The present study centres on an anatomical entity, the dorsal pallium, defined first and foremost by a neurochemical property, the localization of GPT-expressing cells (GPT neuroblasts and neurons). GPT is unusual for its early and continued expression during cell lineage progression from neuroblast to neuron stages (and its absence in other preceding or concurrent precursors invulnerable to the present genetically targeted ablation), as well as its wide horizontal extent in the pallial proliferative matrix (Osheroff and Hatten, 2009
; Xie et al., 2009
). It provides a novel, modern view of one of the largest structural components in mammalian brain consistent with the organizational scheme of Marin-Padilla (1971
. Evidence based on this molecular marker characterizes the most dynamic phase of pallial morphogenesis in mice, but may not be strictly comparable with earlier reports based on different criteria for anatomical boundaries. Nevertheless, measurements of length, width and height of telencephalic vesicles in control mice match corresponding values from same-age examples of normal fetal mice shown by Kaufman (1992)
. External validation of these measurements for P4 control mice also occurs in Valverde's (1998)
masterful Golgi study of P5–P7 mice. Our values are 10% less than those of Valverde, with such slight differences most probably due to the effects of cryoprotection, fixation and age. Since the new measurements of the pallial volume are reasonably accurate, revised estimates of full, hemispheric complements of 2.5×105
dorsal pallial GPT neurons in control mice and 2.3×105
dorsal pallial GPT neurons in severely ablated mice are calculated from peak developmental densities of viable GPT neurons normalized for pallial volume, with the difference due to the lost proliferative output of VZ scars (Xie et al., 2009
). Genetically targeted ablation of GPT cells persistently eliminates up to 81% of the original complement of GPT neurons. The subsequent, comparable peak values demonstrate a near-normal restoration of GPT neurons tightly packed into an unscarred pallium of reduced size in ablated mice. The ablation is remarkable because the targeted cell population is transiently reduced, but not permanently suppressed.
The evidence obtained by the present study proves beyond reasonable doubt that genetically targeted ablation of GPT cells results in progressive and substantial reductions of pallial growth in prenatal mice, despite compensatory responses that rapidly replace the ablated cells (Xie et al., 2009
). Furthermore, these growth defects originate from the proliferative matrix of the pallial VZ, in accord with the rationale of the mechanism of action of the genetically targeted ablation (Xie et al., 2002
). Finally, these growth defects accumulate during prenatal and postnatal development in a constrained dose–response relationship with the extent of specific killing of GPT neurons, concordant with the purported specificity of action of the genetically targeted ablation (Xie et al., 2002
). GPT neurons are, in fact, the principal PP neurons, and developmental delays caused by their early, specific killing and regeneration trigger a cascade of subsequent defects in PP, immature and mature stages of neocortex, the main derivative of the proliferative matrix of the pallial VZ (Bayer and Altman, 1991
). These defects uncover biologically significant aspects of neocortical morphogenesis (determinants of organic growth and form) that are often neglected or inadequately characterized (Jacobson, 1970
; Price and Willshaw, 2000
The hypothesis that the ablation of GPT cells impairs vertical, but not horizontal, growth within the pallium and its neocortical part must be rejected. The evidence obtained by this investigation proves beyond reasonable doubt that growth falters in the horizontal surface area at both the pial and ventricular borders (as well as in the vertical thickness) of the pallium in ablated mice. Simultaneous growth defects in both the horizontal and vertical cytoarchitectural dimensions combine to produce a localized shortfall of dorsal pallial volume in the telencephalic vesicles. These findings expand and explain ambiguous, less detailed signs of impaired forebrain growth reported after ablation of GPT cells (Xie et al., 2002
) and ablation of PP neurons, purportedly with spared progenitors (Ghosh and Shatz, 1993
Three aspects of the present study are unusual and merit further attention.
