Cerebral Cortex Advance Access published September 2, 2009
Cerebral Cortex
doi:10.1093/cercor/bhp168
The Subventricular Zone Is the
Developmental Milestone of a 6-Layered
Neocortex: Comparisons in Metatherian
and Eutherian Mammals
Amanda F. P. Cheung1, Shinichi Kondo1, Omar Abdel-Mannan1,
Rebecca A. Chodroff1, Tamara M. Sirey1, Lisa E. Bluy1,
Natalie Webber1, Jamin DeProto1, Sarah J. Karlen2,
Leah Krubitzer2, Helen B. Stolp1,3, Norman R. Saunders3 and
Zoltán Molnár1
1
Department of Physiology, Anatomy and Genetics, University
of Oxford, Oxford OX1 3QX, UK, 2University of California
Davis, Center for Neuroscience, Davis, CA 95618, USA and
3
Department of Pharmacology, University of Melbourne,
Parkville VIC 3010, Australia
Amanda F. P. Cheung and Shinichi Kondo have contributed
equally to this work.
The major lineages of mammals (Eutheria, Metatheria, and
Monotremata) diverged more than 100 million years ago and have
undergone independent changes in the neocortex. We found that
adult South American gray short-tailed opossum (Monodelphis
domestica) and tammar wallaby (Macropus eugenii) possess
a significantly lower number of cerebral cortical neurons compared
with the mouse (Mus musculus). To determine whether the
difference is reflected in the development of the cortical germinal
zones, the location of progenitor cell divisions was examined in
opossum, tammar wallaby, and rat. The basic pattern of the cell
divisions was conserved, but the emergence of a distinctive band of
dividing cells in the subventricular zone (SVZ) occurred relatively
later in the opossum (postnatal day [P14]) and the tammar wallaby
(P40) than in rodents. The planes of cell divisions in the ventricular
zone (VZ) were similar in all species, with comparable mRNA
expression patterns of Brn2, Cux2, NeuroD6, Tbr2, and Pax6 in
opossum (P12 and P20) and mouse (embryonic day 15 and P0). In
conclusion, the marsupial neurodevelopmental program utilizes an
organized SVZ, as indicated by the presence of intermediate (or
basal) progenitor cell divisions and gene expression patterns,
suggesting that the SVZ emerged prior to the Eutherian--Metatherian split.
Keywords: basal progenitors, cortical neurogenesis, cortical unit column,
evolutionary biology of cerebral cortex, intermediate progenitors,
Monodelphis domestica
Introduction
The hallmark of mammalian brain evolution is the emergence
of a 6-layered neocortex. Although overall lamination and basic
neuronal cell types are largely conserved, mammalian neocortices show dramatic variation of cortical and laminar
thickness, cortical neuron number and density, and cortical
field number and connectivity (Brodmann 1909; Kaas 2006;
Rakic 2008). In one of the most influential and puzzling
observations in comparative studies of the adult mammalian
cerebral cortex, Rockel et al. (1980) argue that the number of
neurons in a 30-lm wide ‘‘unit column’’ in cerebral cortex is
constant across mammals (ca. 110 per radial strip in 25-lm
sections), despite a diversity of cortical thicknesses and relative
proportion of layers . However, this observation does not
include marsupials, and recent studies contend that neuronal
number does differ across cortical areas and between the
species analyzed by Rockel et al. (1980) (Haug 1987; Cheung
et al. 2007; Herculano-Houzel et al. 2008; Rakic 2008).
Ó The Author 2009. Published by Oxford University Press. All rights reserved.
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Mammalian cortical neurons originate from 2 germinal areas.
Projection neurons are born locally along the neuroepithelium
in the dorsal pallium, whereas cortical interneurons are mainly
generated in the medial ganglionic eminence (MGE), lateral
ganglionic eminence (LGE), and caudal ganglionic eminence
(CGE) in the subpallium and travel tangentially to the dorsal
pallium (Parnavelas and Nadarajah 2001; Marı́n and Rubenstein
2003). Tangential migration has been postulated in the
equivalent circuit hypothesis as part of the mammalian--reptilian
transformation (Karten 1969, 1997), but these tangentially
migrating neurons are exclusively inhibitory in mammals and
sauropsids (Cobos et al. 2001; Tuorto et al. 2003; Métin et al.
2007; Moreno et al. 2008). As evidence for tangential migration
of projection neurons from the subpallium to the cortex in
vertebrates is scant, the radial expansion from a 3 to a 6-layered
neocortex and subsequent tangential enlargement of cortical
surface areas are more likely due to increased progenitor
population (Fish et al. 2008) and neural production from the
cortical neuroepithelium.
Although neuroepithelial cells (which later turn into radial
glial cells [RGCs]) in the ventricular zone (VZ) are universal to
all vertebrates, the rate at which they produce progenitors and
the length of time over which neurogenesis occurs can vary
(Rakic 1995; Dehay and Kennedy 2007). These differences can
account for the tremendous variability observed in the size of
the cortical sheet in different species. Furthermore, a distinct
embryonic mammalian progenitor compartment called the
subventricular zone (SVZ) is present, where it is believed the
number of neurons is amplified by increasing the rate and
duration of neurogenesis. Instead of producing one neuron,
RGCs may undergo asymmetric division to produce another
RGC progenitor and one intermediate progenitor cell (IPC, or
basal progenitor cell) that subsequently migrates to the SVZ. In
the SVZ, IPCs undergo 1--3 symmetric divisions to amplify
neuron production (Haubensak et al. 2004; Miyata et al. 2004;
Noctor et al. 2004). The 2-step pattern of neurogenesis could
facilitate the formation of a 6-layered cortex with a larger
surface area (Martı́nez-Cerdeño et al. 2006; Pontious et al.
