The new observations obtained in the present study show: (i) the normal development of GPT cells, (ii) the targeted ablation of GPT cells predicated on the transient, proliferative phase of their normal development, and (iii) the reactions of surviving and replacement GPT cells predicated on the targeted ablation of their predecessors.
GPT neurons in control mice fulfil all of the morphogenetic criteria now employed for the identification of the principal preplate neurons of mammalian neocortex: radial migration, early proliferation, emission of corticofugal pioneer axons from more deeply settled cells, relatively short survival and an ‘outside-in’ vertical gradient of settlement (Marin-Padilla, 1971
; Bayer and Altman, 1990
; Valverde et al., 1995
; Verney and Derer, 1995
; Meyer and Wahle, 1999
; Super and Uylings, 2001
; Zecevic and Rakic, 2001
). By use of the most sensitive methods available to detect the earliest and widest distribution of these cells, their unilaminar organization in preplate neocortex transforms into a trilaminar organization in primitive and mature neocortex, with a superficial lamina embedded in layer I, a prominent middle lamina engulfed by layers V–VI, and a deep lamina that persists in mature subplate were identified (Landry et al., 1998
; Xie et al., 2002
: Jacobs et al., 2007
; Pontious et al., 2008). The superficial lamina of GPT neurons demonstrates a substantial component of the molecular layer derived by radial migration, while true Cajal–Retzius neurons originate mainly by tangential migration (Bielle et al., 2005
). The middle lamina of GPT neurons are definitive preplate neurons, not early-generated infragranular neurons of cortical plate, even in the relatively simple, but highly intercalated, rodent neocortex (Luskin and Shatz, 1985
; Hasling et al., 2003
). The deep lamina of GPT neurons contains the youngest, but most persistent, of the principal preplate neurons. The developmentally emergent laminar organization of these principal preplate neurons may contribute to the vertical ordering of mature neocortex by positioning selective barriers, or targets, for later-generated neurons, as suggested by reorganization in mutant reeler mice (Sheppard and Pearlman, 1997
Ganciclovir treatment of age-vulnerable experimental mice bearing the GPT/HSV-TK transgene rapidly initiates a bilateral pathogenetic sequence of increased apoptosis and suppressed accumulation of GPT neurons in neocortex. Targeted to their GPT progenitors, this attack promotes rapid apoptosis of simultaneously proliferated GPT neuroblasts and GPT neurons. Compensatory reconstitution occurs as earlier, invulnerable non-GPT progenitors in the ventricular zone of the dorsal pallium proliferate replacements for killed GPT neuroblasts, which then proliferate replacements for killed GPT neurons. Near-normal restorations of cell complement delay the settlement of GPT neurons into the preplate, which dramatically limits the outgrowth of their axonal projections. Surviving and replacement GPT neurons have the same morphogenetic features as normal GPT neurons, except for defective axonal outgrowth. The targeted ablation of GPT cells in the dorsal pallium, in concert with the reactions of surviving and replacement cells, yields phenotypic defects of radial migration, laminar framing and afferent guidance, where governance has been attributed to preplate neurons in previous studies (Ghosh and Shatz, 1993
; Allendoerfer and Shatz, 1994
; McConnell et al., 1994
; Molnar and Blakemore, 1995
; Ogawa et al., 1995
; Del Rio et al., 1997
; Molnar et al., 1998
; Super et al., 1998
; Super and Uylings, 2001
; Sarnat and Flores-Sarnat, 2002
; Xie et al., 2002
; Hevner et al., 2003
; Jacobs et al., 2007
). The hypothesis that a permanent, catastrophic extermination of preplate neurons accounts for the targeted ablation must be rejected because the suppression of GPT neurons is transient (i.e. killed cells are replaced) and the apoptosis of GPT neurons is graded (i.e. killed cells are compounded additively by serial ganciclovir treatments during progenitor vulnerability).
