The Journal of Neuroscience, July 1995, 75(7): 4838-4850
The Mouse Mutation Reeler Causes Increased Adhesion within a Subpopulation of Early Postmitotic Cortical Neurons
Raymond M. Hoffarth, Janice G. Johnston, Leslie A. Krushel,” and Derek van der Kooy Neurobiology Research Group, Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario, Canada M5S lA8
Early postmitotic cortical neurons are mostly corticofugal man, 1961; Raedlerand Raedler, 1978; Konig and Marty, 1981; projection neurons that take up positions in deep cortical Caviness,1982; Smart and Smart, 1982; van der Kooy and Fish- laminae. Later postmitotic neurons are preferentially local- ell, 1987; Miller and Nowakowski, 1988). As developmentpro- ized to superficial cortical laminae. In reeler mutant mice it ceeds, the later a neuron becomespostmitotic, then the farther appears that cortical laminar positions with respect to it migratesaway from the proliferative zone to take up a more birthdate are reversed (Caviness, 1982). In a reanalysis of superficial cortical position. reeler lamination we found that early postmitotic cortical It remainsunclear which mechanismsenable neurons fated to neurons labeled by embryonic day (E) 11-13 injections of different layers to migrate through the embryonic milieu of a birthdate marker, or by early postnatal day (PND) 2 ret- neighboring cells, processes,and extracellular matrix to settle at rograde labeling through their output projections, appear an appropriatelaminar position. Individual precursorsin the pro- to take up positions both in the superficial and deep cortex. liferative zone often give rise to progeny all fated to residewith- Neurons born on El 1 and El2 are more likely to be situated in one of the deep or superficial laminae (Crandall and Herrup, superficially in the reeler cortex and neurons born on El3 1990; Fishell et al., 1990; Krushel et al., 1993). On the basisof are more likely to be situated in the deep reeler cortex. transplantation experiments McConnell and Kaznowski (1991) Many corticofugal projection neurons in the deep (but not suggestedthat neuronsdecide their respective laminar fates only superficial) reeler cortex either die or retract their axons during their final division. However, theseexperiments also are before PND 21. open to interpretation in terms of differential survival of lineages We hypothesize that the earliest postmitotic (El l-E12) of fated to reside within deep or superficial cortical positions(van the early postmitotic reeler cortical neurons are overly ad- der Kooy, 1992). During prenatal development superficial and hesive and act as a barrier to later postmitotic migrating deep-layer neurons intermingle and contact each other in the neurons. In vitro cortical aggregation cultures confirmed cortical plate (Cavinessand Rakic, 1978). Each classappears to that early postmitotic (E12) reeler neurons are more adhe- act in homotypic fashion, segregatingthemselves according to sive than early postmitotic (E12) wild-type neurons or late birthdate to produce the laminar mammaliancortex. Mammalian postmitotic (E18) reeler or wild-type cortical neurons. We deep and superficial cortical neuronsalso appearto be able to suggest that the moderate wild-type preferential adhesion separatethemselves in vitro on the basisof an adhesionalhier- of early postmitotic cortical neurons to each other helps archy (Krushel and van der Kooy, 1993). deep and superficially fated lineages to form cortical lam- In mice homozygous for the reeler mutation (Falconer, 1951) inae. the normal deep to superficial pattern of neurogenesisin cortex [Key words: cortical neurogenesis, reeler, cell migration, is apparently inverted, without affecting the appearanceor num- adhesion, corticofugal projections, cell death, axon retrac- ber of any of the various classesof neocortical neurons(Sidman, tion] 1968). Neuronal phenotypes correspondingto deep projection Neo-, or six-layered, cortex is a defining feature of mammalian neuronsin wild-type cortex (that become postmitotic early in forebrain architecture not sharedby other vertebrates (Marin- neurogenesis)are at the top of the reeler cortex, while later Padilla, 1978). In mice the entire complementof cortical neurons postmitotic neurons settle toward the bottom (Sidman, 1968; becomespostmitotic during the final 8 d of a 19 d gestation. Goffinet, 1979; Caviness,1982). Newly postmitotic immature neuronsmigrate radially away from We used both retrograde labeling of corticofugal projection the central lumen (ventricle) to form the cortical plate. Cortical neuronsand birthdate markersin reeler (t-h-1) mutant mice to neurogenesisin mammalsoccurs along a deep-superficial gra- analyze the cortical distributions of thoseneurons that would be dient: deep layer VI and V neuronsare generatedfirst, then layer fated to cortical deep-layer positionsin wild-type mice. The re- IV neurons,and finally layer II/III neurons(Angevine and Sid- sults indicate that only the very earliest postmitotic [embryonic day (E) 11 and El21 of the early postmitotic reeler cortical neu- Received Oct. 26, 1994; revised Feb. 13, 1995; accepted Feb. 15, 1995. rons reside in superficial (ectopic) positions. The later postmi- This work was supported by the Medical Research Council of Canada. totic (E 13) subpopulationof the early postmitotic reeler neurons Correspondence should be addressed to Derek van der Kooy at the above ends up in a wild-type-like deep cortical position; both of the address. early postmitotic reeler subpopulations(deep and superficially “Present address: Department of Neurobiology, Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037. located) contain corticofugal neurons.We hypothesized that the Copyright 0 1995 Society for Neuroscience 0270-6474/95/154838-13$05.00/O reeler mutation disrupts the ability of most postmitotic neurons The Journal of Neuroscience, July 1995, 15(7) 4839 to migrate past the earliest postmitotic neurons to more super- ternoon on El1 (n = 3), or in the morning on El2 (n = 3) or El3 (n ficial positions. Using an in vitro neuronal adhesion assay = 3). Females gave birth to litters containing both heterozygous (rl/+) and homozygous (rllrl) reeler pups. Heterozygous and homozygous (Krushel and van der Kooy, 1993), we tested for differences reeler pups were sacrificed on PND 5 and PND 21 by anesthetic over- between reeler and wild-type animals in the adhesive character- dose and quickly perfused with ice-cold 2% paraformaldehyde, and then istics of the early postmitotic neurons. Our findings suggest that the brains-were removed and postfixed for 2hr in 2% paraformaldehyde. the aberrant reeler cortical architecture results, at least in part, Following oostfix. brains were olaced in 70% EtOH (2 X 1 hr) and then left overnight in 70% EtOH: The next day, brains were dehydrated from a hypermorphic adhesion mechanism peculiar to the ear- through a series of baths of increasing percentages