2007). Conversely, sauropsids have a 3-layered cortex lacking
an organized SVZ (Molnár, Tavare, and Cheung 2006; Cheung
et al. 2007). These studies suggest that a cortical SVZ is an
exclusive hallmark of mammalian neurogenesis. However, this
assumption arises from a limited number of species, and
generalizations could be premature.
Metatheria (marsupials) have evolved independently from
eutheria (often referred incorrectly as ‘‘placental’’ mammals)
for approximately 173--190 million years (Kumar and Hedges
2002; Murphy et al. 2004) and represent an interesting group
for 2 reasons. First, although they possess a 6-layered neocortex, it contains considerably fewer neurons than eutheria
(Haug 1987). Second, morphological studies (Saunders et al.
1989) have hinted that a secondary proliferative zone (the
SVZ) is not seen in the dorsal pallium of the South American
gray short-tailed opossum (Monodelphis domestica), although
such zone has been described in tammar wallaby (Macropus
eugenii) (Reynolds et al. 1985). If an organized SVZ is not
present in the opossum, this suggests that the SVZ is not
universally required for the generation of a 6-layered neocortex
in all mammals. Is it possible that these species have adopted
a different strategy for cortical neurogenesis?
In this study, we compared the number of cortical neurons and
glia in a unit column of adult opossum, wallaby, and mouse. We
examined the pattern of dividing cells and the orientation of mitotic
spindles in the VZ of opossum and wallaby. The expression pattern
of selected genes with known cortical VZ and SVZ expression in
mouse and opossum was also compared. Our data demonstrate that
although there are differences in the number of neurons in
a standardized column unit of cortex (affecting both infragranular
and supragranular layers equally), the general organization of the
SVZ has been conserved across all studied mammals.
Materials and Methods
Animals
All animal experiments were conducted in accordance with the UK
Animals (Scientific Procedures) Act (1986). All protocols for opossum
were approved by the Institutional Animal Care and Use Committee,
the University of Melbourne Animal Ethics Committee, the National
Institutes of Health, and the National Health and Medical Research
Council Australia guidelines.
South American gray short-tailed opossum (M. domestica): Brains
from postnatal days (P) 6, 7, 9, 10, 12, 14, 16, and 20, which covered
early, mid, and late stages of neurogenesis (Table 1), and adult were
obtained from N. Saunders (University of Melbourne) and L. Krubitzer
(University of California--Davis). The mothers were anesthetized with
isoflurane, whereas the pups (n = 3 for each stage) were collected from
their teats. The pups were terminally anesthetized with isoflurane,
decapitated, and fixed in 4% paraformaldehyde (PFA) for 3 days.
Tammar wallaby (M. eugenii): Paraffin sections of Bouin’s fixed brains
from P9, 15, 19, 34, 40, and 50 were obtained from the colony at the
Commonwealth Scientific and Research Organization, Division of
Wildlife and Rangelands Research, Lynham, ACT, Australia, and
described in earlier studies (Dziegielewska et al. 1988). The ages
represent early, mid, and late stages of cortical neurogenesis (Table 1).
Three adult Tammar wallaby was perfused with 4% PFA (see details
below in section on cell quantification in a unit column).
Rat (R. norvegicus): The proliferation patterns in opossum and
tammar wallaby were compared with rat. Our group has previously
described in detail the distribution of phosphohistone H3-immunoreactive cells in rat (Carney et al. 2007). Timed pregnant Wistar rats were
obtained from Harlan Laboratories, UK, and maintained in the Oxford
University Animal Facility. Brains from embryonic days (E) 13, 17, and
19 were fixed by immersion in 4% PFA for 7--10 h, and coronal sections
were cut at 50 lm using a Vibroslicer (Leica VT1000S) for phosphohistone H3 immunohistochemistry.
Mouse (M. musculus): Timed pregnant C57/BL6 mice were obtained
from the Oxford University Animal Facility. Brains from E15 and P0 (n =
3 for each stage) were collected for in situ hybridization.
Cell Quantification in a Unit Column
Three adult mouse, opossum, and wallaby brains were used for
quantification. Animals were perfusion fixed with 4% PFA, and coronal
sections of mouse and opossum brains were cut at 40 lm using
a Vibroslicer (Leica VT1000S). Three interspaced sections (200 lm)
within the primary somatosensory area were stained with cresyl violet.
The mouse and opossum brains and sections were processed in identical
fashion. For wallaby, brains were cut at 60 lm using a cryostat. Cresyl
violet--stained sections within the primary visual area were kindly
provided by L. Marotte (The Australian National University). Section
shrinkage in all specimens, as measured by scanning the z-plane using
confocal microscopy (Zeiss 710), was 75% (40 to 10 lm, 60 to 15 lm; data
not shown). Using Neurolucida 8 (MBF Bioscience, Magdeburg,
Germany), an arbitrary 100-lm wide area (a unit column) was defined
in the primary somatosensory/visual area spanning layers 1--6 (Fig. 1).
