Download Clones in the chick diencephalon contain multiple

Development 122, 65-78 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
DEV8292
65
Clones in the chick diencephalon contain multiple cell types and siblings are
widely dispersed
Jeffrey A. Golden1,2 and Constance L. Cepko1,3
1Department of Genetics, Harvard Medical School, 2Department of Pathology, Brigham and Women’s Hospital, and 3Howard
Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA
SUMMARY
The thalamus, hypothalamus and epithalamus of the vertebrate central nervous system are derived from the
embryonic diencephalon. These regions of the nervous
system function as major relays between the telencephalon
and more caudal regions of the brain. Early in development, the diencephalon morphologically comprises distinct
units known as neuromeres or prosomeres. As development
proceeds, multiple nuclei, the functional and anatomical
units of the diencephalon, derive from the neuromeres. It
was of interest to determine whether progenitors in the
diencephalon give rise to daughters that cross nuclear or
neuromeric boundaries. To this end, a highly complex
retroviral library was used to infect diencephalic progenitors. Retrovirally marked clones were found to contain
neurons, glia and occasionally radial glia. The majority of
clones dispersed in all directions, resulting in sibling cells
populating multiple nuclei within the diencephalon. In
addition, several distinctive patterns of dispersion were
observed. These included clones with siblings distributed
bilaterally across the third ventricle, clones that originated
in the lateral ventricle, clones that crossed neuromeric
boundaries, and clones that crossed major boundaries of
the developing nervous system, such as the diencephalon
and mesencephalon. These findings demonstrate that progenitor cells in the diencephalon are multipotent and that
their daughters can become widely dispersed.
INTRODUCTION
of the diencephalon resembles the more caudal mesencephalon
(midbrain) and rhombencephalon (hindbrain), which are
parceled into discrete nuclei with mostly discrete projections.
How the intermediate structure of the diencephalon develops
is not clear. The development of some of the more rostral and
caudal regions of the brain have been relatively well described
such that we can now appreciate that these areas follow
different rules in key aspects of their development.
The rhombencephalon is transiently parceled into 8 morphologically identifiable units known as rhombomeres. The
neurons in each rhombomere comprise groups of nuclei, each
with defined functions and a stereotypical pattern of axonal
projections (Lumsden and Keynes, 1989; Keynes and
Lumsden, 1990; Lumsden, 1990). A molecular basis for the
morphological and functional specification of rhombomeres
has been established through studies of the expression and misexpression of Hox genes and other transcription factors
(Guthrie and Lumsden, 1991, 1992; Hunt et al., 1991; Guthrie
et al., 1992). Lineage analysis conducted in the hindbrain of
chicks has shown that once the boundaries of the rhombomeres
are established, the majority of clones appear to be restricted
to a single rhombomere (Fraser et al., 1990; Birgbauer and
Fraser, 1994) during the next 48 hours of development. Clones
also appear restricted in cell fate in that most clones comprise
siblings that adopt the same or a related neuronal cell fate
(Lumsden et al., 1994).
One of the hallmarks of the adult nervous system is the
exquisite complexity of its cell types and synaptic connections.
The mechanisms that generate complexity during development
remain largely unknown. Early in development, the neural tube
is thought to be parceled into unique units or segments
(Lumsden, 1990; Puelles and Rubenstein, 1993; Rubenstein et
al., 1994; Rubenstein and Puelles, 1994). Distinct morphological units first appear shortly before neural tube closure when
multiple vesicular outpouchings develop rostrally. The primary
vesicles are termed the prosencephalon, the mesencephalon
and the rhombencephalon. The prosencephalon gives rise to
the telencephalon (cerebral hemispheres) and the diencephalon. The diencephalon is the embryonic precursor to the
hypothalamus, thalamus and epithalamus, and is anatomically
situated between the cerebral hemispheres and more caudal
areas of the brain. The diencephalon appears to be conserved
structurally and functionally throughout evolution, although
there is some debate about whether the organization within the
diencephalon, and the thalamus in particular, is similar across
species (Kappers et al., 1960; Jones, 1985). The embryological origin of the diencephalon appears to be conserved in
disparate species (Kappers et al., 1960).
While functional aspects of the diencephalon appear to
mimic the rostral telencephalon, the anatomical organization
Key words: cell lineage, central nervous system, diencephalon,
thalamus, chick, thalamus, hypothalamus
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J. A. Golden and C. L. Cepko
The telencephalon, the most rostral part of the central
nervous system (CNS), is not separated into morphologically
defined, repeated units. Rather, the cerebral cortex, the largest
component of the telencephalon, is organized into a diffuse
laminated sheet of cells. Although the cortex can be parceled
into functional domains, there is no discrete nuclear organization as is found in the hindbrain and diencephalon. Studies
investigating the expression pattern of a variety of genes,
mostly transcription factors, have uncovered several genes
with nested (Simeone et al., 1992; Bulfone et al., 1993a,b) or
lamina-specific (Frantz et al., 1994a,b; Leifer et al., 1994)
patterns of expression. However, genes investigated thus far
are generally not expressed within morphologically or physiologically defined areas. Lineage analysis in the telencephalon
has demonstrated that siblings spread over great distances to
give rise to neurons in functionally and anatomically unrelated
parts of the cerebral cortex (Walsh and Cepko, 1992, 1993;
Reid et al., 1995). Lineage analysis in the telencephalon has
shown that individual progenitor cells are capable of generating neurons and glia (Price and Thurlow, 1988; Walsh and
Cepko, 1992; Levison et al., 1993; Reid et al., 1995). In the
retina (an outgrowth of the diencephalon) and the chick tectum
(a derivative of the mesencephalon), lineage analysis has also
shown that neurons and glia arise from a common progenitor
cell (Turner and Cepko, 1987; Galileo et al., 1990; Turner et
al., 1990; Gray and Sanes, 1992; Fekete et al., 1995).
The diencephalon is organized into individual nuclei (collections of neurons with a specific projection or defined set of
projections) with some groups of nuclei having similar functional properties and other neighboring nuclei having distinct
functions. Functionally, however, the diencephalon and, particularly, the thalamus closely resemble the telencephalon
(cerebral cortex and basal ganglia). The thalamus functions as
the major integration and projection region to the cerebral
cortex from more caudal structures, including the spinal cord,
cerebellum, hindbrain and midbrain (Jones, 1985). The
neurons of the thalamic nuclei project to different cortical areas
with many thalamic nuclei projecting to overlapping cortical
regions. Furthermore, the thalamocortical projections to the
cortex appear to define the functional cortical units (see Jones,
1985 for review). During development, the thalamocortical
projections show growth into specific cortical areas and thalamocortical axons may provide cues that help define the course
of cortical neuronal projections (reviewed in O’Leary and
Koester, 1993).