Detection of organic growth defects after genetically targeted ablation
Modern combinations of molecular biology and neurochemistry such as genetically targeted ablation can provide a genuine experimental ‘dissection’ of embryological development through the selective elimination or enhancement of its cellular elements (Xie et al., 2002
; Jacobs et al., 2009
). These experiments are predicated on the subsequent, accurate recognition of alterations in phenotype. In practice, large-scale organic differences in structural phenotype can be difficult to identify and thus may be overlooked even by experienced investigators. As shown here, cursory examination is inadequate if it leads to paradoxical and potentially misleading results. We resort to antique, but not antiquated, methods of macroscopic morphometry from haematoxylin-counterstained serial section reconstructions to solve this problem by detection of pallial growth defects, and their place within a developing organic context, in ablated mice.
Serial section reconstructions at appropriate low magnifications allow all aspects of an organ to be viewed simultaneously and as a whole. Structural differences greater than 20–25% are usually needed for immediate recognition of revised phenotype. Instead, barely perceptible growth defects of less than 10% are evenly distributed across all three spatial dimensions of the ablated telencephalic vesicle. Such a widespread distribution of defects sustains the co-ordination of telencephalic and neurocranial growth, a pivotal outcome for successful prenatal development and live birth of ablated mice. Nevertheless, mapping and measurement reveal more substantial differences limited to the dorsal pallial part of the telencephalic vesicle. The volume, area and thickness of control pallium have distinct growth rates and levels of cumulative physical growth due to the different spatial dimensions that contribute to each dependent variable. These indices retain distinct, but suppressed, growth rates and levels of cumulative physical growth in ablated unscarred pallium. All three dependent variables have comparable growth defects relative to their physical magnitude despite measurement by independent means. This normalization for spatial dimensional scaling uncovers a common shortfall of growth, approx. 50% of its potential in control pallium, which propagates simultaneously as horizontal and vertical defects in ablated pallium, as shown by high intercorrelations of its volume, area and thickness.
In control mice, short-lived GPT intermediate progenitor cells in the pallial VZ are the exclusive source of its GPT neurons (Landry et al., 1998
; Xie et al., 2002
; Jacobs et al., 2007
). Between E11 and E13, these cells migrate radially in an ‘outside-in’ pattern and settle as the principal constituent neurons of PP neocortex. Between E13 and E15, they are infiltrated and dispersed by proximal, inverted settlement of early cortical plate neurons (destined for layers V and VI) to form trilaminar immature neocortex. By E16, physical separation of principal PP neurons settled in marginal zone versus cortical plate (i.e. true cleavage) proceeds with distal, inverted settlement of cortical plate neurons (destined for layers II–IV) to form hexalaminar mature neocortex (Bayer and Altman, 1990
). In ablated mice, cellular lamination is normally ordered in PP, immature and mature stages of neocortex. Dyslamination consists of horizontal and vertical growth defects distributed across all laminae, which are characteristically thin, densely cellular and obscurely bordered by projection axons and afferent terminal fields. The delayed settlement and tight packing of a near-normal complement of GPT neurons into this smaller ablated neocortex merely emphasize the presence of the middle lamina of principal PP neurons as usually found during its immature stage (Luskin and Shatz, 1985
; Xie et al., 2009
). Similar growth defects, with thin lamination and tight cell packing, are also distributed across the subcortical laminae of pallium.