Neurons were identified by cytoplasmic and nucleolar staining, whereas
neuroglia stain only the nucleus. Cells touching the bottom (the
boundary between white matter and layer 6) and left boundaries were
counted directly under the microscope, and those touching the top (the
pial surface) and right boundaries were excluded. Neuronal and glial cell
counts from individual layers were obtained by focusing up and down in
the z-plane of the section and expressed as mean ± standard error of the
mean (SEM). As the section of the wallaby cortex was thicker than that of
mouse and opossum, the wallaby data represented in Figure 1D have been
adjusted to two-thirds of the original counts.
Immunohistochemistry and Cell Quantification
Phosphohistone H3 immunohistochemistry was used to reveal the
mitotic division pattern because it is more amenable to quantification
and comparisons. Opossum brains at P10, P14, and P16 (n = 5 for each
stage) were cut coronally at 40 lm using a Vibroslicer, incubated
overnight at 4 °C in rabbit polyclonal antiphosphohistone H3 antibody
(1:500, Millipore, Livingston, UK) prior to incubation in Alexa546conjugated goat antirabbit secondary antibody (1:500, Invitrogen,
Paisley, UK) for 2 h at room temperature. For wallaby, 8-lm paraffin
sections at P15, P40, and P50 (n = 4 for each stage) were used.
Due to the small size of the brains, the total number of H3immunoreactive mitotic figures in the VZ, SVZ, and extraventricular
(EV, which include intermediate zone [IZ], cortical plate [CP], and
marginal zone [MZ]) regions of the entire dorsal cortex of opossums and
tammar wallabies was counted from 3 levels along the rostrocaudal axis of
the cortex (rostral, intermediate, and caudal), averaged, and expressed as
mean ± SEM. Our quantification was performed on the germinal zone of
the entire cortical sector, extending between the cingulate sulcus and the
rhinal fissure. In contrast, data for Wistar rat were quantified by sampling
2 regions in the dorsal cortex of E13, 17, and 19 pups (n = 8, for each
stage, Figs 1B and 2 in Carney et al. 2007). In brief, H3-immunoreactive
mitotic figures in the VZ, SVZ, and EV were counted using an ocular
reticule of 66 000 lm2 under a 340 objective lens in the dorsolateral and
dorsomedial cortex from 3 levels along the rostrocaudal axis.
In a recent study, it has been demonstrated that 2 dense vascular plexi
exist in the VZ and SVZ during cortical development, especially at E14
Table 1
Comparisons of the gestational ages at similar cortical developmental stages for the species used in this study. (Cheung and Kondo et al.)
Gestational period (days)
Mouse
Opossum
Wallaby
19--20
14
27
Page 2 of 11 SVZ in Metatheria and Eutheria
Developmental stage
References
Initial stage (no SVZ)
Early stage (small SVZ)
Later stage (large SVZ)
Final stage (small SVZ)
Before E12
Before P6
Before P2
E13--E15
P7--P12
P5--P15
E16--P0
P14--P24
P20--P50
P1
P64–P100
P67--P213
d
Cheung et al.
Takahashi et al. (1995)
Saunders et al. (1989)
Reynolds et al. (1985)
Figure 1. Quantification of the number of neurons in different species. (A--C) Cresyl violet--stained sections of adult (A) mouse, (B) opossum, and (C) tammar wallaby. An
arbitrary ‘‘unit column’’ (a 100-lm wide, 40-lm thick region [60-lm thick for wallaby] spanning from layer 1 to 6) was marked in the primary somatosensory/visual area (boxed
areas in A--C, higher magnification in A‘--C‘). The number of neurons and glia was quantified in each layer and expressed as mean ± SEM in (D). (E) The mean number of neurons
present in each cortical layer, showing that the number of neuron in a unit column is not constant between different infraclass within mammals. (E‘) The proportion of neurons in
each cortical layer. Scale bar: A--B 5 500 lm, C 5 1 mm.
and 15, when neurogenesis is at its peak (Stubbs et al. 2009). To examine
the neurovascular pattern in opossum, 10-lm thick cryostat sections
from P6, 12, 14, and 16 brains (n = 2) were incubated overnight at 4 °C in
fluorescein isothiocyanate-conjugated Griffonia simplifolica isolectin B4
(1:100, Vector Laboratories Inc, Burlingame, CA).
Mitotic Spindle Orientation
Mayer’s hematoxylin and eosin (H & E) were used to reveal the angle of
mitotic spindle orientation of dividing RGCs. For opossum, a total of 67
cells from 8-lm thick paraffin sections of P7 (n = 2, 8 sections) and P9
animals (n = 2, 5 sections) were measured. For tammar wallaby, a total
of 44 cells from 8-lm thick paraffin sections of P9 (n = 1, 2 sections),
P15 (n = 1, 3 sections), P19 (n = 1, 3 sections), P34 (n = 1, 1 section),
P40 (n = 1, 1 section), and P50 (n = 1, 1 section) were measured. All
sections are interspaced, and the slides were observed under a Leica
DMR upright microscope, and images were captured using a Leica
DFC500 camera and Firecam software. The relative angle between
a line bisecting the 2 chromosome sets and the apical surface (Fig. 3C)
was measured and scored into six 15° bins as described previously
(Konno et al. 2008).