Given that the telencephalon and rhombencephalon appear
to use distinct mechanisms for patterning and perhaps generating cellular diversity, it was of interest to determine clonal
relationships within the diencephalon. Classical embryologists
have described the diencephalon as arising from four horizontal strips (His, 1893; Herrick, 1910; Khulenbeck, 1973) or neuromeric units (Orr, 1887; Bergquist, 1952; Keyser, 1972) based
on the presence of bulges and sulci along the medial walls of
the third ventricle. These morphological units, labeled neuromeres or prosomeres (we have adopted the neuromere
nomenclature in this paper), have been proposed to be
analogous to the rhombomeres (Puelles et al., 1987; Figdor and
Stern, 1993; Puelles and Rubenstein, 1993), although this
analogy has not been completely tested (see Guthrie, 1995).
Examination of the expression patterns of a variety of developmentally regulated genes has shown a correlation with the
neuromeric units (Bulfone et al., 1993a,b; Rubenstein et al.,
1994; Rubenstein and Puelles, 1994), similar to the correlation
noted in the hindbrain. Short-term lineal relationships in the
diencephalon have been analyzed using the method of single
cell microinjection of a fluorescent dye (Figdor and Stern,
1993). The results indicated that the morphological neuromeres
of the diencephalon are akin to the hindbrain rhombomeres in
that clones were restricted to a single neuromere up to 48 hours
after injection, the latest time point analyzed. However, this
technique precludes analysis of the final patterns of clonal dispersion or the mature cell types within any one clone.
Chick/quail chimera studies have also been performed in the
diencephalon (Martinez and Alvarado-Mallart, 1989). In these
studies, quail mesencephalon was transplanted into chick diencephalon, and thus the potential of diencephalic progenitors
was not tested. Nonetheless, these studies indicated that mesencephalic progenitors dispersed to populate several, but not
all, nuclei in the diencephalon. One interesting pattern of
spread for the mesencephalic progenitors in these studies was
that they selectively populated primary visual nuclei, suggesting a relationship with the tectum, the normal derivative of the
mesencephalon.
Using a complex retroviral library comprising DNA tags as
lineage markers (Golden et al., 1995), we have evaluated 275
clones in the chick diencephalon. This technique allows
analysis of clones in the mature diencephalon, after clonal dispersion is complete. We found that sibling cells can spread
extensively within the diencephalon, sometimes occupying
nuclei derived from more than one neuromere. Dispersion
occasionally led to cells being located in both the diencephalon
and the mesencephalon. Furthermore, siblings were found on
both the left and right sides of the third ventricle in approximately 16% of the clones. We also found that progenitors of
diencephalic cells were located in the ventricular zone of the
third ventricle, as well as in the ventricular zone of the lateral
ventricle. Clones frequently contained neurons, glia and radial
glia, supporting the existence of multipotent progenitor cells.
MATERIALS AND METHODS
Production of the retroviral library CHAPOL
Construction and characterization of the retroviral library used in this
study have been described (Golden et al., 1995). Briefly, the library
of retroviral vectors was constructed from a replication defective
avian retrovirus, CHAP (Ryder and Cepko, 1994), that encodes the
human placental alkaline phosphatase gene (PLAP). A pool of
synthetic degenerate oligonucleotide tags with a theoretical complexity of >107 were cloned into the vector to create the vector library,
CHAPOL. A large scale viral preparation yielded a stock with a concentration of 1.1×107 cfu/ml that was used for all experiments (Golden
et al., 1995). Numerous experiments with this same stock of CHAPOL
have led to recovery of >350 unique inserts (current paper and
(Golden et al., 1995)). These data indicate that the CHAPOL library
has a complexity of at least 105 members (Walsh et al., 1992). Furthermore, each insert has been recovered from only one infection
event (i.e. only 1 brain or 1 well of an infected microtiter plate of
tissue culture cells), indicating the library has an approximately equal
distribution of inserts.
In vivo infection
The neural tube of fertilized virus-free White Leghorn chicken
embryos was injected as previously described (Fekete and Cepko,
Clones in the chick diencephalon
1993) using CHAPOL. Injections were performed at either stage 1012 or stage 16-17 (all staging according to Hamburger and Hamilton,
1951). Approximately 0.3-0.5 µl of CHAPOL stock was injected into
the neural tube at stage 10-12 and 1.0-1.5 µl at stage 16-17. Infected
embryos were incubated in a humidified, 37°C chamber until
embryonic day 18 (E18) at which time the brains were harvested in
PBS. After fixation overnight in 4% paraformaldehyde (in PBS, pH
7.4) at 4°C, brains were washed with three changes of PBS over 24
hours and then cryoprotected in 30% sucrose in PBS. Brains were
oriented for coronal sections, embedded in OCT medium and cut on
a Reichart-Jung CM3000 cryostat at 60 µm. Each section was
collected and mounted sequentially on Superfrost/Plus microscopic
slides (Fisher Scientific). On average, 3 sections/brain (approximately
45 sections included the diencephalon) were lost and the ordered
position of each missed section was documented. Infected cells were
identified histochemically by incubating the sections with X-phos
(Research Organics, Inc.) and NBT (Research Organics, Inc.) for 60
to 240 minutes according to previously published protocols (Ryder
and Cepko, 1994; Golden et al., 1995). Cells infected with the retrovirus were identified by the purple formazan precipitate.
Coronal sections that included the diencephalon, from the anterior
commissure (anterior limit of analysis) to the red nucleus (posterior
limit of analysis) were photographed at 2× magnification with a Nikon
SMZ-U stereomicroscope equipped with a Nikon DX FX-35WA
Camera. Representative cells were photographed at higher magnification on a Zeiss Axiophot microscope. In addition, camera-lucida
drawings of selected cells were made from a drawing tube mounted
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on a Zeiss Axioskop. The 2× magnification Kodachromes were
scanned into a Macintosh Quadra 650 using a Kodak 2035 plus slide
scanner and Adobe Photoshop software. The location and cell type of
each alkaline phosphatase positive (AP+) cell and/or cluster of cells
was given a unique identification based upon the section number.