The present study provides support for additional conclusions of biological significance by integration of the cellular origins, morphogenesis and population dynamics of normal and ablated GPT cells. These conclusions address persistent issues regarding the specificity of the targeted ablation and the regulated development of the principal preplate neurons.
Specific cell killing eliminates a major fraction of GPT neurons in ablated mice
Binary poisoning by HSV-TK/ganciclovir can yield specific killing of target cells that express HSV-TK during S-phase, and non-specific killing of adjacent cells that do not express HSV-TK (Mesnil and Yamasaki, 2000
). GPT neurons are one of the principal specific targets for ablation, as demonstrated directly by the co-localization of apoptosis and GPT reporters. Despite the dissemination of cellular lesions and the progressive failure of target gene expression in dying cells, the extent of apoptotic GPT neurons can be determined from the preponderance of morphological evidence and key statistical observations of densities of viable GPT neurons and apoptotic cells in control and ablated mice during and shortly after ganciclovir exposure
Half the peak density of non-phagocytic cells killed during early-onset apoptosis is a predictable shortfall of GPT neurons due to the killing mechanism of the targeted ablation initiated during asymmetric cell division. The remaining half consists of specifically killed GPT neuroblasts. Consistent with these interpretations, the sum of 50% of the apoptotic cell density and the total GPT neuron density observed on E13 shortly after ganciclovir treatment in the main group of ablated mice predictably and nearly attains the peak density of GPT neurons in littermate control mice, with similar results obtained throughout the first phase of apoptosis. Furthermore, the fraction of GPT neurons that escapes specific killing at E11–E12 is predictable from durations of effective ganciclovir action (5 h), S-phase (5 h), cell cycle (10 h) and intertreatment interval (12 h) (Kauffman, 1968
; Takahashi et al., 1996
). The observed survival of 11% in the main group of ablated mice matches the predicted survival of 10% for surviving GPT neurons generated during the E11–E12 period of the targeted ablation.
Based on this evidence, we conclude that the conditions tested in the present study specifically eliminate up to 90% of the normal complement of GPT neurons. Using the schedule of origin of BrdU-labelled GPT neurons in control mice, these methods can be extended to estimate the dose-dependency of specific cell killing in ancillary ablation groups. For example, three ganciclovir injections at E11–E12 eliminate up to 70%, two ganciclovir injections on E11 eliminate up to 36% and one ganciclovir injection on E11 eliminates up to 18% of the original unablated complement of GPT neurons. Qualitative observations of the magnitude of the defective neocortical phenotype of ablated mice match this ordered sequence.
Non-specific cell killing is insignificant, but structurally distinctive, in ablated mice
HSV-TK/ganciclovir ablations are often associated with non-specific ‘bystander’ killing, an apoptotic amplification by three possible mechanisms: intercellular toxin transfer via gap junctions, intercellular toxin transfer by endocytosis and/or local immunological activation by extracellular toxin. The extent of bystander killing in the predominant unscarred dorsal pallium can be determined with reasonable certainty by key statistical observations of ratios of apoptotic cell densities in ablated mice to GPT neuron densities in control mice during and shortly after ganciclovir exposure.
Excess dying cells in ablated mice accumulate after the rapid saturation of clearance mechanisms (Thomaidou et al., 1997
). High ratios of dying to normal cells would reveal amplification, particularly via gap junctions, with a range of predicted values between 6:1 (oncological studies) and 100:1 (dye-coupling of neuronal progenitors) (LoTurco and Kriegstein, 1991
; Nadarajah et al., 1997
; Mesnil and Yamasaki, 2000
; Bahrey and Moody, 2003
). The observed ratio at E13 is 2:1 (131×103
), a value incongruent with apoptotic amplification, but consistent with the specific killing of either symmetrically divided pairs of GPT neurons derived from a GPT progenitor, or asymmetrically divided pairs of GPT neurons and GPT neuroblasts derived from a GPT progenitor. The characteristic reorganization of the proliferative matrix in the ventricular zone and the proliferative replacement of GPT neurons in ablated mice are in accordance with only the latter condition. Based on this evidence, and comparable results throughout the first phase of apoptosis, we conclude that bystander killing plays an insignificant role in the unscarred neocortex of ablated mice.