Probe Fragment Isolation and In Situ Hybridization
E15 and P0 mouse brains and P12 and P20 opossum brains were
embedded and flash frozen in Tissue Tek O.C.T. compound. Sections
were cut at 14 lm using a cryostat (Leica Jung CM3000) and stored at
–80 °C. Tissues were then processed for in situ hybridization with
digoxigenin-labeled riboprobes for the following genes: Brn2
(NM_008899), Cux2 (NM_007804), NeuroD6 (NM_009717), Tbr2
(NM_010136), and Pax6 (NM_013627). Primers for amplification of
mouse probe fragments were designed to sequences retrieved from
GenBank. Opossum orthologs of the above genes were identified by
basic alignment search tool of the opossum genome assembly (The
Broad Institute, Cambridge, MA) on the UCSC and Ensembl Genome
browsers. The identified opossum fragments were then aligned with
known mammalian and chicken sequences using Clustal X (Thompson
et al. 1997) and primers designed to regions of conservation outside
gene family motifs using Primer3 v0.4.0 (Supplementary Table 1). To
define the VZ, SVZ, IZ/subplate (SP), CP, and the MZ, mouse and
opossum brain sections stained with cresyl violet were compared.
Results
The Number of Neurons in a Standardized Unit Column
of Adult Marsupials Is Fewer than Mouse
To compare the number of neurons per unit volume in the
cerebral cortex, the number of neurons of adult mouse,
Cerebral Cortex Page 3 of 11
Figure 2. Distribution of dividing cells in the developing opossum cortex. (A, B) At P10, the majority of H3-immunopositive (H3þ) cells were in the VZ, although a few H3þ cells
were in the extraventricular zone (EZ, which includes IZ, CP, and MZ). At this stage, there were no H3þ cells in the cortical SVZ; however, there were numerous SVZ divisions in
the subpallium. Only at P14 onward were H3þ cells located in the SVZ (arrows). (C, C’) Cell division in the SVZ could be not only detected by H3 immunohistochemistry but also
on Nissl-stained sections (arrow). (C’) is a higher magnification of the boxed region in (C). (D, E) The anatomical boundary for VZ, SVZ, and EZ region at P10, 14, and 16 was
defined using Nissl-stained sections (D, right 3 panels), and the number of H3þ cells in each region (D, left 3 panels) was then quantified and expressed as mean ± SEM in (E).
(F) The relative ratio of H3þ cells in different anatomical compartments of opossum. Whereas the total number of cell division decreases as development progresses, cell
divisions in the SVZ become more prominent, a feature very similar to rat and wallaby (Fig. 4E). Asterisks represent a significant difference between opossum and rat (P \ 0.05,
Student’s t-test). Scale bar: A--C 5 200 lm; C‘ 5 30 lm; D 5 20 lm.
opossum, and wallaby was counted (Fig. 1). There was
a significant difference in the number of neurons between
the 3 species (P < 0.05, one-way analysis of variance). On
average, there were 743 ± 9 neurons in mouse; however, there
were fewer neurons in both marsupials. Wallaby has 544 ± 13
neurons, whereas opossum has even fewer (418 ± 29),
although it could be attributed to different cortical areas
selected for quantification. These data indicate that although
marsupials have a very similar neocortical organization, they
Page 4 of 11 SVZ in Metatheria and Eutheria
d
Cheung et al.
have lower neuronal numbers overall and different neuron/glia
ratios than observed in a mouse unit column. It has been
suggested that the population of IPCs might be responsible for
the increased neuronal numbers in mammalian cerebral cortex
(Kriegstein et al. 2006; Molnár, Métin, et al. 2006; Molnár,
Tavare, and Cheung 2006; Pontious et al. 2007; Fish et al. 2008).
Therefore, differences in adult cortical neuronal numbers
suggest that the structure and organization of the germinal
zone differs between marsupials and mouse.
Figure 3. Orientation of mitotic spindle in the developing opossum cortex. (A, B) H & E was used to reveal neuroepithelial cells that were in anaphase (arrows) in order to
measure the angle between the cleavage plane and the ventricular surface. (A‘) is a higher magnification of the boxed region in (A). (B) A dividing cell with typical planar division,
whereas (B‘) shows a dividing cell with an oblique spindle orientation. Dotted lines represent the cleavage plane. (C) Schematic diagram of a mitotic neuroepithelial cell. The
cleavage plane was defined as the orthogonal plane bisecting the line between the center of each chromosome (adapted from Konno et al. 2008). (D) Distribution of the cleavage
plane orientation for mitotic neuroepithelial cells measured at P7 and P9 and averaged. Similar to mouse, most planar divisions were within 15° of the apicobasal axis. Scale bar:
A 5 200 lm; A‘ 5 50 lm; B 5 10 lm.