Once documented, each cell or cluster of cells (with a small group of
surrounding white AP− cells) was removed using heat-pulled glass
micropipettes and transferred to a 96-well PCR plate with 10 µl of
400 µg/ml proteinase K solution (Golden et al., 1995). Approximately
50-60 AP+ cells were picked from sections and analysed on each 96well PCR plate. One or two picks from a similar stage uninfected brain
that had been processed in parallel with the experimental brain were
included on each plate as negative contrals. The cells were then
digested, amplified and sequenced as previously described (Golden et
al., 1995). Once all sequences were collected and stored, all common
sequences could be grouped and the position and cell type(s) for
sibling cells revealed. Each AP+ cell or cluster or AP+ cells was then
reidentified on the computer images to obtain a map of the distribution of sibling cells in the diencephalon. The position of each clone
was also transposed onto an atlas of the chick diencephalon (Kuenzel
and Masson, 1988). AP+ cells were anatomically positioned on atlas
sections by first defining their location within nuclei or white matter
tracts on histological sections (Fig. 1). Since all nuclei and white
matter tracts could not be defined on the AP-stained sections, cells
outside anatomically definable structures were localized according to
their position relative to defined structures. However, the majority of
cells could be definitively assigned based upon the tissue morphol-
Fig. 1. Examples of distinct
cell types and histological
identification of diencephalic
nuclei. (A-F) Representative
examples of cell types
identified on the basis of
morphology. Neurons were
identified by the presence of
long thin processes. Glia
(astrocytes) were identified
by intense staining and
indistinct borders, with
occasional thick short
processes. Radial glia were
identified by a cell body
apposed to the ventricular
surface and a long thin
process radiating into the
parenchyma. Cells that could
not be identified were
generally small and round
with no clear morphological
feature. (G,H) Two
representive sections of the
diencephalon stained for AP
activity. The left half of each
figure is a schematic outline
of the nuclei and white
matter tracts that could be
defined as a result of the AP
histochemistry and
anatomical landmarks. Note
the AP+ cells in the nucleus
rotundus in G and the
nucleus ovoidalis in H. Scale bar 100 µm for A, B and E; 50 µm for C; 267 µm for D; 200 µm for F; and 450 µm for G and H. AL, ansa
lenticularis posterior; DLAl, nucleus dorsolateralis anterior thalami (lateral); DLAm, nucleus dorsolateralis anterior thalami (medial); FPL,
fasciculus prossencephali lateralis (lateral forebrain bundle); GLv, nucleus geniculatus lateralis, pars ventralis; ICT, nucleus intercalatus
thalami; OM, tractus occipitomesencephalicus; OT, optic tract; OV, nucleus ovoidalis; ROT, nucleus rotundus.
68
J. A. Golden and C. L. Cepko
ogy. The following structures were identified on histological sections:
ansa lenticularis, anterior commissure, nucleus anterior medialis
hypothalami, posterior commissure, nucleus dorsointermedialis
posterior thalami, nucleus dorsolateralis anterior thalami (lateral),
nucleus dorsolateralis anterior thalami (medial), nucleus dorsolateralis posterior thalami, nucleus dorsomedialis anterior thalami, nucleus
dorsomedialis posterior thalami, fasciculus prossencephali lateralis
(lateral forebrain bundle), nucleus geniculatus lateralis, pars ventralis,
nucleus intercalatus thalami, optic chiasm, tractus occipitomesencephalicus, optic tract, nucleus ovoidalis, nucleus paraventricularis
hypothalami, nucleus rotundus, tractus tectothalamicus. Three-dimensional reconstruction of the diencephalon was performed by transforming the Photoshop two-dimensional images using Spyglass Dicer.
DiI labeling
Radial glia were labeled as previously described (Gray and Sanes,
1992). Briefly, brains from uninfected E15-E18 chicks were dissected
in PBS and fixed overnight in 4% paraformaldehyde. After washing
in PBS, approximately 0.1 ml of a 2.5 mg/ml solution of DiI
(Molecular Probes, Inc.) in 100% ethanol was injected into the right
lateral ventricle of the brain using a 30-gauge needle. The DiI solution
was allowed to passively fill the entire ventricular system. The brains
were then placed in a 60 mm Petri dish filled with sterile water and
0.03% sodium azide and incubated for two weeks at 37°C. After incubation the brains were imbedded in 5% agar (in H2O) and sectioned
on a vibratome at 100-200 µm. The sections were mounted on Superfrost/Plus microscopic slides, coverslipped with gelvatol and viewed
with rhodamine fluorescent filters on a Zeiss Axiophot microscope.
Photos were taken with Kodak Elite 100 film.
RESULTS
Summary of brains analyzed and recovery rates of
infected cells
The clones described here were isolated from seven E18
brains. Injection of the retroviral library was at stage 10 (n=1),
stage 11 (n=1) or stage 16-17 (n=5). Small tissue samples containing a single alkaline phosphatase (AP+) cell or small
clusters of AP+ cells along with surrounding AP− tissue were
removed (each sample is referred to as a ‘pick’). The total
number of picks was 1,537 from the diencephalon of the 7
brains. An additional 263 picks were collected from areas with
no visible AP+ cells. PCR amplification yielded the predicted
121 base pair product from 1,088 (71%) of the 1,537 picks.
Sequencing of the PCR products revealed that 70% (761) of
the AP+ regions isolated contained a single insert and 17%
(186) contained multiple inserts. (Those with multiple inserts
were not analyzed further since identification of more than one
sequence implies that cells that are members of more than one
clone were present in the tissue sample analyzed. If one AP+
cell was present, it was impossible to define which sequence
belonged to the AP+ cell and which belonged to the AP−
cell(s). Similarly, if two or more AP+ cells were present, it was
again impossible to determine which sequence belonged to
which cell or cells. Furthermore, technical limitations have
made it difficult to separate multiple PCR products, so that the
sequence of each can be defined (Golden et al., 1995).) No
sequence was obtained in 141 (13%) of the cases. Efficiency
of recovery of PCR products and sequences was similar from
single cells and small clusters of cells (data not shown, and
Golden and Cepko, 1995).
The 761 picks that gave a single sequence were classified
into 275 clones (the same insert has never been recovered from
more than one brain). Of these, 154 picks contained an insert
that was recovered only once and were therefore defined as
‘single pick clones’. These 154 clones had from 1 to 8 cells
(i.e. each pick had from 1 to 8 cells), 118 having a single cell.
121 insert sequences were obtained 2 or more times from the
761 picks and these were defined as ‘multiple pick clones’.
Each pick from the multiple pick clones also had from 1 to 8
cells. The single pick clones and multiple pick clones are
analyzed separately below.
Single pick clones
The 154 single pick clones contained an average of 1.52 cells
of which 0.4 were neurons, 0.4 were glia and 0.1 were radial
glia. An average of 0.6 cells in each clone was not classifiable
on morphological criteria alone (Fig. 1). Oligodendrocytes
were rarely found and difficult to identify morphologically in
this study. Since they represented such a small subset of cell
types identified, they have been lumped with astrocytes in the
category of glial cells. The data derived from the single cell
clones provide an approximation of the birthdates for different
cell types (see Table 1 and Discussion). For example, a greater
percentage of the single pick clones with more than one cell,
compared to single pick clones with one cell, comprised glia
only (31% vs 5%). This suggests that progenitors that ultimately gave rise only to glia were making some mitotic
daughters at the time that they were infected with the retrovirus. In contrast, the percentage of neurons from single pick
clones with multiple cells, compared to those with a single cell,
was relatively similar (22% vs 27%). Thus neurons were being
generated at the time of injection more often then glia and some
progenitors produced mitotic daughters that gave rise only to
neurons. Furthermore, the percentage of single pick clones
with a single cell that was a neuron (27%) compared to the percentage of clones containing neurons in the entire data set
(60%) was greater than the comparable numbers of clones containing glia (5% compared to 65%). Thus, neurons were far
more frequently represented in the single cell clone category.