Growth defects in the pallial VZ precede and are highly intercorrelated with subsequent growth defects in more distal pallial laminae. During the first phase of ablation, VZ growth stalls due to ganciclovir-induced cell killing, which evokes a profound structural reorganization of new GPT cells and mitotic precursors in pallial VZ. As dying cells down-size the original periventricular mitotic array, newly generated mitotic cells reconstitute as a paraventricular mitotic array (e.g. a thickened, porous plate akin to later-formed SVZ; Smart, 1973
; Noctor et al., 2001
; Xie et al., 2009
). Despite its horizontal and vertical compaction, the reconstituted matrix increases cell density in the VZ, with a concomitant increase in proliferative output sufficient to compensate for killed GPT cells during the second phase of the ablation. The reconstituted matrix maintains the normal sequence of generation for PP, proximal and distal cortical plate neurons, and replenishes by de novo
proliferation the normal complement of GPT neurons. The compensatory response is necessary for the resumption of pallial growth, which indicates that principal PP neurons are essential building blocks for the physical foundation of the neocortical assembly (Ghosh and Shatz, 1993
). The subsequent cost of growth reduction indicates that the VZ proliferative matrix encodes, at least in part, the physical dimensions of the pallium (Rakic, 2005
). The original VZ template is reduced by specific cell killing in ablated mice. The ‘gathering up’ of the reconstituted matrix limits the outward spread of the regenerated VZ template, diminishes the mobility of precursor and progenitor cells within it and translates into a horizontal growth defect in the somatic compartment of its neocortical derivatives (Walsh and Cepko, 1993
). Concurrent destruction of radial fibres contributes to horizontal and vertical growth defects in the somatic and neuropil tissue compartments. Dying GPT neuroblasts lose their radial fibres. RC2-immunoreactive radial fibres, believed to originate from neuronal precursors but not intermediate progenitors at this stage of development (Misson et al., 1988
; Hasling et al., 2003
; Molyneaux et al., 2007
), are then grossly reduced as ganciclovir-invulnerable, non-GPT-expressing precursors are recruited toward production of GPT neuroblasts. The decimation and disarray of the radial fibre network impair the distal disaggregation of settling neurons and reduce their migratory trajectory, but do not block radial migration or alter the normal settlement sequences of PP and cortical plate neurons.
Interaction of development and genetically targeted ablation in growth defects
If the only effect of genetically targeted ablation is permanent elimination of principal PP neurons, then pallial growth defects at P4 should be negligible because principal PP neurons normally account for only 1–2% of neocortical neurons at that age. Instead, the pattern of substantial, progressive reduction of growth in the ablated pallium indicates that the initial defect introduced by the specific killing of GPT cells is amplified by subsequent, faulty cellular interactions. Large-scale biological processes such as assembly of mammalian neocortex are obviously the developmental product of many concerted factors (Waddington, 1956
; Debat and David, 2001
). We use a composite ablation×age
interaction in a robust, two-factor statistical model to represent their cumulative effect. However, a stereotyped sequence of faulty, interrelated cellular interactions, where each step contributes to the composite interaction, is also beginning to emerge. The natural occurrence of both the composite interaction and the sequence of its component cellular interactions is best observed during the in situ
development of whole brain, since many of the component cellular interactions may be unavailable or inoperative in more restricted cell and tissue models.
As outlined above, specific killing of GPT cells produces only a few immediate changes, which occur mainly in pallial VZ. Excess dying GPT cells are temporarily stranded there, and their radial fibres are destroyed. These abnormal circumstances down-size and decelerate horizontal and vertical pallial growth within somatic and neuropil tissue compartments. Of greater importance, they also initiate a maturational delay for the settlement of principal PP neurons and introduce a morphogenetic discontinuity into the radial fibre network. Their destructive impact persists in the statistical model as the ablation main effect, which subtracts across age groups from constructive development normally represented by the age main effect.
main effect triggers subsequent defects of inductive, morphogenic and tropic cellular interactions (Edelman, 1988
), which join the composite ablation×age
interaction that increasingly subtracts between age groups from the age
main effect. The earliest fault in the sequence is the distortion of a close-proximity, inductive interaction between dying GPT cells and non-GPT precursors in the VZ, which accelerates and prolongs precursor differentiation toward an intermediate progenitor, replacement GPT neuroblasts. This compensatory recruitment is accompanied by faulty morphogenic interactions that reconstitute the VZ proliferative array and further disrupt the radial fibre network (Xie et al., 2002
). Reduction of horizontal and vertical growth arises from the restriction of the replacement VZ template, and the constriction of its physical translation into neocortex, as mediated by replacement PP neurons. The onset of these faults, and the ablation
main effect, coincide with and are reflected by the early E12–E13 growth deflection.