of EtOH (75%, 80%, liest postmitotic cortical neuron population, rendering them im- 90%, 95%, 100%) for 1 hr each. Each brain was then placed in EtOW permissive to the migration of later postmitotic cortical neurons. p-dioxaine (50:50) for 2 hr and left overnight in 100% p-dioxaine. The majority of later postmitotic neurons are likely affected sec- Brains were then embedded in paraffin, and ‘I-pm-thick sections were ondarily, only through their inability to migrate past the very cut on a microtome and mounted on gelatin-coated slides. earliest postmitotic population. The reeler mutation reveals that Sections were deparaffined and prepared for BrDU immunocyto- chemistry. The fixed tissue was first treated with 0.1% trypsin in 0.1 M selective neuronal adhesion may be an important mechanism Tris buffer with 0.1% CaCl, at 37°C for 20 min. Slides were then rinsed helping to segregate deep versus superficial cortical neuronal with phosphate-buffered saline PBS (pH 7.4) and denatured with 2N lineages to distinct laminae in wild-type mice. HCl (1 hr), quick rinsed in PBS (pH 6.0), and incubated in mouse anti- BrDU antibody (Becton Dickinson) 1:25 in PBS (pH 7.4) for 1 hr. Methods and Materials Sections were washed (5 min) in PBS (pH 7.4) and further processed Reeler mice. Heterozygous (rZ/+) reeler (the Orleans allele on a using a Vector ABC Labeling Kit (Vector Labs), and then the labeling BALB/C background) females were housed overnight with a homozy- was demonstrated with 5% diaminobenzidine (DAB) in PBS (pH 7.4) gous (rlhl) reeler male. Females were examined at 9:00 A.M. for the plus 0.005% hydrogen peroxide. Some sections were enhanced by add- presence of a vaginal plug and positive dams were then housed sepa- ing cobalt chloride (1%) and nickel ammonium sulfate (1%) in 0.1 M rately, and this day was taken to be EO. Littermates from these matings PBS (pH 7.4) to the DAB solution. Reaction product from the above result in both heterozygous (r-Z/+) and homozygous (r/M) offspring. protocol stained BrDU-positive cell nuclei dark brown (Takahashi et al., Given that reeler mice have a recessive autosomal (chromosome 5) 1992) or, when enhanced, black. Both procedures resulted in compa- mutation, only homozygous (rl/rl) animals display the misaligned cor- rable staining, and the data from both were pooled for analysis. tical (reeler) phenotype, while heterozygous (rl/+) animals have ap- Sections were viewed under a bright-field microscope and camera parently normal cortices and will be referred to here as heterozygous lucida drawings were made using a 20X objective and a drawing tube. controls. The aggregation experiments required all homozygous (rlhl) Labeled neurons were charted, and the drawings of the PND 21 cortex embryos, so homozygous (r/h-Z) females were housed overnight with subsequently were divided into ten equal bins spanning the radial dis- homozygous (r&l) males, while wild-type (+/+) females were housed tance from the pia to the underlying white matter. Counts of BrDU- with wild-type (+/+) males on the same days. The controls for the labeled cortical neurons were made in areas 400 pm in width, and the aggregation experiments were wild-type BALB/C mice also timed-bred height of the bins was the same for each bin within a section but according to the above procedure, and their embryos are referred to changed across sections depending upon the radial distances from white here as wild type. matter to pia. The total numbers of labeled neurons within each of the Retrograde labeling of corticofugal neurons. Deep cortical projection 10 bins were expressed as percentages of the total numbers of labeled neurons were retrogradely labeled (Fishell and van der Kooy, 1987) by neurons within all 10 bins counted within a section. Counts were av- microinjections of 0.2 pl true blue (Sigma) into the cerebral peduncles eraged from two separate and nonadjacent sections chosen from the (to selectively label layer V neurons), and in some animals layer VI same approximate rostrocaudal level through the somatic sensorimotor neurons were labeled retrogradely using injections of 0.2 pl diamidino cortex. In PND 5 cortices similar counts of BrDU-labeled neurons were yellow (Sigma) into the thalamus. Postnatal day (PND) 2 mouse pups made in comparable areas. were placed in a freezer (- 10°C) for cold anesthetization and PND 19 In vitro aggregation. Early postmitotic neurons were differentially pups were anesthetized with sodium pentobarbital by intraperitoneal labeled in the cortices of both reeler (r/h-l) and wild type (+/+) mice. injection (65 mg/kg). Three types of microinjections were performed in Pregnant reeler (rl/rl) mice (n = 2) were given intraperitoneal injec- which separate mouse pups from the same litters were (1) iniected on tions of BrDU (140 mg/kg) dissolved in 0.007% NaOH on the morning PND 2 and sacrificed on PND 5 (n = 17), (2) injected on PND 2 and of E12, at the same time their wild-type El2 counterparts (n = 2) sacrificed on PND 21 (n = 6), or (3) injected on PND 19 and sacrificed received 0.1 mCi injections of 3H-thymidine (thy) (specific activity, 44 on PND 21 (n = 16). On the day of sacrifice pups were anesthetized Ci/mmol). Four days later (E16) laparotomies were performed on in- with sodium pentobarbital and transcardially perfused with ice-cold dividual dams. This allowed time for the early postmitotic neurons to (4°C) oaraformaldehvde (4%). Brains were oostfixed for 1 hr afterward migrate into the cortical plate, as well as allowing the remaining ven- in paraformaldehyde (4%), then overnight-in paraformaldehyde (4%) tricular zone precursors to continue to cycle and dilute out the birthdate and sucrose (30%). Cryostat coronal sections (30 pm) were mounted markers. In a separate set of animals the injection protocol was reversed on gelatin-coated slides and viewed under fluorescence illumination for the groups: a pregnant reeler dam was injected with 3H-thy on E16, with a 360 nm filter. All the true blue retrogradely labeled neurons while an age-matched wild-type (+/+) counterpart was injected with within each of 10 bins dividing up the cortex equally between pia and BrDU. In this group, laparotomies were performed 2 d later on El& white matter were examined in sections through the somatic sensori- allowing for the dilution of the birthdate markers in cells continuing to motor cortex of both heterozygous control (rll+) and homozygous (r-Z/ divide and ensuring that neurons were cultured prepartum to ensure rl) littermates sacrificed on PND 5 and PND 21. The width of the good survival and growth in vitro. Fetal pups were removed under cortical areas was 400 pm and the height of the bins was the same for sterile conditions and decapitated, and their brains removed. Cortices each bin within a section but changed across sections depending upon of the embryos were dissected, minced, and placed in D-MEMIF14 the expanded radial distances