The products of non-specific killing deserve close attention for their probative value, as well as for their required inclusion in a comprehensive survey of the impact of targeted ablation. Based on evidence presented by these distinct forms of cellular damage, we conclude that the specificity of the targeted ablation method, while not absolute, is sufficient to permit meaningful developmental analyses. Only one early form of non-specific killing can be recognized in the unscarred dorsal pallium of all ablated mice: dying macrophages, which ingest toxin by endocytosis from specifically killed neurons. A second early form of bystander killing is seen in scars, which are neither necessary nor sufficient to account for the unscarred ablation phenotype. Scars provide an alternative outcome where both GPT and non-GPT cells are clearly subject to ganciclovir-initiated apoptosis. These scars have two key features. First, they are limited in extent, unlike the widespread neuroepithelial collapse produced by early ionizing radiation (Bayer and Altman, 1991
). Secondly, their location is restricted to the dorsomedial peak of the dorsal pallium, a site that divides capillary beds from anterior and middle cerebral arteries. This site is unvascularized in mice until E15, although capillaries penetrate all other dorsal pallial ventricular zones on E11 (Conradi and Sourander, 1980
; Marin-Padilla, 1985
). Here, serial ganciclovir treatments and slow toxin clearance may promote non-specific bystander killing due to inherent vascular defects that resemble those found in neoplastic tumours, the most intensely studied tissue model for bystander killing. Scars encompass killing of all available cell types, which suggests mediation by toxin endocytosis. Neuroepithelial collapse within the scars releases toxins into the ventricular CSF (cerebrospinal fluid). Endocytosis of toxin-contaminated CSF from residual processes at the ventricular lumen or ‘leaky’ non-ciliated tanycytes may then non-specifically kill neurons in circumventricular organs. Hydrocephalus may originate later from the inherent structural weakness and impaired vascular drainage of the scars, a progressive organic effect often found in association with neoplastic forebrain tumours.
Replacement GPT neurons proliferate from GPT neuroblasts in ablated mice
The capacity for near-normal replacement of the complement of GPT neurons in ablated mice is unexpected and may shed light on the difficult problem of control of proliferation during neocortical development (Price and Willshaw, 2000
). The key observation is the expression of HSV-TK in both GPT neuroblasts and neurons, a finding inferred, but not demonstrated, in our previous study (Xie et al., 2002
), and congruent with molecular biological indicators of GPT expression (Landry et al., 1998
). These asymmetrically divided progeny show a very early localization of the reporter-target for ganciclovir action during a shared S-phase within their common GPT progenitors, conditions required for apoptotic induction, but not coincident in ganciclovir-insensitive, G0
-phase (post-mitotic) GPT neurons or non-GPT cells (Moolten, 1986
GPT neuroblasts are located entirely in the ventricular zone of the dorsal pallium for a short developmental period in normal and ablated mice. The occurrence and extent of addition of GPT neurons is determined by preceding levels of proliferation of GPT neuroblasts in these restricted sites. Newborn GPT neurons migrate radially to settle into the mantle and marginal zones, where they can be detected by less efficient τ-eGFP and lacZ reporters. The origin, migration and settlement patterns of GPT cells shown by HSV-TK match the morphological trail of apoptotic cells in ablated mice. Also, birthdays of apoptotic cells and GPT neurons in ablated mice fit these patterns when lacZ expression is corrected to the earlier, more accurate, timing of HSV-TK expression. Finally, replacement of GPT neuroblasts and neurons fails only where the instructive artefact of neuroepithelial collapse of a ventricular zone scar in the dorsal pallium extirpates the proliferative source and radial migratory framework for newly generated cells.