Table 1. Cell type composition of single and multiple pick
clones
Single pick
Multiple
pick
multiple
cells
single
cell
total
121
36
118
154
59(49)
6(5)
16(44)
8(22)
−
32(27)
48(31)
40(26)
Include glia
Glia only
74(61)
9(7)
17(47)
11(31)
−
6(5)
23(15)
17(11)
Include radial glia
Radial glia only
20(17)
0
3(8)
2(6)
−
10(8)
13(8)
12(8)
(a) Number of inserts
recovered
(b) Number (percent) clones
with cell types
Include neurons
Neurons only
The number (and percent) of clones that include the indicated cell type are
grouped according to whether they are multiple pick or single pick clones.
Multiple pick clones are the clones that gave the same insert from multiple
tissue picks in the same brain. Single pick clones are clones in which an insert
was recovered only once. The single pick clones are further divided into the
picks comprising a single cell or multiple cells.
Clones in the chick diencephalon
Analysis of the composition of small clones reveals that
there are more radial glia than would be predicted based upon
the overall frequency of radial glia in the entire data set. 10 of
the 33 (30%) clones that contained radial glial cells were single
cell clones. An additional 2 multiple cell clones from the single
pick clones were composed of radial glia only, making a total
of 12 of 33 (36%) clones with only radial glia. These two
clones that contained radial glial cells as their only members
indicate that there may be progenitor cells that can divide to
give rise to 2 or more radial glia (also see Gray and Sanes,
1992). However, since there were no multiple pick clones
composed entirely of radial glia, such a progenitor cell, if it
exists, does not produce many radial glia.
Analysis of the distribution of the 118 single cell clones in
three dimensions suggested no particular bias for an anteriorto-posterior, medial-to-lateral, or dorsal-to-ventral birth order.
However, the limited number of clones and injection times
preclude definitive assessment of birthdating gradients in the
diencephalon.
Multiple pick clones
121 inserts were recovered from two or more picks in the same
brain. The average number of cells in these clones was 7.8. The
average numbers of neurons, glia and unidentified cell types in
these clones were 1.4, 3.6 and 2.8 respectively. In contrast to
the single pick clones, multiple pick clones frequently had
multiple cell types (Table 1). It remains possible that some of
the clones that contained unidentifiable cells would have been
classified as clones with only one cell type. Nonetheless, 43 of
the 121 (36%) clones had phenotypically distinguishable cells
(Table 2), either neurons and glia, neurons and radial glia, glia
and radial glia, or all three. These data indicate that multipotent progenitor cells were frequently infected.
101 of the clones were found exclusively on either the left
or right side of the lateral ventricle. Table 2 is a list of all 121
multiple pick clones and includes the composition of cell types
and the extent of dispersion in the anterior-posterior, mediallateral, and dorsal-ventral planes. Figs 2-4 are composite maps
showing the distribution of cells within clones from a subset
(44/121) of the multiple pick clones and Fig. 5 shows the
anterior to posterior spread (also see Fig. 2, clone 3 and Fig.
3, clone 7).
Clones were observed to spread from 2% (60 µm) to 54%
(1140 µm) of the anterior to posterior extent of the entire diencephalon, with the average clone extending 11% of the
anterior-posterior distance. Thus cells were separated by
approximately 350 µm on average along the anterior-posterior
axis. Spread of sibling cells was most pronounced in the
medial-lateral axis, where the average extent of spread was
57.5% (between 1500 and 2000 µm; table 2), and spread
ranged from none (cells located along the third ventricle) to
spread across virtually the entire diencephalon from the third
ventricle to the lateral margin (in Fig. 3G compare clone 8 to
clone 5).
Cells also spread along the dorsal-ventral axis across an
average of 19% of the entire diencephalon, corresponding to
approximately 1000 µm. The range of spread was from 1% (5
µm) to 74% (2900 µm; Table 2). The spread of cells in the
dorsal-ventral plane showed a common bias toward the ventral
side with progressively more laterally displaced cells. Siblings
leaving the third ventricle, the most common site of birth,
69
appeared to disperse both laterally and ventrally (e.g. Fig. 2,
clones 2 and 3; Fig. 3, clone 9; and Fig. 4, clones 12 and 14).
Some clones showed a more limited dorsal to ventral dispersion with extensive lateral dispersion (e.g. Fig. 2, clone 12 and
Fig. 3 clone 5), but few showed extensive dorsal-ventral dispersion without accompanying lateral dispersion. While the
vast majority of clones showed lateral and ventral spread,
several clones violated this general trend. Two clones were
found to spread a significantly greater percentage along the
dorsal to ventral axis compared to the medial to lateral axis
(Table 2, asterisked clones).
The dispersion of clones in three planes indicates that cells
from an individual clone occupy >1 nucleus of the diencephalon (e.g. Fig. 2, clones 1 and 10; Fig. 3, clones 1 and 14;
and Fig. 4, clone 3). Clonally related neurons and glia could
be found in nuclei that are both adjacent to and distant from
each other. Some clones remained localized to a single nucleus
or cluster of nuclei (e.g. Fig. 2, clone 6; and Fig. 4, clone 13).
Since sibling cells populated multiple nuclei, we attempted
to define whether clones crossed neuromeric boundaries.
However, detailed maps delineating which mature structures
are derived from the developmentally defined neuromeres are
not available for any species. Thus, in order to provide an
estimate of the number of clones that occupy derivatives of >1
neuromere, we extrapolated from the data of Figdor and Stern
(1993), identifying only those clones that clearly crossed neuromeric boundaries (Table 3). Cells that were in the region of
a proposed boundary (Puelles and Zabala, 1982) were placed
in the ‘could not determine’ category of Table 3. Based on this
analysis, at least 8% of all clones crossed neuromeric boundaries. Because the precise boundaries have not been defined for
the mature diencephalon, this represents the most conservative
estimate possible. It is likely that some of the 47% of the clones
that could not be definitively assessed did cross a neuromeric
boundary. Furthermore, the fact that many clones were found
within a single neuromere does not mean that they were
restricted by the boundaries of that neuromere.