from white matter to pia (especially be- media (GIBCO) supplemented with 5% horse serum (GIBCO), 5% fetal tween pups sacrificed at PND 5 and PND 21). The total numbers of bovine serum (GIBCO), 25 mg/ml transferrin, 25 mg/ml insulin, 25 ngl true blue labeled neurons within each of the 10 bins were expressed as ml sodium selenite (Sigma). Each dam supplied six to eight pups from percentages of the total numbers of labeled neurons within all 10 bins which cortical tissue was collected and mechanically dissociated by within a section. Counts were averaged from two separate and nonad- titrating through a series of pipettes with decreasing diameters. Pups jacent sections chosen from the same approximate level rostra1 to the from one reeler (G-1) and one wild-type (+/+) mated dam, collected crossing of the anterior commissure in each mouse. separately, supplied the tissue for use in a single coculture. Cell viability BrDU birthdating. Using injections of bromodeoxyuridine (BrDU) to (as judged by -trypan blue exclusion) was approximately 87% in the label embryonic neurons in their last divisions before becoming post- E12-iniected tissue and 75% in the E16-iniected tissue. Cells were olat- mitotic, we plotted the distribution of early postmitotic (presumptive ed in growth medium at a density of 1 X 1”07cells/60 mm dish (Falc’on), deep layers V and VI) neurons in homozygous (rllrl ) and heterozygous with each of reeler and wild-type cells supplying half (0.5 X 10’ cells/ (rllf) cortices at PND 21 and PND 5. Pregnant dams were injected dish) of the population in a tissue culture dish. Dishes were incubated with BrDU (140 mg/kg) dissolved in 0.007% NaOH, either in the af- at 37” C with 5% CO, on a rotary shaker at 70 rpm for 5 d. Each dish 4840 Hoffarth et al. - Reeler Causes Increased Corhcai Adhesion
Figure 1. Photomicrographs of PND 5 cortical neurons in control heterozygous (flrl) (A) and homozygous (rllrl) (B) reeler littermates retro- gradely labeled by injections of true blue (blue label) into the cerebral pcduncles and diamidino yellow (yellow/green label) into the thalamus on PND 2. Labeled neurons in control heterozygous (+lrl) cortex (A) are confined to bands (true blue, layer V, diamidino yellow, layer VI) within the deep half of the cortex. However, labeled neurons in the reeler (rllrl) (B) are dispersed radially across the cortex with tight clustering of some labeled neurons at the most superficial margin of the cortex. str, striatum; ctx, cortex. Scale bar, 60 pm.
was inspected immediately after plating to ensure that neurons were Results completely dissociated. Cultures were supplemented with fresh growth media after 2 d in vitro. Retrogrudely labeled cortical projection neurons are situated After 5 d the resulting aggregates were rapidly fixed with ice-cold in both superJicia1 and deep reeler cortex 70% EtOH and stored overnight in the refrigerator (4°C) in 70% EtOH. Neurons retrogradely labeled from cerebral peduncleinjections Aggregates were collected the following day and dehydrated through a with true blue and from thalamic injections with diamidino yel- series of alcohol baths. They were then processed with p-dioxane (as low in heterozygous control (rll+) mice were invariably con- described above) and embedded in paraffin. Microtome sections of 5- 7 pm were mounted on gelatin or 2% 3-aminopropyltriethoxysilane fined within the deep half of the cortex to narrvw bandscorre- (Sigma)-coated slides and processed for BrDU as described above. spondingto layer V and layer VI, respectively (Fig. IA). True Slides then were coated with Kodak NTB-2 nuclear track emulsion in blue retrogradely labeled corticofugal neurons(Figs. lA, 2A,C) a darkroom and exposed between 5 and 9 weeks at 4°C. The emulsion were always in a distinct layer V bandregardless of ageat time was developed in Kodak D-19 at WC, rinsed, and fixed with 25% of injection or sacrifice. However, in the reeler (rllrl) cortex sodium thiosulfate. presumptive layer V and layer VI neurons were initially broadly Camera lucida drawings were made of cross sections through the middle of randomly selected, individual, medium-sized aggregates. Ag- distributed (Fig. lB), both superficially and deeply in the cortex. gregates from the two El2 injection cocultures (n = 50 aggregates) Many of the true blue and diamidino yellow retrogradely labeled were analyzed according to the three different cell types observed: (1) neurons were tightly clustered at the superficial aspect of the BrDU (reeler) E12-labeled neurons, (2) Wthy (wild-type) E12-labeled reeler cortex. Interestingly and unlike in wild-type cortex, with neurons, and (3) unlabeled (reeler and wild-type) cells. Since El2 is further development the distribution of true blue-labeled (pre- very early in cortical development the vast majority of unlabeled cells sumptive layer V) neuronsin the reeler changed.Early injection (both reeler and wild-type) must have become postmitotic at later times. Thus, unlabeled cells were the progeny of precursors that continued to (PND 2) and sacrifice (PND 5) true blue-labeled neuronsap- undergo mitosis after injection of either BrDU or ?H-thy and therefore pearedto be prevalent in both the deep and superficialportions diluted out the birthdate markers, or never took up the markers in the of the cortex (Fig. 1B). Later injection (PND 19) and sacrifice first place. In the El6 injection coculture experiment (n = 25 aggre- (PND 21) revealed an inverted distribution (true blue-labeled gates), the three cell types included for analysis were (1) BrDU-labeled neuronswere situated superficially) (Fig. 20) comparedto that (wild-type) E16-labeled neurons, (2) ‘H-thy-labeled (reeler) E16-la- beled neurons, and (3) unlabeled (wild-type and reeler) neurons. Since seenin heterozygouscontrol (rZ/+) cortex, which accordswith the animals were not sacrificed until El8 (allowing 2 d for the dividing conventional descriptionsthat the reeler mutation results in a cells to dilute out the birthdate markers), unlabeled cells in the El6 linear inversion (superficial-to-deep)among cortical phenotypes injection aggregates would likely be a mixture of earlier (
Figure 2. Fluorescence photomicrographs of true blue-labeled neurons in control heterozygous (+/rZ) (A and C) and homozygous (d/d) (B and 0) reeler littermates retrogradely labeled by injections into the cerebral peduncles. Labeled neurons in heterozygous (+/t-Z) mice injected on PND 2 and sacrificed on PND 21 (A), and injected on PND 19 and sacrificed on PND 21 (C), are confined to bands (layer V) within the deep half of the cortex. True blue-labeled cortical neurons in homozygous reeler (rllrl) mice injected on PND 19 and sacrificed on PND 21 (D) were most frequent in the superficial cortex and diminished in numbers toward the deep cortex. B shows a more diffuse distribution of true blue-labeled cortical neurons in a homozygous (rZ/rZ) reeler injected on PND 2 and sacrificed on PND 21 that is similar to the distribution of labeled neurons observed in (rZ/rZ) reeler PND 2 injectionEND 5 sacrifice mice (see Fig. 1B). Arrows in A and B point to striatal patches that are labeled by early but not late (notediffuse striatal labeling in C and D) midbrain injections of true blue. str, striatum; ctx, cortex. Scale bars, 200 pm. 4842 Hoffarth et al. * Reeler Causes Increased Cortical Adhesion