Based on this evidence, we conclude that, like normal GPT neurons, replacement GPT neurons in ablated mice arise entirely by extended de novo
proliferation from GPT neuroblasts. Regulation, modulation and technical sensitivity may contribute to differences in reporter efficiency, but de novo
expression and/or up-regulation of GPT in post-mitotic neurons that normally do not localize GPT are neither apparent nor consistent with the data, unlike trophic determinations of transmitter identity in sympathetic ganglion neurons (Patterson, 1978
Replacement GPT neuroblasts originate from non-GPT progenitors in ablated mice
GPT neuroblasts first arise from non-GPT progenitors, then self-replicate for a limited period by asymmetric division as shown by HSV-TK expression in unablated experimental mice. Self-replication of GPT neuroblasts is largely eliminated by specific cell killing during the period of ganciclovir-induced apoptosis in ablated mice, when neocortical development falters, but does not fail. Simultaneously, dying GPT neurons are accumulated in an additive, continuous fashion. The key observations are the reconstitution and extended replacement of GPT neuroblasts during and shortly after their targeted ablation, which uncover a mechanism of proliferative plasticity for the subsequent replacement of killed GPT neurons.
Based on this evidence, we conclude that, like the initial pool of normal GPT neuroblasts, a substantial component of replacement GPT neuroblasts seen during ganciclovir treatment in ablated mice originate and replenish by proliferation from prior progenitors. These progenitors are invulnerable to ganciclovir-induced ablation due to lack of GPT expression, and neither do they seem to be prone to bystander killing from dying GPT cells within the ablated ventricular zone. This progenitor–neuroblast–neuron lineage sequence identifies the GPT neuroblasts as intermediate progenitor cells. Their programmed death after three to five cycles of asymmetric division would account, at least in part, for the regulated accumulation of viable GPT neurons and the early high levels of apoptosis normally encountered in the ventricular zone of the dorsal pallium. The lineage sequence appears to be obligatory and unidirectional, with no apparent bypass towards cortical plate instead of preplate assembly or reversal from cortical plate to preplate assembly.
Such lineage sequences contribute to the amplification, regulation and diversification of cell division in neocortex (Kriegstein et al., 2004
; Pontious et al., 2008
). The best known example occurs in the late generation of the subventricular zone and supragranular neurons (Noctor et al., 2001
). The new data indicate that a comparable sequence may also be employed in the ventricular zone by early neuronal progenitors. The harnessing of HSV-TK/ganciclovir ablation to GPT expression is a significant technical improvement for studies of intact brain because, unlike previous ablative agents such as ionizing radiation or the anti-mitotic methylazoxymethanol (Bayer and Altman, 1991
; Cattabeni and DiLuca, 1997
), the new attack spares prior progenitors, specifically kills intermediate progenitors of GPT neurons and largely saves the proliferative matrix of the ventricular zone from neuroepithelial collapse.
The complement of GPT neurons is inductively regulated in ablated mice
Ablated and normal mice eventually attain similar peak total complements of viable GPT neurons. A key observation is obtained from ablated mice, which display a proliferative capacity to replenish and replace killed cells by generating nearly twice the total complement of GPT neurons found in normal mice, with no alteration of the cell-cycle duration among GPT neuroblasts. Based on this evidence, we conclude that the total complement of viable GPT neurons in ablated mice is inductively regulated by the fates of their cell lineage predecessors (Edelman, 1988
). This regulation, also embedded in control mice, involves both positive and negative feedback, since substantial shortfalls or excesses of the total population of viable GPT neurons are avoided. The regulation appears to be global for the dorsal pallium, which may reflect numerous local cellular interactions widely distributed throughout its proliferative ventricular zone. Despite previous speculation (Noebels et al., 1991
; Johnson, 1993
; Chenn and McConnell, 1995
; Sestan et al., 1999
), its mechanisms are presently uncharted.