Unusual clone types
Several clones were found with unusual distributions of cells
or an unexpected site of genesis. One type of clone had cells
on both sides of the midline surrounding the 3rd ventricle. 20
clones (16%) in the data set were found with a bilateral distribution (Table 2; Fig. 3, clones 3, 6, 9, 11, 13 and Fig. 4, clone
2). These clones contained both glia and neurons, including
neurons on both the right and left sides of the third ventricle
in individual cases. Overall, the distribution of cell types was
similar to that of the unilateral clones. Several presumptive
clones in which cells appeared to be migrating across the
anterior or posterior commissure have been found in brains
infected at stage 16-17 with CHAP or CHAPOL and harvested
at E8-E10 (Szele F, Golden J, and Cepko C, unpublished data
and Fig. 6A). Analysis of the anterior-to-posterior distribution
of bilateral clones showed no specific localization.
Although most studies have focused upon the germinal zone
of the third ventricle as the source of cells that populate the
diencephalon, another rare clone type appeared to originate in
the lateral ventricle. Several of these clones included radial
glial cells projecting from the lateral ventricle medially and
ventrally into the diencephalon (Fig. 4 clone 10; note that the
cerebral hemispheres and lateral ventricles, located along the
3A.
J. A. Golden and C. L. Cepko
2A.
70
4A.
Fig. 5. Three-dimensional
reconstruction of clones
within the diencephalon.
Representative clones have
been transposed to the
surface of a reconstructed
diencephalon. Each cell was
localized according to its
anterior-posterior and
dorsal-ventral position;
medial-lateral position is
not shown. Several clones
spread widely in the
anterior-posterior plane,
while others remain
relatively constricted. Dispersion along the dorsal-ventral axis also varied. The overall
extent of dispersion of a clone did not always reflect the number of cells within the
clone, as small clones showed similar dispersion patterns to large clones.
Figs 2-4. Schematic representation of 44 clones. Diagrams of the diencephalon and
associated structures were traced from an atlas of the chick brain (Kuenzel and
Masson, 1988). Cells from approximately 7 serial 60 µm sections were entered onto
each of seven coronal images, (A-G) oriented from anterior to posterior, respectively,
based on the location of the cells in the histological sections (see Methods). Each color
represents a separate clone that has been randomly numbered. All of the cells within a
clone are represented regardless of cell type. For illustrative purposes, the clones from
several brains, regardless of their location in the brain, have been placed on the left
side of each figure except bilateral clones which are shown on both sides. White matter
tracts are colored light grey and nuclei are clear. AC, anterior commissure; ALP,
nucleus ansae lenticularis posterior; AM, nucleus anterior medialis hypothalami; CP,
posterior commissure; CPa, commissura pallii; DIP, nucleus dorsointermedialis
posterior thalami; DLAl, nucleus dorsolateralis anterior thalami (lateral); DLAm,
nucleus dorsolateralis anterior thalami (medial); DLP, nucleus dorsolateralis posterior
thalami; DMA, nucleus dorsomedialis anterior thalami; DMP, nucleus dorsomedialis
posterior thalami; FPL, fasciculus prossencephali lateralis (lateral forebrain bundle);
GCt, substantia grisea centralis; GLdp, nucleus geniculatus lateralis, pars dorsalis;
GLv, nucleus geniculatus lateralis, pars ventralis; ICT, nucleus intercalatus thalami;
IH, nucleus inferioris hypothalami; LA, nucleus lateralis anterior thalami; LHy, lateral
hypothalamic nucleus; OC, optic chiasm; OM, tractus occipitomesencephalicus; OT,
optic tract; OV, nucleus ovoidalis; PHN, nuclus periventricularis hypothalami; PVN,
nucleus paraventricularis hypothalami; ROT, nucleus rotundus; SCE, stratum cellulare
externum; SL, nucleus septalis lateralis; SM, nucleus septalis medialis; T, nucleus
triangularis; Te, tectum; TeO, optic tectum; TT, tractus tectothalamicus; VLT, nucleus
ventrolateralis thalami.
Clones in the chick diencephalon
71
72
J. A. Golden and C. L. Cepko
Table 2. Cell type composition and dispersion of all multiple pick clones
*
*
B
B
B
B
B
B
B
B
B
B
N
0
2
2
8
0
0
0
0
0
3
0
1
1
1
1
0
0
1
1
0
3
1
1
6
5
0
0
1
0
0
3
2
0
3
1
0
0
8
0
0
0
0
0
1
3
5
18
0
0
0
4
1
0
1
0
4
7
0
0
0
2
G(RG)
0
0
18(1)
5
0(2)
1
8(1)
0
1
0(2)
4
0
1
1
4
3
0
10
10
1(4)
28
0(1)
5
2
4
2
13
0
5(3)
15
0
0
2
21
17
2(1)
0
0
1
1
2
6
3(2)
13(1)
3
0
0
0
0
0
2
6
5
6
3
7
1
3
0
1
0
U
APµm(%)
2
60(2)
0
120(4)
4 540(16)
1 660(20)
2
60(2)
2
120(4)
0 360(11)
2
60(2)
1
0
60(2)
1
300(9)
2 360(11)
1
0
60(2)
0 840(25)
1
120(4)
3
60(2)
0
120(4)
0
300(9)
0
0 480(15)
0
60(2)
0
300(9)
2 1020(31)
0
60(2)
0
60(2)
2
300(9)
1 480(15)
2 420(13)
2 1140(35)
0
60(2)
0 420(13)
2
60(2)
1
300(9)
0 1020(31)
0
120(4)
5 420(13)
2
300(9)
1 360(11)
2 360(11)
0
60(2)
1 360(11)
2 720(22)
0 360(11)
2 3 900(40)
5 900(40)
42
60(3)
7 480(20)
4
60(3)
1 4 540(25)
7
180(8)
2
0
0
1
0
22
10
4
1
1
n= 1 6 4 405(30) 3 4 0
Range
µm
%
ave%
DVµm(%)
1515(42)
61(3)
667(16)
1152(19)
2273(54)
152(3)
667(15)
212(5)
MLµm(%)
3030(74)
3152(83)
1667(70)
2273(71)
1758(48)
2667(80)
1212(36)
1970(52)
455(10)
2121(56)
91(2)
364(10)
2879(63)
1000(30)
91(2)
1758(39)
1152(25)
242(8)
697(14)
2667(70)
1121(30)
2000(73)
1424(39)
1364(50)
2697(70)
2727(45)
1515(36)
2212(46)
303(6)
2273(47)
667(16)
60(1)
1485(32)
1303(28)
1091(22)
1727(38)
394(10)
1394(30)
154(4)
3030(68)
1000(21)
2636(82)
2182(64)
2273(63)
2576(79)
1364(59)
1667(46)
1606(57)
2848(76)
1606(27)
1970(57)
2152(62)
3333(92)
3364(74)
2667(97)
2242(62)
636(13)
303(7)
394(8)
1364(31)
364(8)
818(23)
1788(87)
1455(44)
1970(54)
2636(76)
1000(22)
455(11)
1300(30)
185(4)
460(18)
185(4)
185(4)
850(30)
370(15)
3061(76)
1152(32)
2200(90)