Unlike the single band of .true blue-labeled reeler neuronsin the heterozygous control (rll+) mice, counts of true blue-la- beled neuronsin reeler (rllrl) cortices revealed that the labeled reeler neuronswere distributed bimodally on PND 5. Although in reeler (rllrl) animalstrue blue-labeled (presumptivelayer V) W 19iI21s neuronsappeared across the entire radial cortical axis (Fig. lB), there was a significant effect of the distribution of true blue- 0 2il5s labeled neuronsacross the 10 bins spanningthe radial cortical axis (F9,80= 11.3, p < 0.05). True blue retrogradely labeled neurons were most prevalent in two distinct bands (Fig. 3B). Neuman-Keulstests revealed that the superficialmostbins 1 and 2 individually containedsignificantly (p < 0.05) more true blue- labeledneurons than any individual intermediatebins 3-8. The deepestbins 9 and 10 also each contained significantly (p < 0.05) more true blue-labeled neuronsthan individual interme- diate bins 3-8, but the counts in the two deepestbins were not different (p > 0.05) from those in bins 1 and 2 (Fig. 3B). The distribution of retrogradely labeled neuronsalong the ra- dial axis of the homozygous (rllrl) at PND 5 wasdifferent from that seenafter true blue injections on PND 19 and sacrificedon PND 21. True blue-labeled (presumptivelayer V) neuronswere i0 20 30 40 50 60 70 located primarily in the dorsalhalf of the reeler cortex (bins l- percentage of total True Blue labeled neurons 5) after PND 19 injections, and their numbersdiminished steadi- ly with closerapproach to the white matter (Fig. 20). More than B half of the true blue-labeled (presumptivelayer V) neuronswere .5 located in superficialbins l-3 (Fig. 3B). The bottom-mostthree a 1 bins in the PND 19 true blue injection/PND 21 sacrifice reeler cortices contained only a very small proportion of all the true 2 blue-labeledcells. An ANOVA revealed a significantinteraction Vim = 37.7, p < 0.05) between the distributions of neurons 3 along the radial axis and the early reeler (PND 2 true blue in- 4 jection/PND 5 sacrifice) versus late reeler (PND 19 true blue injection/PND 21 sacrifice) cortices. These results suggestthat 5 layer V neuronsare initially prominent in both deep and super- A / n 19iI21s ficial PND 2 reeler cortex, but by PND 21 relatively few neu- 6 r rons in the deep reeler cortex maintain projections as far as the 0 7 cerebralpeduncles. \ * 2il21s Resultsfrom a single reeler animal injected with true blue on 8 PND 2 and sacrificedon PND 21 revealed a pattern of labeling correspondingto that seenin cortices on PND 5 (Fig. 3B). Reel- er neuronsretrogradely labeledby early true blue injections are still present on PND 21 in a distribution more representativeof .g 10 x their early widespread,than of their late inverted distribution B 0 5 10 15 ,,,, , , , ( 20 25 (Fig. 1B). A secondreeler mouseinjected with true blue on PND 2 and sacrificed on PND 21 had weak labeling that, while not percentage of total True Blue labeled neurons sufficient to quantify, showed the same distribution of retro- Figure 3. Distributionsof true blue-labeledcortical cells in control gradely labeledneurons across both superficial and deepcortex. heterozygousreeler (+lrZ) (A) andhomozygous reeler (d/d) mice(B) The distributionsof true blue-labeled neuronsin the cortices of iniected(PND 2) andsacrificed (PND 5) early (n = 8 +/rl and9 rl/ PND 2 injection/PND 21 sacrifice control heterozygous (+/t-l) rlj, injectedlate’ (PND 19)and sacrificedlate (PND 21) (n = 8 +lrl
pups (n = 4) were indistinguishablefrom thoseof PND 19 true and8 rl/rl). ,, andiniected _I earlv (PND 2) andsacrificed late (PND 21) blue injection/PND 21 sacrifice and PND 21 heterozygouscon- (n = 4 +/rZ and1 d/d). Measurementsare expressed as the percentages ( + SEM) of the total labeledpopulation in eachof 10 binsrepresented trol (d/f) animals (compareFig. 2A with Figs. lA, 2C). on the ordinateas extendingfrom the pial surface(bin 1) to white Injections of true blue into the cerebral pedunclesoften re- matter(bin 10). sulted in retrogradelabeling of striatal neurons,presumably by uptake through their projectionsto the substantianigra that the cerebralpeduncle true blue injections sometimesinfringed upon. using an early postnatal ( superficial the early agesat time of BrDU injection (Ell, E12, or E13), labeledneurons in control heterozygous(rl/+) cortices were re- u deep stricted to the deepcortex (Figs. 5A, 6A-C). Later-labeled(E13) cells occupiedmore of the superficialbins within the (rllf) deep cortex than did the earlier-labeledcells. The numbersof BrDU- labeledneurons were relatively consistentacross all three label- ing times (El 1, E12, and E13), and the numberslabeled at one of theseages were never more than three times greater than at any of the other ages.The results indicate that our injections aimed to label deep-fated neurons in the control heterozygous (rllf) mice were different enough acrossinjection age groups to mark positional variabilities within deep cortex associated with birthdate date. No attempt was made to analyze the distri- butions according to control heterozygous (rllf) cortical layers P2i I P5s P19i / P21s P2i I P21s1 (II-VI), and insteada radial division of the cortex into equal- reeler True Blue labeled neurons sized bins allowed direct spatial comparisonsbetween homo- Figure 4. Counts of true blue-labeled neurons in the superficial and zygous reeler (rllrl) and control heterozygous (Al+) cortices. deep halves of 400-pm-width areas of the cortex of homozygous (rll In accordancewith the known wild-type deep-superficialneu- rl) reeler mice injected on PND 2 and sacrificed on PND 5 (n = 9), rogenic gradient (Angevine andSidman, 1961; Smart and Smart, injected on PND 19 and sacrificed on PND 21 (n = S), and injected 1982; van der Kooy and Fishell, 1987; Miller and Nowakowski, on PND 2 and sacrificed on PND 21 (n = 1). Counts were corrected (rl/+) for section thickness using the Abercrombie (1946) correction factor. 