Dividing Cells Appear in SVZ at a Later Stage of Cortical
Development in Opossum
In P10 opossum brains, the majority of phosphohistone H3-immunoreactive mitotic (H3+) cells were in the VZ. A few H3+
scattered cells located in the CP and IZ, but no H3+ cells were
found in the SVZ (Fig. 2A). Starting at P14 and by P16, much
later than expected, a number of H3+ cells were present in the
SVZ (Fig. 2B,E). The average number of H3+ cells in different
anatomical compartments was calculated in the entire dorsal
cortex of opossums, and the relative ratio of H3+ cells in SVZ
between opossum and rat was shown in Figure 2F. Although
the total number of H3+ cells gradually decreases as development progresses, the proportion of SVZ divisions
increases during development in both species (Carney et al.
2007). There was no clear difference in the pattern of VZ and
SVZ division between opossum and rat (see Fig. 2 legends for
statistical comparisons).
The Spindle Orientation of Neuroepithelial Cells of
Opossum Is Similar to Mouse
Given the differences in gestational length and total neuronal
output between mouse and marsupials, it is likely that the
neural stem cell pool size is different. We hypothesized that
mitotic spindle orientation, an indicator for neuroprogenitors
undergoing symmetric or asymmetric division, might also differ
at the early stages of brain development (Fig. 3A,B). The angle
between the cleavage plane and the apicobasal axis (Fig. 3C)
was scored into six 15° bins (Fig. 3D). The majority (86.6%) of
dividing cells of the opossum dorsal cortex were planar
divisions in which cleavage planes were within 15° of the
apicobasal axis. This percentage of planar division was similar
to that of E13.5--E15.5 mouse data reported previously (Kosodo
et al. 2004; Konno et al. 2008). These results suggest that
despite over 100 million years of independent evolution, there
is no difference in the proportion of spindle orientation of
neuroprogenitor cells between opossum and mouse.
The Developmental Organization of VZ and SVZ Division
Is Similar between Wallaby and Opossum
In another marsupial, the tammar wallaby, the distribution of
H3+ mitotic cells in the dorsal cortex was examined at P15,
P40, and P50 (Fig. 4A--C). Similar to opossum, the number of
H3+ cells in the VZ, SVZ, and EZ was counted (Fig. 4D). No H3+
cells were detected in the SVZ until P40 (arrows in Fig. 4B).
Although the total number of H3+ cells has gradually decreased,
the SVZ has shown an increase of H3+ cells at P50. The relative
proportion of H3+ cells present in VZ, SVZ, and EV of wallaby
is presented in Figure 4E. As observed in opossum and rat
(Fig. 2F), the proportion of H3+ cells in the SVZ of the wallaby
dorsal cortex gradually increased throughout brain development. We also analyzed the spindle orientation of neuroepithelial cells in P9--P50 wallaby and observed that the
percentage of planar divisions (Fig. 4F,G) was similar to
opossum (Fig. 3D) and mouse (Kosodo et al. 2004; Konno
et al. 2008). These results suggest that the proportion of IPCs in
the SVZ and the dividing neuroepithelial cells in the VZ are
very similar in rat, opossum, and wallaby (see Fig. 4 legend for
statistical analysis) despite considerable difference in neuronal
numbers in an arbitrary unit cortical column and the much
more protracted period of cortical development of marsupials.
mRNA Expression Pattern of VZ and SVZ Marker Genes in
Opossum Is Similar to Mouse
To confirm that the SVZ of opossum is similar to that of other
mammals, in situ hybridization for Pax6 (VZ marker), Brn2,
Cerebral Cortex Page 5 of 11
Figure 4. Distribution of dividing cells and mitotic spindle orientation in the developing wallaby cortex. (A--C) At P15, the majority of H3þ cells were in the VZ, although a few
H3þ cells were in the IZ. There was no H3þ division in the SVZ until P40 (arrows in B), and by P50, numerous H3þ cells were located in the SVZ. The anatomical boundary for
VZ, SVZ, and EZ was defined using H & E--stained sections (A‘--C‘), and the number of H3þ cells in each region was then quantified and expressed as mean ± SEM in (D). (E) The
relative ratio of H3þ cells in different anatomical regions of wallaby. Although the total number of cell division decreases as development progresses, cell divisions in the SVZ
become more prominent, a feature very similar to opossum and rat. Asterisks represent that there is a significant difference between wallaby and rat (P \ 0.05, Student’s t-test).
(F) A dividing cell with a typical planar division. Dotted lines represent the cleavage plane used to measure the orientation of mitotic spindle. (G) Distribution of the cleavage plane
orientation for mitotic neuroepithelial cells measured at P9, 15, 40, and 50 and averaged. Similar to opossum, most planar divisions were within 15° of the apicobasal axis. Scale
bar: A--C 5 300 lm; F 5 10 lm.
Cux2, NeuroD6, and Tbr2 (SVZ markers) was carried out. Of
note, attempts at immunohistochemistry were not successful
with Cux1 (1:1000 Santa Cruz Biotechnology, Santa Cruz, CA),
Tbr2 (1:2000 generous gift from R. Hevner, University of
Washington), and Pax6 (1:100 Developmental Studies Hybridoma Bank, University of Iowa) antibodies. At the earlier stage of
brain development (E15 mouse and P12 opossum), both the
Page 6 of 11 SVZ in Metatheria and Eutheria
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Cheung et al.
SVZ and VZ marker genes were expressed in corresponding
compartments of the germinal zone in a similar fashion (Fig.