750(27)
1850(95)
400(15)
N
G(RG)
U
APµm(%)
0
0
2
2
2
0
60(2)
1
0
1
60(2)
0
7(1)
3
0
5
0
120(4)
0
0
3
0
3
3
120(2)
0
0
1
0
8
0 300(11)
1
0
2
60(2)
0
4
1
120(5)
8
0
7
240(9)
1
0
1 120(5)
0
0
2
60(2)
0
0
2
60(2)
1
0(1)
0
60(2)
0
3
1
180(7)
1
0(1)
0 300(11)
*
1
2
0 840(32)
0
0
3 360(14)
1
0
3 300(11)
1
0
1
60(2)
1
2
3 360(14)
0
4
0
120(6)
0
2
0
60(3)
Many
Many
180(9)
0
2
4 1140(54)
11
14
2 4 480(23)
2
15
0 240(10)
0
2
1
60(2)
0
3
1
60(2)
2 20(2)
1 3 660(24)
0
0
2
210(7)
3
0(2)
1
60(2)
0
4
0
240(9)
8
1
5
120(4)
2
0
0
60(2)
1
0
2 360(13)
1
0(1)
1
240(9)
2
3(1)
4 900(33)
0
3
1
240(9)
0
2
3 840(31)
0
2
0
60(2)
0
0
3
2
0
0
60(2)
0
9
3 1020(38)
0
2
3
60(2)
3
0
0
60(2)
0
0
4
60(2)
0
0
2
60(2)
2000(98)
1750(67)
B
B
B
B
B
B
B
B
B
B
1 13(1)
1
0(1)
0
3
0
0
0
2
0
8
1
4
3
5
1
0(1)
0
0
Clones:
60-1140
2%-54%
11.10%
5-2939
1%-74%
18.90%
DVµm(%)
MLµm(%)
520(12)
273(7)
2881(75)
2515(75)
182(5)
636(28)
394(8)
455(14)
394(10)
515(12)
152(3)
303(7)
2394(53)
61(1)
91(2)
1576(63)
1606(76)
2515(73)
1061(78)
1455(55)
485(19)
545(29)
667(16)
1303(31)
2939(69)
2061(74)
212(5)
273(6)
2455(54)
50(1)
250(5)
550(13)
930(20)
930(20)
580(15)
2939(94)
91(8)
1606(65)
2455(79)
1303(41)
667(22)
2606(82)
93(26)
355(53)
2930(76)
2470(67)
2900(82)
2090(77)
600(22)
500(12)
300(7)
200(7)
300(7)
650(15)
0
200(7)
300(8)
1900(48)
80(3)
200(5)
150(4)
1900(72)
2350(80)
1850(63)
500(37)
800(26)
600(24)
0
1700(58)
500(19)
1900(62)
1950(70)
1900(70)
2350(84)
180(4)
1000(25)
150(5)
410(11)
1000(24)
300(7)
1750(66)
1470(54)
0
400(14)
1320(69)
1320(41)
2
5
2
3
1
3
2
11
4
1
n=121(44)
0-3550
0-98%
57.50%
The table is a complete listing of the cell types and the dispersion of all multiple pick clones (n=121). Dispersion is
given in µm and in parenthesis is the percent of the distance from anterior to posterior (AP), dorsal to ventral (DV), and
medial to lateral (ML) at the level of the brain where the clone was located. If dispersion distances are not listed, this
indicates that either these clones arose from the lateral ventricle, or includes cells from AP− areas that also gave the
same insert and thus the full extent of dispersion could not be accurately determined. An asterisk identifies the clones
that showed a greater dorsal-to-ventral spread compared to the medial to lateral. B indicates that the clone is bilateral.
N, neurons; G, glia; RG, radial glia; U, unidentified cell.
Clones in the chick diencephalon
Table 3. Clones crossing neuromeric boundaries
Clone type
n
Cross
Don’t cross
Could not
determine
Unilateral
Bilateral
90
18
5 (6%)
4 (22%)
47 (52%)
1 (6%)
38 (42%)
13 (72%)
108
9 (8%)
48 (44%)
51 (47%)
Total
The number (and percent) of clones that were observed in >1 neuromere
(crossed) was determined by comparing the location of all members of a
clone to the boundaries proposed by Figdor and Stern (1993). Only clones
with siblings clearly located in more than one neuromere were included as
crossing. All clones that had cells near borders were classified as “could not
determine”.
dorsal border, are not included on the illustration). Other cells
from these clones included neurons and glia that were within
the body of the diencephalon. A total of 5 of the 275 (2%)
clones appeared to have arisen from the lateral ventricles,
making the contribution of progenitors from the lateral ventricles to the final cell numbers in the diencephalon relatively
small during the time period studied. No specific composition
of cell types within these clones was found (Table 2).
Another unusual clone type had siblings in both the diencephalon and the mesencephalon (tectum) (Fig. 3, clone 12 and
Fig. 4, clone 10). Since only a few AP+ cells were analyzed
from the mesencephalon, and no cells from the telencephalon,
an estimation of the frequency of clones that cross from the
diencephalon to adjacent brain regions cannot be made.
However, the presence of siblings in both the diencephalon and
mesencephalon indicate that the earliest subdivisions of the
CNS do not form absolute lineage boundaries, as all infections
were made after the prosencephalon and mesencephalon
boundaries were established. One of the clones with cells in
the diencephalon and mesencephalon appeared to have its
origin in the lateral ventricle. Thus it is possible that cells
generated in either the lateral ventricle or third ventricle can
migrate between these two brain regions. Examination of
whole-mount E8 chick brains showed a relatively simple and
direct possible pathway for the migration of cells into these
two structures (diagramed in Fig. 6, cell C).
The pattern of radial glia in the diencephalon
The most common pattern of clonal dispersion in the diencephalon was from medial to lateral with a progressively more
ventral location as members of the clone migrated more
laterally. One common pathway for migrating newborn
neurons in the CNS is along radial glial fibers. To investigate
whether radial glia could account for the most common pattern
of dispersion seen here, DiI was used to label radial glial cells.
Fig. 7 shows the pattern of fibers revealed by the DiI staining
in the diencephalon. From the third ventricle, radial glial fibers
course laterally and ventrally in a pattern paralleling the distribution of sibling cells. Clones that did not disperse in this
common pattern may be using other guides that were not identified in this study.
Radial glial cells were also noted in the lateral ventricle
extending into the diencephalon (Fig. 7). The processes of
these cells showed a general downward trend as they moved
from the lateral ventricle into the substance of the diencephalon. This pattern reflects the distribution of cells found
in a few of the clones that appeared to originate in the germinal
73
zone of the lateral ventricle, supporting the finding that progenitors located in the lateral ventricle can contribute to the
diencephalon.