1988), Ell-labeled neuronsin the control heterozygous Data represent means (? SEM). cortices were located entirely within bins 5-10, with more than 40% in bin 10 alone. Heterozygouscontrol (AS) E12- and E13- labeled neuronswere also present primarily in deep bins, with mutation does not affect striatal compartmentation.Despite the E13-labeledneurons the most superficially representedpopula- similar adhesivemechanisms operating in deep cortical neurons tion within the deep bins. and striatal patch neurons(Krushel and van der Kooy, 1993), In homozygous (rllrl) cortex El l- and ElZlabeled neurons theseresults in reeler suggestthat additional(perhaps redundant) made up significantly larger proportions of the superficialmost mechanismshelp to compartmentalizethe striatum. cortical populations than did the E13-labeled neurons. In ho- Sample counts of true blue-labeled neuronsrevealed a sig- mozygous (rllrl) pups, regardlessof age at the time of BrDU nificant (F,,,, = 100.5, p < 0.05) lossof approximately 40% of injections, some labeled early postmitotic (presumptive deep- the labeledcells in both heterozygouscontrol (rl/+) and reeler layer) neuronswere prevalent acrossthe entire radial expanse (rllrl) cortices in the PND 19 injection/PND 21 sacrifice mice of the cortex (Figs. 5B, 6A-C), reminiscent of early (PND 2 comparedto the number of labeledcells in the PND 2 injection/ injection/PND 5 sacrifice) true blue labeling. Both of the two PND 5 sacrifice mice. In the control heterozygous (rl/+) cortex earliestBrDU injections (El 1 and E12) resultedin distributions this loss in labeling presumablyresults from cell death and/or of labeled cells primarily in two prominent bands in the reeler axon retraction occurring within the band of layer V neuronsin cortex (Figs. 5B, 6A,B). One band was located superficially to- the deep cortex. In reeler (rllrl) mice there was a comparable ward the pia, and one band deep near the white matter, with lossin numbersto that in the control heterozygous (rllf) mice more of the El l- and E12-labeled reeler neuronsin the super- except that the losscould be accountedfor primarily by the loss ficial band. The areabetween these two bands(the intermediate of labeled neuronsin the deephalf of the cortex (Fig. 4). True bins) had a relatively smaller number of labeledneurons. Reeler blue-labeled corticofugal reeler neurons were prevalent neuronslabeled as postmitotic on El3 were distributedmore like throughout the superficial and deep halves of the cortex at PND their control heterozygous (rl/+) cohorts (with the majority sit- 5, but PND 19 injections were no longer able to label neurons uated deep in the reeler cortex) than neuronslabeled on either in the deep cortical half. However, preliminary results from a El 1 or E12, implying that the reeler deficit differentially affects single animal reveal that somecorticofugal neuronslabeled early early versus later postmitotic cortical neurons within the early (PND 2) were still present on PND 21 (Fig. 2B). Basedon the postmitotic population. Thus, only a subpopulationof presump- numbers of retrogradely labeled corticofugal neurons in this tive deep-layerneurons are affected by the reeler mutation, and reeler (rllrl) mouse,we suggestthat perhaps40% of the lossof the majority of thoseaffected neuronsbecome postmitotic at the deep reeler neuronsis due to axon retraction and 60% of the earliest times in cortical neurogenesis. loss is attributable to cell death. Indeed, these deep projection A comparisonof genotype with the distribution of labeled neuronsthat regressin reeler can be said to be in an ectopic neuronsacross bins at PND 2 1 and with ageat the time of BrDU position based on thalamic inputs (Caviness and Frost, 1983; injection using an ANOVA revealed a significant three-way in- Molnar and Blakemore, 1992) in the inverted architectureof the teraction (F,, 250= 64.8, p < 0.05). The deepestbin (10) clearly reeler (rllrl) cortex. reveals the difference between the distributions of early- and later-labeled early postmitotic neurons in homozygous (rllrl) The earliest-born reeler cortical neurons are located when comparedto control heterozygous(r-Z/+) cortices. Counts superficially of the labeledneurons in bin 10 revealed a significantinteraction The difference in the distributions of early-born BrDU-labeled (Fz.25= 161.4,p < 0.05) betweenage at time of BrDU injection neurons between reeler (rllrl) and control heterozygous (rllf) and genotype. In control heterozygous(rllf) cortex about 40% cortices at PND 21 mimicked the dispersedpattern observed in of El l-labeled neuronswere located in bin 10. These earliest the early (PND 5 sacrifice) true blue labeling. Independentof postmitotic cortical plate neurons correspondin both time of 4844 Hoffarth et al. l Reeler Causes Increased Cortical Adhesion Figure 5. Photomicrographs of El 1 BrDU-labeled cortical neurons from control heterozygous (+lrZ) (A) and homozygous reeler (d/d) (B) littermates. Labeled neurons in heterozygous (flrl) mice (A) are primarily restricted to the deepest portion of the cortex. A slightly higher magnification of the cortex of a El1 homozygous reeler (G-Z) (B) reveals the presence of labeled cells in both the superficial and deep cortex. Arrowheads point to top of corpus callosum in both A and B. Arrows in A point to BrDU-labeled presumptive subplate cells in the control heterozygous (rZ/+) cortex. str, striatum; ctx, cortex. Scale bars, 100 pm. origin and position within the mature cortex with deep layer VI beled neuronscorrespond in fate to deep layer VI and subplate and subplateneurons (Raedler and Raedler, 1978; Konig and neurons (as judged by their correspondingposition in control Marty, 1981; Bayer and Altman, 1990; McConnell et al., 1994). heterozygous (rZ/+) littermates), then we suggestthat theseear- Newman-Keuls tests revealed that a significantly @ < 0.05) liest of the cortical plate and subplateneurons are preferentially larger proportion of heterozygous control (rZ/+) El l- and E12- affected by the reeler mutation. Later (E13) postmitotic reeler labeledneurons were at the bottom of the cortex in bin 10 com- neurons(with a preferential wild-type fate to residein more of pared to Ell- and E12-labeled neuronsin homozygous (t-Z/d) the superficial positions within the deep cortex; Fig. 3C) were cortex, but there was no significant (p > 0.05) difference in the preferentially located in deep cortical positions [relatively nor- proportion of the E13-labeledneurons in bin 10 comparing het- mal in relation to control heterozygous (d/S) littermates]. How- erozygous control (rl/+) and homozygous (rllrl) cortices (Fig. ever, someE13-labeled reeler neuronswere still positionedmore 6A-C). Together with the long-term (PND 2 injection/PND 21 superficially, scattered in the cortex subjacent to the earliest sacrifice)true blue labeling of presumptivedeep-layer projection postmitotic neurons. It is important to note that birthdate is a neurons,these results indicate that during reeler cortical devel- good but not an absolutepredictor of which neuronswill end opmenta significant number of neuronsthat becomepostmitotic up superficially in the homozygous (d/d) cortex (a few El 1 and on Ell, E12, and El3 survive in the deep cortex until at least El2 neuronsend up deep in the reeler cortex), just as birthdate PND 21. is not an absolutepredictor of laminar position in the control A comparisonin reeler cortex of El l-, E12-, and E13-labeled