5B--F, N--R). Whereas the mRNAs of Pax6 and Tbr2 were more
homogeneously expressed within VZ and SVZ (Fig. 5B,F,N, and
R), Cux2, NeuroD6, and Brn2 were more abundant in the SVZ
of both mouse and opossum (Fig. 5C--E, O--Q). At the later stage
of brain development (P0 mouse and P20 opossum), Cux2,
Figure 5. Comparison of mRNA patterns of VZ (Pax6) and SVZ (Cux2, NeuroD6, Brn2, and Tbr2) marker genes in mouse and opossum dorsal cortex at early (E15 and P12) and
later stages (P0 and P20) of neurogenesis. The expression patterns of VZ and SVZ marker genes in opossum (N--R, T--X) are similar to those of the mouse (B--F, H--L). (A, G, M, S)
Nissl-stained sections were used to identify VZ, SVZ, IZ or subplate, CP, and MZ. Scale bar: A--F, M--X 5 100 lm; G--L 5 200 lm.
Brn2, and Tbr2 mRNAs were expressed in the upper layer of
the cortex (Fig. 5I,K,L,U,W, and X). The proportion of the
upper CP that expresses these varied, for example, Tbr2 in
the P0 mouse is only at the very top of the CP, whereas in the
opossum, it takes up about the top half of the CP. These
differences might be genuine or due to the slightly different
stages selected for comparisons. Nevertheless, these results
suggest that the overall VZ and SVZ marker gene expression is
highly conserved and that these transcription factors may have
a similar function in both mouse and opossum.
The Neurovasuclar Pattern Is Similar between Mouse and
Opossum
Our previous observations in mouse revealed that cortical
blood vessels form a characteristic plexus in the SVZ during
cortical development (Stubbs et al. 2009). We performed
similar staining in opossum at various developmental stages and
observed identical patterns of vascular plexi in the SVZ
(Supplementary Figure 1). Analyses on both the anatomical
and molecular levels suggest that marsupials do have a distinct
SVZ, with dividing IPCs.
hamster, cat, ferret, and macaque. These studies have excluded
orders of mammals such as marsupials and monotremes where
substantially less is known about cortical development. The
metatherian (marsupial) lineage split from the last common
ancestor of eutherian mammals more than 100 million years
ago (Murphy et al. 2004). They are a highly diverse order of
mammals and, like eutherian mammals, have undergone
dramatic radiations and have diversified to fill a variety of
environmental niches. For example, marsupials possess fewer
neurons in their cortex (Haug 1987; Saunders et al. 1989), have
a large anterior commissure instead of a corpus callosum
(Reynolds et al. 1985), and have a more protracted period of
cortical development (Reynolds et al. 1985; Saunders et al.
1989; Mark and Marotte 1992; Molnár et al. 1998) with an
apparent absence of a distinct motor cortex (Karlen and
Krubitzer 2007). Furthermore, it has been suggested that their
cortical germinal zone organization might differ (Saunders et al.
1989; Abdel-Mannan et al. 2008) from their eutherian counterparts. To determine whether cortical development, as observed
in rodents and primates, is conserved among all mammals, as
opposed to a derivation of eutherian mammals, we compared
several aspects of organization and development in 2 representatives from each group.
Discussion
Although the process of cortical development is believed to be
fundamentally similar in all mammals (Rakic 1988; Bystron et al.
2008), the data on which this supposition is based have been
obtained from only a handful of species such as mouse, rat,
Reduced Neuronal Numbers in all Cortical Layers in
Adult Marsupial Cortex Compared with Mouse
Marsupials possess a 6-layered cerebral cortex with similar
basic organization to eutherian mammals (Northcutt and Kaas
Cerebral Cortex Page 7 of 11
1995); however, there are some marked differences in
marsupial cortex. Haug (1987) first compared the neuronal
numbers in several mammalian species, including opossum.
Our data confirmed his observation that the number of cortical
neurons arranged in a unit column is fewer in opossum and
extends to tammar wallaby as well. This suggests that even with
a protracted period of neurogenesis, marsupials still have less
cell division during cortical development. It has been reported
that several eutherian mammals possess the same number of
neurons (congruent to 112) in a 30-lm wide unit column,
except area 17 of primates, which contains 2.5 times more
neurons (Rockel et al. 1980). However, it remains unclear
whether all marsupials maintain a constant, albeit fewer,
neuronal number. Our data suggest that opossum has fewer
neurons than wallaby. We only had access to visual cortical
sections from wallaby, and we acknowledge that such
difference could be due to the cortical area selected for
counting. The primary visual area of wallaby is clearly
demarcated by the Line of Gennari (Vidyasagar et al. 1992),
a feature also present in primates. Thus, it is likely that the
primary visual area of wallaby is another exception in that it
possesses more neurons than other cortical areas.