A faulty morphogenic interaction next takes place, most likely between the degenerated radial fibre network and the delayed replacement GPT neurons, which then fail to emit long projection axons (Xie et al., 2009
). These pioneer axons normally guide neocortical penetration of long afferent, particularly thalamocortical, axons (Molnar and Blakemore, 1995
; Molnar et al., 1998
; Hevner et al., 2002
; Jacobs et al., 2007
). In their absence, thalamocortical projections, most notably ‘specific’ thalamic relay connections for sensory systems, do not penetrate neocortical targets in an orderly fashion to elaborate characteristic terminal fields, usually initiated in immature neocortex (Auladell et al., 2000
; Lopez-Bendito and Molnar, 2003
). The onset of this defect coincides with and is reflected by the late E15–E16 growth deflection. The defect also extends to include associational and commissural corticocortical projections. Reduction of horizontal and vertical growth arises from the decreased frequency of axons in the neuropil tissue compartment and fibre tracts.
A faulty trophic interaction then occurs between the impoverished terminal fields of long axonal projections and neurons in mature neocortex, which curtail the outgrowth and branching of their dendrites and local axonal collaterals (Adams et al., 1997
; Xie et al., 2009
). Reduction of horizontal and vertical growth arises from the decreased frequency of neuronal processes in the neuropil tissue compartment. A final faulty trophic interaction proceeds between the inadequate synaptic connectivity conveyed by axons (afferent projection and local fibers) and neocortical neurons, which accelerates the pace of neuronal apoptosis and reduces growth in the somatic and neuropil tissue compartments during the third phase of the ablation (Cowan et al., 1984
; Xie et al., 2002
). It is interesting to note that this phase is accompanied by gross neuronal degeneration in subcortical sites, such as claustrum, normally predominated by reciprocal connections with neocortex.
This is not a full and exhaustive account of all abnormal cellular interactions that can take place in the ablated pallium, where the normal pattern of gradual, smooth and linear physical growth is largely maintained but progressively decelerated. In normal forebrain, such extensive, sequential interactions may buffer, regulate and synchronize development. In ablated forebrain, sequential cellular interactions underlie a constructive compensation that sustains pallial development, but sets in motion destructive consequences that limit its long-term success. In any event, the pathogenic amplification achieved by composite interaction shows that even modest delays of 24–48 h in the settlement of principal PP neurons can lead to many, if not all, of the same devastating structural defects claimed to be due to their selective, permanent elimination (Ghosh and Shatz, 1993
Persistence of growth defects after genetically targeted ablation
Specifically killed GPT cells are clearly the nexus and intervening variable between experimental conditions (genotype, ganciclovir treatment and age at ganciclovir treatment) that cause genetically targeted ablation and its long-term phenotypic expression (Xie et al., 2002
). We delineate a previously uncharted, immediate dose–response relationship between the ablation conditions and killed GPT neurons. Contingent on this outcome, a subsequent dose–response relationship unfolds between killed GPT neurons and persistent pallial growth defects. Both dose–response relationships are important demonstrations of the specificity of the genetically targeted ablation. However, the second relationship is technically constrained in at least three respects.
First, values for the independent variable, killed GPT neurons, are derived as a constant for each ablation group, not measured for each case within a group (although such observations prove to be feasible, and consistent with derived predicted values). In view of this limitation, all between-case, within-group variance of dependent variables is currently included in statistical error terms. This conservative solution underestimates the variance that should properly be attributed to killed GPT neurons. It also precludes multiple regression and factor analyses.
Secondly, the trigger episodes for the killing of GPT neurons are superimposed on normal morphogenetic processes, which require time to marshal the combination of constructive and destructive recovery mechanisms that lead to continued development and persistent, progressive growth defects. Extension of the post-ablation survival period allows the amplification of growth defects through sequential developmental interactions, but their increasing complexity tends to dilute the dose–response relationship between killed GPT neurons and growth defects. The step-like, stochastic clustering of outcomes for ablation groups with different levels of killed GPT cells suggests that some faulty cellular interactions do not contribute significantly to growth defects unless a minimum level of prior damage is attained (e.g. a threshold effect), while some faulty cellular interactions fail completely if a minimum level of prior damage is surpassed (e.g. a ceiling effect). Thus it is reasonable to propose that the ‘severe ablation’ cluster represents the full expression of the true ablation phenotype, while the ‘mild ablation’ cluster represents an intermediate version of the same ablation phenotype.