populations in the superficialmostbin 1 revealed a significant heterozygous (rZ/+) cortex. Perhapsthe reeler mutation works through a processthat correlateswith, but is not the sameas, (Fzz = 47.8, p < 0.05) effect of birthdate. The percentagesof El l-labeled (36%) and ElZlabeled (25%) neuronsin bin 1 were becomingpostmitotic. significantly @ < 0.05) larger than the percentageof the E13- Sample counts of BrDU-labeled cortical neurons in control labeled (10%) neuronsin bin 1. There were no significant dif- heterozygous (rl/+) and reeler mice sacrificed on PND 5 and ferences(p > 0.05) in reeler with respectto the populationsof PND 21 indicate a significant (F,,,, = 14.2, p < 0.05) loss of El 1, E12, or El3 BrDU-labeled cells within intermediatebins labeled cells. Both control heterozygous (rll+) (n = 24) and 3-6 (Fig. 6A-C). Given that the majority of Ell- and E12-la- homozygous(rllrl) (n = 33) mice showedan equivalent decline The Journal of Neuroscience, July 1995, 75(7) 4845 0 5 10 15 20 25 30 35 40 percentage of total BrDU labeled cortical neurons Figure 7. High-power photomicrograph through the center of a cor- tical aggregate containing wild-type and reeler neurons labeled on El2 with 3H-thymidine and BrDU, respectively, and cocultured on El6 for I 5 d. The focus is on silver grains, and thus the BrDU and unlabeled ,““,““,““,““,““,‘.~~,‘~‘.,..I cells appear out of focus. Arrows point to 3H-thymidine-labeled neu- 0 5 10 15 20 25 30 35 40 rons. Arrowheads point to BrDU-labeled neurons. Scale bar, 20 pm. percentage of total BrDU labeled cortical neurons (-25%) in labeledcells, presumablydue to cell death that takes C place between PND 5 and PND 21. In vitro aggregation of reeler and wild-type neurons BrDU-labeled cells were readily distinguished by their dark brown (or black when intensified) nuclear staining, and only El3 cells heavily labeled with silver grains (minimum five times more silver grains than the background number over unlabeled cells) were regardedas ‘H-thymidine labeled (Fig. 7). Labeled cells in both El2 injection and El6 injection aggregatesare like- ly to consist entirely of neuronsand not glia, becausein rodents glial cells becomepostmitotic only later in embryogenesisand postnatally (Das, 1979; van der Kooy and Fishell, 1987). Thus, glial precursorscontinue to divide and dilute out birthdate mark- ers prior to sacrifice. There did not appearto be any difference in the sizes of the El2 injection or El6 injection aggregates. Aggregate diameter rangedfrom 250 Frn to > 1000 km. Aggre- percentage of total BrDU labeled cortical neurons gatesof between 400 and 600 pm were examined in this study. The aggregatesrevealed equivalent labeling indexes (labeled Figure 6. . Distributions of BrDU-labeled cells in control heterozygous cells as a percentageof total cells) for the two different birthdate (d/f) and homozygous (rllrl) reeler PND 21 dorsolateral cortex ex- pressed as percentages ( + SEM) of the total labeled populations across t 10 bins represented on the ordinate as extending from the pial surface (bin 1) to the white matter (bin IO). Data represent means (k SEM) labeled cortices; B, El2 (+lrl) (n = 4) and (d/d) (n = 7) labeled of BrDU-labeled cells in, for A, El 1 (+lrl) (n = 4) and (r&-Z) (n = 6) cortices; and C, El3 (+lrl) (n = 4) and (rllrl) (n = 6) labeledcortices. 4848 Hoffarth et al. * Reeler Causes Increased Cortical Adhesion markers across both El2 injection and El6 injection cultures. The BrDU labeling indexes were 5.9% in the El2 injection cul- A tures, and 6% in the El6 injection cultures, while the 3H-thy labeling indexes were only slightly lower at 4.7% and 5.1%, respectively. These close correspondences between the labeling indexes across conditions suggest that there were no differences between reeler and wild-type cortical neuronsin terms of their ability to incorporate either of the birthdate markers. Crosssections of the aggregatesrevealed that labeled neuron- al populationsfrom eachgenotype (i.e., reeler or wild-type) dif- fered with regard to their radial positions within an aggregate. To measurethe distributions of labeled neurons,camera lucida drawings of aggregateswere divided into three nonoverlapping concentric circles. Measured radially from the center of each aggregateto the periphery, the center ring covered O-30%, the middle 31-70%, and the outside71-100% of the distance.The ratio of reeler and wild-type labeledneurons to the total number of cells in each aggregatewas plotted for all of the aggregates (Fig. 8A,B). An ANOVA on the positional densitiesof the E12- labeledpopulations revealed a significant main effect (F2,294= 41.O, p < 0.05) of position demonstratingthe preferenceof both reeler and wild-type neurons for more central positions (Fig. SA). As might be expected if the E12-labeled reeler neurons were more highly adhesivethan the E12-labeledwild-type neu- rons (Steinberg, 1970; Seeds,1983), there was a significant in- a teraction (Fz,294= 10.8,p < 0.05) of position with reeler labeled 25 o.os- versus wild-type labeled neurons,showing the increasedpref- erenceof reeler (rllrl) cells for more central positions(Fig. 8A). ; 0.06- Y Newman-Keulstests revealed that E12- reeler labeled neurons a made up a significantly higher @ < 0.05) ratio of neuronsin $ 0.04- the centers of the aggregatesthan E12-labeledwild-type cells. 0 Consistent with the present findings, De Long and Sidman fj 0.02- (1970) found more neuronsin the centersof reeler cortical ag- gregatesthan in the centersof wild-type cortical aggregates. 04- - 1 Comparingthe positional densitiesof labeledcells within the El6 injection aggregatesusing an ANOVA revealed no signif- icant interaction (F,,,,, = 1.0, p > 0.05) between position and Figure 8. Ratio of the numbersof reeler(rllrl) andwild-type (+/+) genotype (Fig. 8B). Nevertheless,there was a significant main labeledneurons to the total numbersof cells within threeconcentric effect (Fz,f44= 21.5, p < 0.05) of position, demonstratingthat circularareas encompassing O-30%, 31-70%, and 71-100% of the ra- the proportions of both reeler and wild-type labeledcell popu- dial distancesfrom the centerto the peripheryof the aggregates.Cor- lationsin the El6 injection aggregateswere lower in the centers tical neuronsfrom wild-type and reeler mice labeledon El2 andco- culturedon El6 (A) arelocalized toward the centerof aggregates.La- than in the middle and outsideareas of the aggregates.Less than beledreeler (d/d) neuronswere more prevalentin the centersthan 20% of all labeled cells were within the central area acrossthe labeledwild-type (+/+) neurons.Cortical neurons from wild-type and El6 injection aggregates(compared to 50% acrossEl2 injection reeler micelabeled on El6 andcocultured on El8 (B) arelocated more aggregates),suggesting that El6 neuronsof either genotype are towardthe peripheriesof the aggregates.Data represent means (? SEM) lessadhesive than El2 neuronsof either genotype (Fig. 8B). from 2281labeled neurons for the El2 reeler + El2 wild-typecultures, and 1114labeled neurons in the El6 reeler + El6 wild-type culture. The averagediameter of a cell within an El2 aggregatewas about 7 pm. Nearly 55% of E12-labeled reeler neuronswere within 7 pm (one cell diameter) of another labeled reeler neu- another reeler El6 injection-labeled neuron (comparedto 55% ron, and this percentagedeclined as the distance(bins) between in El2 injection aggregates),and only 13% of wild-type labeled like-labeledcells increased(Fig. 9A). Only about 13% of all the neuronswere within 7 Frn of other El6 injection-labeled neu- E12-labeledreeler neuronswere more than 28 p.m from another rons (comparedto 3540% in El2 injection aggregates).More labeled reeler neuron. E12-labeledwild-type neuronswere not than of 60% of the El6 injection-labeled neuronswere more quite as closely associatedwith other labeled neurons(wt-wt, than 21 p,rn from other labeledneurons (compared to <40% in wt-rl, rl-wt), making up smaller percentages(35-40%) of the the highest El2 injection case)(Figs. 9A,B). nearest-neighbor-measurebin (7 pm). This suggeststhat wild- A nearest-neighboranalysis (Clark and Evans, 1954) was type El2 neuronsare lessable to form homotypic associations used to statistically describethe relative distancesbetween la- than reeler El2 neurons. Compared to the distancesbetween beled neuronsof the sameand different genotypes.The shortest labeledneurons in the E12injection aggregates,in El6 injection distanceto another labeled neuron was scoredfor each labeled aggregatesneither reeler nor wild-type labeled neurons were neuron in an aggregate.Measures were madeof homotypic (rl- associatedwith other labeled neurons(Fig. 9B). Less than 8% rl and wt-wt) and heterotypic (rl-wt and wt-rl) distances.Within of reeler El6 injection-labeled neuronswere within 7 p,rn of the E12rl + E12wt aggregates,reeler neuronswere found to be The Journal of Neuroscience, July 1995, 75(7) 4847 A El2i n d-d q wt-wt rl-wt 0 wt-rl i 28 35 42+ distance from each labeled cell to nearest labeled neighbor (p) Figure 10. Low-power photomicrograph of a El6 wild-type + El6 reeler cortical aggregate in which the wild-type (+I+) cells were la- beled with BrDU (durkly labeled cells). At this magnification only the BrDU-labeled wild-type (+/+) [but ‘not the 3H-thymidine-labeled (rll rl)] cells can be visualized to be distributed relatively evenly across the entire aggregate. Scale bar, 100 pm. 7 14 21 28 35 42+ distance from each labeled cell to nearest labeled neighbor Q.L) is interestingto note that while E12-labeledreeler neuronswere Figure 9. A, Distances between each E12-labeled wild-type (+/+) observedto be clumped (h-l) more than ElZlabeled wild-type neuron and its nearest labeled wild-typeand reeler neighbors and be- neurons(wt-wt), the two different genotypic populations(rl-rl tween each ElZ-labeled reeler (rllrl) neuron and its nearest labeled reeler and wild-type neighbors were measured in 50 El2 wild-type + and wt-wt) in the El6 injection aggregatesdid not differ from El2 reeler aggregates. Measurement distances were grouped into equi- each other (Fig. 9B). This suggeststhat the E16-labeled reeler distant bins and expressed as percentages (-+ SEM) of each of the dif- and wild-type neuronsdo not differ in their adhesion.The lack ferent pairs of nearest-neighbor measurements. A total of 1201 wild- of adhesivedifferences between these two genotypesin the El6 type and 1080 reeler neurons were analyzed. B, Distances between each injection aggregateswas also evident in the equivalent likeli- E16-labeled wild-type neuron and its nearest E16-labeled wild-type and reeler neighbor and between each labeled reeler neuron and its nearest hood of each to show heterotypic clumping (i.e., wt-rl and rl- labeled reeler and wild-type neighbor were measured in 25 El6 wild- wt). type + El6 reeler aggregates. A total of 605 wild-type and 509 reeler neurons were analyzed. Discussion Although reeler neocortical neuronsare misorganizedspatially, there appearto be the normal types and numbersthat are present clumped together more often than wild-type neurons. The near- in the wild-type six-layered cortex (Goffinet, 1984).Despite the est-neighbor measurements revealed that reeler E12-labeled cortical misorganization, functional anatomical circuits arise neurons showed significant 0, < 0.05) homotypic (rl-rl) clump- such as those formed by whisker barrel afferents (Cavinessand ing in 74% of the aggregates. In contrast, significant homotypic Frost, 1983). Physiological features of reeler neurons,such as (wt-wt) clumping of ElZlabeled wild-type neurons occurred in those in the visual cortex, retain properties appropriateto their only 37% of the aggregates. Significant heterotypic clumping counterpartsin wild-type animals(Drager, 1976, 1981). The po- (rl-wt, wt-rl) in the sameaggregates was seenin 30% and 26% sitions of cortical neurons along the superficial-deep (radial) of their respective cases.In El6 injection aggregates,statistical axis are apparently inverted with respectto birthdate when com- analysis of nearest-neighbor measurements revealed that signif- pared to wild-type or heterozygous reeler mice (Sidman, 1968; icant 0, < 0.05) clumping of homotypic or heterotypic labeled Caviness and Sidman, 1973; Goffinet, 1979). This observation neurons never occurred in more than 24% of the aggregates. It led to the hypothesisthat reeler homozygouscortex resultsfrom 4848 Hoffarth et al. * Reeler Causes Increased Cortical Adhesion the inability of later postmitotic neurons to migrate past earlier In early postmitotic (E12) reeler and wild-type aggregatesla- postmitotic neurons into more superficial positions. The hypoth- beled neuronswere more clumped than labeledneurons in El6 esis, as yet untested, holds that neurons do not exit the radial aggregatesof wild-type and reeler neurons,but the early post- glial highway, resulting in a “log-jam” distribution, whereby mitotic (E12) wild-type neuronsappear to lack the hyperadhe- later-born neurons pile up and occupy deeper and deeper posi- sive quality of El2 reeler neurons.In E12-labeled,but not E16- tions (Pinto-Lord et al., 1982; see Goffinet, 1984). labeled, aggregatesreeler (rllrl) labeled neurons established The present evidence is not compatible with the view that the more homotypic associations,and were consistentlycloser to the reeler mutation affects the migration of all neuronsin the cortex, aggregate centers, than their wild-type cohorts. Late (E16-la- either directly or through their interaction with radial glial cells. beled) postmitotic reeler (rllrl) neuronsdo not appearto act any The log-jam hypothesis predicts that the distribution of reeler differently than their wild-type counterparts; neither show evi- neuronsalong the superficial-deepaxis shouldbe correlated in dence of selective adhesion.The retrograde tracing of presump- an outside-in