Analysis of alkaline phosphatase negative (AP−)
areas
A potential problem in retroviral mediated lineage analysis is
the failure to detect infected cells. For example, transcription
from the LTR promoter could be weak or absent or the AP
protein could be mutant or unstable. To investigate this issue,
the entire diencephalon from one heavily infected brain was
systematically analyzed for the presence of non-expressing
cells. 263 regions were isolated from histochemically negative
areas (i.e. no purple cells). These picks were defined as either
small (n=52), approximately the size of the picks from single
AP+ cells, or large (n=211). The large picks involved removing
from 1/8 to 1/4 of a section of diencephalon from a slide and
subjecting the tissue to PCR, as was done with the AP+ areas.
For the small AP− picks, an insert was amplified from 19
(37%) picks. Sequence analysis of the inserts from the small
picks revealed a single insert in 17 of the 19 amplified
products, and multiple inserts in 2 of the 19. The large picks
had a higher recovery rate for inserts after amplification
(178/211, 84%). Sequence analysis revealed 133 of the 178
inserts were actually multiple sequences and only 25 of the 178
inserts were single sequences. 20 amplified products yielded
no sequencing product. The higher recovery rate and presence
of multiple inserts in a greater proportion of the large picks
was attributed to the large tissue volumes collected. In some
cases, the sequences from the histochemically negative regions
were unique indicating that entire clones either did not express
active PLAP or turned off the expression at some time prior to
E18. Among other clones (5/93, 5.4%), a subset of cells
exhibited alkaline phosphatase activity while other cells did not
at the time of analysis. In addition, 25 samples from uninfected
brains were analyzed and an insert was never recovered from
these brains. Several explanations for the AP− cells exist. Since
we completed this study, we observed that some cells, particularly some neurons, require a staining time longer than used
here to become positive by light microscopy (J. Lin, F. Szele,
J. Zitz, J. Golden and C. Cepko, unpublished data). Studies
from other brain regions have also shown that some cells can
turn off AP activity (Halliday and Cepko, 1992) while other
cells appear never to exhibit AP activity. Although we cannot
determine the contribution of any one mechanism in our data,
it remains likely that a combination of these factors play a role
in recovering viral genomes from AP− areas.
DISCUSSION
We have analyzed lineal relationships in the chick diencephalon using a highly complex retroviral library. Although
the rodent has served as a model organism for lineage analysis
in the cerebral cortex (Price et al., 1987; Walsh and Cepko,
1988, 1992; Austin and Cepko, 1990; Parnavelas et al., 1991;
Levison et al., 1993) and retina (Turner and Cepko, 1987;
Turner et al., 1990), technical limitations prevent the use of
rodents for studying structures such as the diencephalon that
develop before E12-E13, the earliest age that the neuroepithelium is accessable for viral injections. The timing of
74
J. A. Golden and C. L. Cepko
no characteristics that distinguished them from the clones of
the animals injected later, the data from all 7 animals were
pooled.
Fig. 6. Generation of bilateral clones and clones that span the
diencephalon and mesencephalon. (A) A 60 µm coronal section at
the level of the anterior commissure (AC) of an E9.5 chick brain
injected with CHAP at stage 17 is shown. A nearly continuous
population of cells appears to trace back to an origin in the
ventricular zone of the third ventricle. Migrating cells, with long
leading processes, appear to move away from the ventricle, then
appear to turn and enter the AC. A bundle of fibers crossing the
midline clearly defines the dorsal and ventral limits of the AC. (B) A
whole-mount of an E8 chick brain viewed from the dorsal side with
the midline opened to display the ventricular surfaces. The left side
of the brain was traced and the major subdivisions colored and
labeled. The position of the anterior and posterior commissures (AC
and PC, respectively) is shown on the diagram (arrows) as possible
routes for cells to cross the midline. Cell A depicts one potential
route in which a cell could migrate around one end of the third
ventricle. Although this is only shown at the anterior end, there is a
continuous surface from the anterior end to the posterior limit of the
diencephalon, along the ventral surface. Cell B illustrates a model in
which a midline progenitor cell is the source of cells that migrate
into each side of the diencephalon. Cell C shows where a clone
might originate that spans the diencephalon and the mesencephalon.
Scale bar 200 µm.
injection of the retrovirus was selected according to the time
when neuromeric boundaries were established in the diencephalon (Figdor and Stern, 1993). 5 of the 7 chicks were
injected at stage 17 after the major boundaries were established
and the other two were injected prior to the formation of diencephalic boundaries. Since the two brains infected at stage 1011 contained a total of only 9 clones, and these clones showed
Neuromeres and patterning of the diencephalon
The developmental mechanisms governing segregation of the
diencephalon into nuclei remain largely unknown. One hypothesis is that the diencephalon is parceled into segments, each
giving rise to a set of functionally related nuclei, analogous to
the rhombomeres in the hindbrain. Two models have been
proposed for parcelling the diencephalon into segments. The
first model divides the diencephalon into three units (prosomeres) and has been most extensively studied in the mouse
(see Rubenstein and Puelles, 1994 and Rubenstein et al., 1994
for reviews). In the second model, four units (neuromeres)
have been identified, with the most caudal two in this second
model corresponding to the first prosomere in the first model
(Figdor and Stern, 1993). Since the second model of four neuromeres has been proposed for the chick and the first primarily
established in the mouse, we have chosen to use the neuromeric
model in analysis of the data.
Figdor and Stern (1993), utilizing single cell injection and
DiI and DiO labeling techniques, found that clones did not
cross the morphological boundaries between the neuromeres of
the diencephalon by stage 25 in development of the chick.
These findings are similar to those of lineage analysis in the
hindbrain rhombomeres. In contrast, however, using the
boundaries of neuromeres defined by Figdor and Stern in the
stage 40 chick (see Fig. 2 in Figdor and Stern, 1993) at least
8% (a minimum defined by conservative criteria) (see Table 3)
of the clones in the current study cross the boundaries of neuromeres. Furthermore, in the prosomeric model (Rubenstein
and Puelles, 1994; Rubenstein et al., 1994), the hypothalamus
and thalamus are derived from distinct prosomeres, thus the
clones spanning these two regions identified in this paper
would also have to be considered boundary crossers (see
below). In the calculations performed for Table 3, hypothalamic-thalamic clones were not included as crossing boundaries.
Several explanations exist for the difference in dispersion
between the two studies. The first, and most likely, is that the
tracer labeling studies were analyzed after only 48-72 hours,
whereas in the current study, embryos were harvested 16 days
beyond the time of infection. Thus it seems likely that progenitor cells give rise to daughters that are initially restricted
to within a single neuromere, but that later some siblings
escape restrictions and move according to other mechanisms
or cues. Alternatively, a relatively small subset of clones cross
neuromeric boundaries early in development; such a subset of
clones may have been missed by the techniques used in the
previous study. This scenario is similar to the recognition of a
small percentage of clones that violate rhombomere boundaries
early in development (Birgbauer and Fraser, 1994).