The Basic Organization of the Cortical Germinal Zone Is
Similar in all Mammals
It has been suggested that the elaboration of the germinal zone
might compensate for the time constraints imposed on the
rapid development of the cerebral cortex in mammals (Smart
et al. 2002; Fish et al. 2008) and birds (Striedter and Charvet
2008). Although the elaboration of the dorsal cortical SVZ is
one of the hallmarks of the mammalian dorsal cortex, there are
several observations that suggest that further elaboration of the
SVZ may be the driving force of an expanded cortical sheet
observed in some groups of mammals. Before the demonstration of the presence of the SVZ in marsupials, it was an
interesting possibility that the lower neuronal numbers and the
extended cortical development in marsupials correlated with
an altered germinal zone. However, our study has shown that
the spatial and temporal organization of mitotic figures in the
developing cerebral cortex of marsupials, such as opossum and
wallaby, are fundamentally similar to previously studied
eutherian species such as rodents and primates (Lukaszewicz
et al. 2005; Kriegstein et al. 2006; Bystron et al. 2008). We used
several independent approaches to investigate the SVZ
divisions: 1) H3 staining to look for mitotic cells corresponding
to an SVZ, 2) morphological differences based on Nissl and H &
E staining and quantitative analysis of the mitotic spindle
orientation in VZ, 3) VZ/SVZ--specific mRNA expression of
selected genes, and 4) patterns of vascular plexi in the SVZ. The
results obtained from these experiments all suggest that like
other studied mammalian species with 6-layered dorsal cortex,
opossum and wallaby have a SVZ with IPCs during cortical
development. Although the number of neurons was proportionally lower in both upper and lower layers in a standard unit
column, these species achieve the same cortical arrangement
with similar gene expression within a comparable structure.
Does the Evolution of a 6-layered Mammalian Cerebral
Cortex Coincide with the Appearance of the Organized
Cortical SVZ during Cortical Development?
In the telencephalon of reptiles, birds, and mammals, embryonic neurogenesis occurs in the VZ along the lateral ventricle
Page 8 of 11 SVZ in Metatheria and Eutheria
d
Cheung et al.
and in abventricular divisions at various distances from the VZ
(Martı́nez-Cerdeño et al. 2006). It is believed that developmental mechanisms in the mammalian telencephalon are distinguished from those in reptiles and birds by the presence of
a neuron-generating SVZ in the germinal zone of dorsal cortex
(Cheung et al. 2007). Previous analysis of the variation in
mitotic cell frequency with distance from VZ in chicken brains
demonstrated a second proliferation peak corresponding to an
organized SVZ in the basal ganglia, nidopallium, and mesopallium at E8 and E10 but was absent in the hyperpallium
(equivalent to mammalian dorsal cortex) (Cheung et al. 2007).
Mammals also have a large SVZ with scattered mitotic profiles
throughout the depth of the LGE, MGE, and CGE of the
subpallium (Bhide 1996). Mitotic cells are abundant at similar
levels throughout the bulged ganglionic eminences, but they
do not line up into a discrete band parallel to the ventricular
surface (Bhide 1996; Carney 2005). The subpallial SVZ is
believed to produce tangentially migrating interneurons
(Anderson et al. 2001), whereas the IPCs in the cortical SVZ
generate excitatory neurons (Haubensak et al. 2004; Miyata
et al. 2004; Noctor et al. 2004). In marsupials, dorsal pallial
abventricular proliferation was infrequent and less organized at
early stages, although the ratio of abventricular to ventricular
H3+ cells is not significantly different at P10 in opossum and
P15 in wallaby between the pallium and subpallium. In the
turtle, VZ mitosis peaks in earlier stages (S18 and 20) before
shifting to an increasingly abventricular site of proliferation
(Molnár, Tavare, and Cheung 2006; Cheung et al. 2007).
However, abventricular division remains infrequent and scattered in the turtle dorsal cortex, whereas they align along
a distinct zone at P14 in opossum and P40 in wallaby, and this is
consistent with the hypothesis that IPCs in the organized pallial
SVZ contribute to the expansion of the cortex from sauropsids
to mammals. By demonstrating the existence of organized
pallial SVZ in the developing marsupial cortex, we strongly
support the notion that the evolution of a 6-layered mammalian
cerebral cortex coincides with the appearance of the organized
cortical SVZ. However, our study included representative species
from only 1 of the 2 subclasses of mammals, that is, Theria, which
includes the infraclasses Metatheria and Eutheria. The other
subclass of mammals—Prototheria (monotremes)—has not been
examined. This egg-laying group of mammals may represent
a more primitive condition and thus may very well not follow
the pattern of SVZ development common to Eutherians and
the Metatherians examined in this study. It will be important
to carry out similar work on Prototheria.
The Role of Intermediate Progenitors
Currently, the developmental or evolutionary role of IPCs is not
known. The expansion in cortical surface area accompanying
mammalian evolution might have arisen from an increase in the
intermediate progenitor compartment (intermediate progenitor hypothesis; Tarabykin et al. 2001; Haubensak et al. 2004;
Kriegstein et al. 2006). IPCs were further implicated in the
generation of upper cortical layers (upper layer hypothesis;
e.g., Tarabykin et al. 2001; Wu et al. 2005). IPCs and upper
layers indeed share several genes. A precise molecular
expression sequence for the transition of neuroepithelial cells
to RGCs to IPCs to neurons might have played a major role in
the diversification of the mammalian telencephalon. In mammals, several transcription factors label the pallial SVZ during
neurogenesis including Svet1, Cux1 and Cux2, and Tbr2
(Tarabykin et al. 2001; Nieto et al. 2004; Englund et al. 2005,
respectively), and upper layer differentiation defects were
reported in Cux2 (Cubelos et al. 2008) and Tbr2 (Arnold et al.