fashion with early-to-late postmitotic dates. This tive layer V and VI projection neuronsdemonstrated tight clus- hypothesispredicts that the distribution in reeler mice of pre- tering of the retrogradely labeled neuronsselectively in the su- sumptive layer V and VI neuronsretrogradely labeled from in- perficial reeler cortex, which may reflect the high adhesionpe- jections into the cerebralpeduncles and thalamus(respectively) culiar to the earliest-bornreeler neurons. should always be highest in the superficial cortex. with few in Many neuronsmay be fated to deepor superficialfates prior the deep half of the cortex toward the white matter. The distri- to exiting the proliferative cycle in the ventricular zone (Crandall butions of retrogradely labeled neuronsin reeler cortex clearly and Herrup, 1990; Fishell et al., 1990; McConnell and Kaz- do not support this prediction. In reeler animals sacrificed on nowski, 1991; van der Kooy, 1992; Krushel et al., 1993). In PND 5 the distribution of cerebral peduncle neurons was not mammalianforebrain developmentlayer I neuronsare the first linear but rather bimodal with large collections of labeled neu- postmitotic neurons of the cortical plate (Bayer and Altman, rons appearingtoward both the deep and superficial marginsof 1990) and they remain (primarily as Cajal-Retzius cells) in the the cortex. This suggeststhat rather than an inversion, the reeler maturecortex as the most superficialneurons (Konig and Marty, mutation causesa split within the deep-layer population, result- 1981; Luskin and Shatz, 1985). Following the birth of layer I ing in a portion of the deep-fated neuronsbeing pushedto the cells the earliest postmitotic cortical neuronsare fated to differ- top of the cortex, and a portion remainingdeep. Further evidence entiate into subplate and deep-layer cortical plate neurons againsta primary deficit in radial glial cells in reeler mice comes (McConnell et al., 1994). Indeed, the earliest BrDU-labeled from the presentaggregation studies where differencesin reeler postmitotic neuronsin control heterozygous (rl/+) corticeswere versuswild-type neuronalpositioning can be detectedin the ab- in the deepestportions of the cortex; however, in the homozy- senceof any radially positioned glial cells in such cortical ag- gous reeler (rllrl) the earliest-labeledpostmitotic neurons ap- gregates(Garber et al., 1980). pearedprimarily in the superficialcortex. This suggeststhat sub- We hypothesize that the split of early postmitotic reeler neu- plate and deep-layercortical plate neuronsmay sharethe same rons might be due to abnormally high adhesionamong a sub- adhesivecomponent, and therefore the samepositional fate. If population of the earliest postmitotic neuronsthat then become hyperadhesionensures that the relatively small number of layer impermissiveto the migration of later postmitotic neurons.By I neuronsmaintain the superficialmostposition in the developing primarily affecting the earliest postmitotic deep-layer cortical cortex, then the reeler mutation might convert the fates of sub- neurons, reeler results in a phenotypic cortical split into a su- plate and early postmitotic deep-layer cortical plate neuronsto perficial subpopulation(the earliest-bornof the early postmitotic a layer I neuronaladhesive fate. The earliestcortical neuronsof neurons)and a deep subpopulation(the later-born of the early the reeler preplate are misoriented (Crandall and Caviness, postmitotic neurons). Later postmitotic subpopulationsof the 1991), which may be a result of hypermorphic adhesionamong early postmitotic neurons (as well as late postmitotic popula- this population making them impermissibleto the passageof the tions) are relatively unaffected by the reeler mutation. Thus, later postmitotic cortical plate neurons. theselater postmitotic subpopulationsmay be able to sort them- N-cadherinmRNA is selectively expressedin deep-layerneu- selvesout in a relatively wild-type, deep-superficialpattern, be- rons, and specifically during the perinatal period when deepand low the hypermorphically adhesiveearliest postmitotic neurons superficial neuronsare segregating(Redies and Takeichi, 1993). that are located together at the superficial boundary of the cor- We suggestthat the reeler mutation may affect an intrinsic ad- tex. Consistentwith the proposed adhesive changesin reeler hesionprogram in cortical neuronsby hypermorphic regulation cortex, Shur (1982) noted a decreasein galactosyltransferase of an adhesivefactor that is peculiar to deep-layer neuronssuch activity in reeler cortex; however, this change was measured as N-cadherin. The adhesionmechanism of the earliest postmi- postnatally and may be a secondarysequela or the early embry- totic neuronsmight be expressedto a greater degreeor earlier onic changesthat we suggestare critical for the misorganization than normal. In wild-type developmentdeep-layer adhesion may of reeler cortical laminae. turn on during or after migration to clump laminae, or before The presentBrDU birthdating data supportthe hypothesisthat migration of later postmitotic neuronsto keep the earliestpost- during reeler cortical developmentthe earliestpostmitotic (El 1 mitotic neuronsin place while later postmitotic populationsmi- and E12) populationsof deep-layerneurons tend to be in ectopic grate past. Reeler may act in the N-cadherinregulatory pathway (superficial) positions, Later postmitotic (E13) labeled reeler causingeither an over- or early expression,resulting in the ec- neurons(still presumptivedeep-layer neurons)had lessectopic topic positioning of the earliest postmitotic deep-layer neurons. distributions [compared to control heterozygous (rl/+) litter- Nevertheless the reeler mutation (mapped to chromosome5; mates], which may indicate that they are less affected by the Goffinet, 1992) is not a hypermorphic mutation of the N-ca- reeler mutation. Early postmitotic deep-layer cortical neurons dherin gene itself (mappedto chromosome18; Miyatani et al., have been shown to possessan adhesive affinity that is absent 1992). The cerebellumof reeler mutantsis characterized by the in superficialcortical neurons(Krushel and van der Kooy, 1993). malpositioning and loss of Purkinje neurons and a substantial The Journal of Neuroscience, July 1995, 75(7) 4849 reduction in the granule cell population (Goffinet, 1984; Heck- Caviness VS. Sidman RL (1973) Time of origin of corresponding cell classes in the cerebral cortex of normal and reeler mutant mice: an roth et al., 1989; Goffinet, 1992). The arrested migration of reel- autoradiographic analysis. J Comp Neurol 148: 141-152. er Purkinje cells also could be explained by a hypermorphic Clark PJ, Evans FC (1954) Distance to nearest neighbor as a measure homotypic cell adhesion mechanism among Purkinje cells, lim- of spatial relationships in populations. Ecology 35:445453. iting their migration. 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