It is important to recall that neocortical growth is abridged in ablated mice, which leads to excessive, transient local accumulations of viable GPT neurons. The scale of this supranormal local replacement of GPT cells is paradoxical with regard to ganciclovir-induced reductions of mitotic activity immediately adjacent to the ventricle in the dorsal pallium of ablated mice. However, ablative harrowing of the proliferative matrix of the ventricular zone evokes its structural reorganization. The periventricular mitotic array converts from a flat, thin configuration into a perforated, thick configuration, which expands the vertical complement of mitotic progenitors, enlarges regenerative capacity and apparently restricts horizontal growth. This structural accommodation could disrupt progenitor fluidity within the proliferative matrix and misdirect radial migration unless locally corrected (Rakic, 1988
; Walsh and Cepko, 1993
). Ganciclovir-induced apoptosis is accompanied by a transient shift in the dominant cleavage plane of mitotic cells in the dorsal pallial ventricular zone, which may contribute to the early replacement of GPT neuroblasts in ablated mice by the symmetric division of their non-GPT progenitors (Sanada and Tsai, 2005
). However, this shift may also reflect a dissociation of cellular cleavage and division planes (Konno et al., 2008
) that subserves a compensatory modification of radial migration. Dying GPT neurons in ablated mice enter an impaired migratory framework within a regenerating neuroepithelial matrix (Xie et al., 2002
). The vertical cleavage plane may derail them from the outward-bound track of radial migration, confine them to the malleable ventricular zone and benefit the laminar assembly of ablated neocortex by sparing the marginal and mantle zones from the clearance of excessive killed cells.
Delayed preplate formation blocks projection axons from GPT neurons in ablated mice
A second unexpected finding is that GPT neurons in ablated mice conserve all of the definitive morphogenetic properties of the principal prelate neurons except one: they emit few pioneer projection axons, which consist mainly of corticothalamic fibres in normal mice (Jacobs et al., 2007
). Sparse residual projections arise from GPT neurons generated before ganciclovir treatment, whereas surviving and replacement GPT neurons retain the capacity to emit local infracortical axons. Preplate formation is delayed in ablated mice due to replacement of killed GPT neurons. By inference, we conclude that this delayed settlement is an unavoidable timing error that desynchronizes the development of neocortex and thalamus, and arrests the outgrowth of axonal projections by GPT neurons. The mechanism underlying projection blockade is uncertain, but this outcome has significant implications for the ‘handshake hypothesis’ often used to explain the midcourse contact and subsequent guidance to cellular targets of developing, reciprocal corticothalamic and thalamocortical projections (Lopez-Bendito and Molnar, 2003
). The primary defect of ablated mice is restricted to the principal preplate neurons, unlike Tbr1
mutants (Stoykova and Gruss, 1994
; Hevner et al., 2002
). The principal preplate neurons in ablated mice do not emit substantial, albeit unstable, projections that approach the pallial-subpallial boundary, unlike Coup-tfi
mutants (Zhou et al., 1999
). Thus ablated mice provide a novel opportunity to dissect apart the elements of the ‘handshake hypothesis’ because preplate neurons, a distal target for thalamocortical axons, are restored at an excessive local density, whereas preplate projections, a proximal target for thalamocortical axons, are never established.
The middle and deep laminae of the principal preplate neurons in control mice are well-situated to guide ingrowth of ‘specific’ thalamocortical axons for termination in neuropil arcades at the superficial and deep borders of infragranular pyramidal neurons in sensory neocortex (Molnar and Blakemore, 1995
; Adams et al., 1997
; Molnar et al., 1998
). Both afferents and arcades are grossly diminished in ablated mice, which contribute to the dyslaminated appearance of neocortex despite the conserved laminar organization of its neuronal cell bodies. It is reasonable to speculate that inadequate thalamocortical connections, as well as their lack of pioneer corticothalamic projection axons, accelerate the diffuse, late-onset apoptosis of GPT neurons (Cowan et al., 1984
), whereas spared infracortical connections are sufficient to support the survival of many non-GPT pyramidal neurons. These surviving neurons do not generate significant seizure activity in ablated mice, perhaps because of their isolation within an impaired neocortex that is, in turn, isolated from the rest of the brain.