2008; Sessa et al. 2008) knock-outs. However, there are several
observations that question the IPC --upper layer hypothesis.
Haubensak et al. (2004) reported on the appearance of
neurogenic IPCs in the mouse cortex as early as E10.5,
implying that the role of IPC cannot be confined to upper
layer neuron production only. Moreover, Kowalczyk et al.
(2009) recently demonstrated that IPCs contribute pyramidal
projection neurons to all layers in the mouse cerebral cortex.
Conditional inactivation of Tbr2 in the central nervous system
of mice selectively reduces the pool of IPCs (Arnold et al.
2008). Although the differentiation of upper layer neurons is
more prominently affected in the Tbr2 conditional knock-out,
the layer thickness of both supra- and infragranular layers are
abridged and the number of both supra and infragranular
neurons are equally reduced (AFP Cheung, S Arnold, M Groszer,
Z Molnár, unpublished data). Consistent with the fundamental
radial unit concept of Rakic (1988), Farkas et al. (2008) have
shown that in the Insm1 knock-out mouse, there is cortical
expansion in the lateral dimension concomitant with the
reduction in IPCs and radial thickness. Hevner and colleagues
suggest that changes in IPC abundance alter cortical thickness
and not necessarily cortical surface area in various mouse
mutants and propose a modified radial unit hypothesis
(Pontious et al. 2007), where IPCs would act as radial amplifiers
for the neuronal output from the ontogenic units (Rakic 1988).
Further Elaboration of the Cortical Germinal Zone in
Mammals
Although our study emphasizes the universal presence of SVZ
in all mammalian cerebral cortices, we would like to stress the
considerable differences in the elaboration of the cortical
mitotic compartments. The elaboration of the germinal zone is
more apparent in macaque (Smart et al. 2002; Lukaszewicz
et al. 2005) with 3 distinct zones: VZ, inner SVZ, and outer SVZ
(OSVZ), each with a characteristic gene expression pattern
(Smart et al. 2002; Fish et al. 2008). Recently, the interesting
‘‘epithelial progenitor hypothesis’’ was proposed (Fish et al.
2008). This hypothesis argues that evolutionary changes, which
promote the maintenance of epithelial features in neural
progenitors, including OSVZ progenitors, have been instrumental in the expansion of the cerebral cortex in primates.
Therefore, by extrapolation, it is plausible that the mitotic
spindle orientation of neuroepithelial cells in marsupials
(which has a less complex cortical organization than primates)
is not as tightly regulated as in primates.
More species with gyrencephalic and lissencephalic brains
should be investigated to be able to correlate the presence or
the absence of sulci and gyri with the compartmentalization of
the germinal zones (Cheung et al. 2007). However, our result
suggests that spindle orientation of neuroepithelial cells of
marsupials was similar to that of mouse, thus extending the
concept to other mammalian infraclass. Further studies are
needed to clarify the role of spindle orientation in regulating
sibling cell fate. Future experiments with electroporation of
green fluorescent protein-expressing plasmids into the pallium
for time-lapse microscopy of cell dynamics in various vertebrates could shed light on the intrinsic and environmental
factors involved in regulation of cell division in the SVZ.
Neuron Numbers in Mammalian Cerebral Cortex
The marsupial cortex has a protracted development that would
seemingly reduce pressure on the timing of neuronal production compared with some small eutherians such as mouse.
Our current study revealed the relatively late appearance of the
SVZ with slightly altered proportions of VZ and SVZ cell
division especially at early stages of marsupial cortical development. The prolonged cell cycle duration and the increased number of cell divisions of cortical progenitors in
macaque when compared with rodents are hypothesized to
underlie the evolutionary expansion of the neocortex (Rakic
1995). Interestingly, the protracted development in opossum
and wallaby does not result in an expanded cortical sheet. It
would be interesting to examine other marsupials, such as the
striped possum, which have an encephalization quotient that
rivals that of many primates to determine if similar mechanisms
account for the expansion of the cortical sheet. Because
comparative developmental neurobiology depends on observations across numerous species, additional marsupial, eutherian,
and monotreme species will need to be examined to determine
the differential role of the pallial SVZ division.
Conclusion
Our description of the organized IPC in the SVZ, the
comparable orientation of mitotic spindles in the VZ, and
similar expression of selected genes with known cortical VZ
and SVZ expression in mouse, opossum, and tammar wallaby
further support the hypothesis that a cortical SVZ is a hallmark
of 6-layered cortex in all mammals. The role of the SVZ as a key
mitotic compartment in mammalian cortical evolution is still
not fully understood, but SVZ divisions might be necessary for
the generation of extra neurons for both radial and tangential
expansion of the mammalian cerebral cortex.
Supplementary Material
Supplementary Table 1 and Figure 1 can be found at: http://
www.cercor.oxfordjournals.org/.
Notes
Medical Research Council (G0300200, G0700377 to Z.M.); Biotechnology and Biological Sciences Research Council (BB/F003285/1 to Z.M.);
National Science Foundation (IOS-0743924 to L.K.).Conflict of Interest :
None declared.
Address correspondence to Zoltán Molnár, Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros
Clark Building, South Parks Road, Oxford OX1 3QX, UK. Email:
zoltan.molnar@dpag.ox.ac.uk.
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