus, bleedings in the GE may also impair the differentiation of oligodendrocytes. PVL is a common accompaniment to ICH. In these cases the impairment of oligodendrocytes and myelin formation is more pronounced because the protective effect of CNTF produced in the GE on white matter oligodendrocytes is reduced. This notion is supported by the observation that associated brain lesions (PVL + ICH) may lead to a more pronounced reduction in developmental outcome.
The migration of GE neurons towards the cerebral cortex and the dorsal thalamus may be of relevance in the pathogenesis of schizophrenia. Decreased neuronal density has been reported in the cerebral cortex (particularly in the frontal lobe (Popken et al. 2000)) and in the thalamus (particularly in the mediodorsal nucleus (Akbarian et al., 1995). This reduction may be linked to an alteration in GE cell migration. The latter is dependent on the presence of PSA-NCAM. In line with the aforementioned hypothesis, the level of PSA-NCAM expression is decreased in schizophrenic brains (Barbeau et al., 1995).