Thirdly, the genetically targeted ablation used in our longitudinal studies is actually a serial lesion of GPT cells related to each ganciclovir treatment, which further contributes to the clustered outcomes of different ablation groups (Finger et al., 1973
). The sequence and timing of episodic killing of GPT neurons recruit distinct levels of defective interactions. GPT intermediate progenitors have a short cell cycle of approx. 10 h and generate six to seven cohorts of GPT neurons over a period of approx. 72 h (Xie et al., 2009
). Killing of two or more consecutive, early cohorts of GPT neurons (36–81% losses of original complement), with minimum delays of 24 h for settlement of replacements, yields the catastrophic outcome of groups in the severe ablation cluster. In contrast, killing of a single early cohort of GPT neurons (18% loss) or two non-consecutive cohorts of GPT neurons (41% loss), with a minimum delay of 12 h for settlement of replacements, yields the survivable outcome of groups in the mild ablation cluster. Groups in the severe ablation cluster have impaired pioneer axons and afferent projections, which appear to be largely intact in groups in the mild ablation cluster. This difference suggests that cellular interactions underlying the outgrowth of sufficient pioneer axons for afferent guidance can tolerate short, but not long, delays of settlement of replacement GPT neurons, which constitute the permissive benefit actually obtained from the plasticity of precursors in the ablated VZ. The killing of two consecutive, late cohorts of GPT neurons on E13 (9% loss) leads to a mild ablation outcome because cellular interactions underlying the outgrowth of sufficient pioneer axons occur mostly before the cells are attacked.
We focus on the unscarred pallium of ablated mice because it retains sufficient structural integrity to preserve at least some functional role in neural processing and transmission. Yet, the pallium of severely ablated mice often has a second structural component composed of scar tissue. As shown previously (Xie et al., 2009
) and now in extensive maps of the ablated neocortical surface, these scars arise as a product of non-specific bystander killing related to the early, avascular arrangement of a limited site marking the pallial boundaries of distribution for the anterior and middle cerebral arteries (Marin-Padilla, 1985
). Scars, when present, do not alter the conclusions of the present study, since measurements based on total pallium (scarred plus unscarred tissue) and unscarred pallium yield consistent and comparable results. However, scars exhibit a low level of growth distinct from adjacent unscarred tissue. They fill a benign but non-essential gap in the development of ablated pallium until the late advent of hydrocephalus, after which the cellular fabric of both scarred and unscarred tissue is so compromised as to be unable to resist extreme thinning and ultimately fatal disruption (Xie et al., 2002
Taken together, the new evidence shows that genetically targeted ablations of the principal PP neurons can cost up to half of the growth potential of the dorsal pallium in mice, a species with modest neocortical evolution. It is certain that such losses produce significant functional impairments consistent with the principle of mass action and the high degree of cytoarchitectural organization normally achieved in neocortex (Lashley, 1929
; Jones, 1988
). The apparent absence of neurological seizures is not surprising due to the global distribution, symmetric localization and insular nature of impoverished synaptic connectivity in the ablated neocortical phenotype (Cepeda et al., 2010
). Because of the extensive evolutionary elaboration of neocortex, a structural loss in humans comparable with the severe ablation phenotype is likely to result in gross microtelencephaly, with marginal chances for fetal survival to birth (Jones, 1997
; Striedter, 2005
). However, structural loss in humans comparable with the mild ablation intermediate phenotype is more likely to result in survival. If asymmetrically biased to locations in one hemisphere, such mild forms of damage could distort the radial fibre network and contribute to the laminar alteration of cell density, neuronal disarray and pyramidal neuron misorientation found in clinical cases of (neo)cortical dysplasia associated with intractable pediatric epilepsy (Andre et al., 2007
). It has not escaped our notice that such early defects are unpromising candidates for amelioration by stem cell replacement therapies, since even modest delays of settlement of well-sited, intrinsically generated replacements for principal PP neurons, derived from the same cell lineage as the original killed cells, lead to faulty cellular interactions and progressive growth defects in a comparable neocortical phenotype now characterized after genetically targeted ablation.