We have also analyzed retrovirally marked clones in the
ventral forebrain, focusing on the hypothalamus, in brains
harvested at E8-E10 (Arnold-Aldea and Cepko, 1995).
Approximately 96% of the clones were simple radial columns,
which appeared to respect neuromeric boundaries. However,
approximately 4% of the retrovirally marked clones were
widely dispersed, with distinctive patterns of dispersion at E8E10, including bilaterally symmetric clones in the hypothalamus. Ongoing experiments in our laboratory are seeking to
Clones in the chick diencephalon
75
Fig. 7. DiI labeling of radial glia.
(A) Overview of a DiI-labeled, 200 µm
section through the diencephalon of E15
brain. The third (3 V) and lateral (LV)
ventricles of a fixed embryo were filled with
a DiI solution; periventricular regions show
bright labeling. Single fibers are seen
radiating into the body of the diencephalon
from both ventricular locations. (B) A
schematic diagram of (A) with a tracing of
several of the radial glia arising along the
third and lateral ventricles. The two boxes
indicate the location of both C and D. (C) A
view at higher magnification from the
region where the radial glial fibers
decussated. Fibers are seen coming from the
lateral ventricle (upper left) and third
ventricle (upper right). (D) The radial glial
fibers located along the third ventricle
showed a slight ventral slope as they
projected from medial to lateral. Scale bar
1000 µm for A; 125 µm for C; and 250 µm
for D.
identify the timing of the dispersion that occurs in the majority
of clones reported in the current study, which should clarify
the relationships of the patterns reported in all of the aforementioned studies
Clonal dispersion and the generation of
diencephalic nuclei
Data from birthdating studies using [3H]thymidine have been
used as the basis for a proposal of gradients of cell generation
and other properties of the germinal zone of the third ventricle.
A study in Xenopus led to a proposal of an overall gradient
from ventral-lateral to dorsal-medial in the generation of
neurons in the diencephalon (Tay and Straznicky, 1982). In
mammals, where the majority of work on the diencephalon has
been conducted, a detailed analysis of the birthdates of each
nucleus or small cluster of nuclei has been reported (Angevine,
1970; Altman and Bayer, 1978a-c, 1979a-c, 1988a-c, 1989ac). Together these studies have indicated that some nuclei
within the diencephalon have fairly short periods of genesis
while the neurons of other nuclei are born over a longer period
of time. Furthermore, the birthdays of particular nuclei did not
precisely correlate with the anatomical location of the nuclei.
These birthdating studies led to the hypothesis that the
germinal epithelium is a mosaic of patches, each giving rise to
neurons in a nucleus or specific region of the diencephalon
(Altman and Bayer, 1988a). The clonal distributions reported
here included clones where the sibling cells tended to cluster
in one or a small group of nuclei of the diencephalon. These
clones would be consistent with the [3H]thymidine data, supporting the idea that progenitors within the ventricular zone of
the third ventricle generate specific regions in the diencephalon. However, other members of some of these clones as
well as many other clones were widely dispersed, indicating
that most progenitors are not dedicated to producing cells for
one nucleus or a small group of nuclei.
Since siblings did not strictly populate one region of the
diencephalon, we wanted to investigate how clonally related
cells disperse. To examine one possible mechanism, the architecture of the radial glia in the diencephalon was explored
using DiI labeling. The labeling of radial glial fibers arising
from the third ventricle revealed that fibers projected into the
diencephalon from the medial-to-lateral direction, with a
dorsal-to-ventral slope, paralleling the most common patterns
of distribution within clones. It is worth noting that not all
clones showed this pattern of dispersion. Several clones
showed marked displacement in the dorsal-ventral plane with
relatively little dispersion from medial to lateral. This suggests
that other mechanisms for clonal dispersion in the dorsalventral plane exist in the diencephalon. In addition, a
mechanism that could explain the marked anterior-to-posterior
dispersion of clonally related cells is currently unknown.
Generation of cell types within clones
The majority of large clones and approximately 50% of small
clones contained more than one cell type. This indicates that
progenitor cells are frequently multipotent, capable of
producing both neurons and glia, similar to progenitors within
the rodent retina (Turner and Cepko, 1987; Turner et al., 1990)
and telencephalon (Price and Thurlow, 1988; Walsh and
Cepko, 1992; Levison et al., 1993; Reid et al., 1995) and the
chick tectum (Galileo et al., 1990; Gray and Sanes, 1992). The
distribution of cell types indicates that most progenitors
generate relatively small numbers of neurons and large
numbers of glia (see Table 2). This was not always the case,
as several clones had large numbers of neurons with relatively
few, or no, glia. The large number of glia in many clones, along
with the small numbers of neurons in some of these same
clones, is consistent with a multipotent progenitor that divides
and first gives rise to one neuron at each cell division. Multiple
cell divisions with one neuronal daughter at each division
would account for the clones with multiple neurons. One or
more mitotic daughters from these same progenitors could
continue to proliferate and later produce one or many glial
cells. Analysis of single cell clones (see below) supports this
76
J. A. Golden and C. L. Cepko
order of genesis, as does [3H]thymidine birthdating (Angevine,
1970). The observation of two cell clones with a radial glial
cell and a neuron might suggest that progenitor cells remain
multipotent up to the last cell division, although cell death
and/or inefficiency in amplification or sequencing could result
in missing cells from such clones.
Analysis of the single cell clones also provides insights into
the timing of genesis of different cell types in the diencephalon.
The 118 single cell clones could be derived by one of several
mechanisms, which are not mutually exclusive. As retroviruses
integrate during the M phase of the cell cycle, integration into
a single chromosome during M-phase means that only one
daughter cell from the first cell division will be marked (Roe
et al., 1993). If this daughter cell does not re-enter the cell
cycle, it will result in a 1-cell clone. Alternatively, clones of
greater than 1 cell would appear as single cell clones if some
siblings were AP−, died, did not amplify with PCR, or did not
sequence. However, it appears that no particular cell type
amplified or sequenced preferentially and thus these potential
problems would not bias the data set. An estimation of the
number of clones that contained AP+ cells and AP− cells is
approximately 5% (see Results and unpublished data).
Therefore, single cell clones can be interpreted as an approximation of cell birthdays.
27% of all single cell clones were neurons, indicating that
neurons were being born near the time of injection of the retrovirus at stage 16-17 in development. No single cell clones of
neurons were found in the two brains analyzed from the stage
10-12 injections, but only 9 clones were analyzed, making the
data set too small to permit a definite conclusion as to whether
neurons were being born at this time. In contrast, glial cells
were rarely born at the stages of development that injections
were performed. Despite their high frequency in the total data
set, only 6% of the single cell clones comprised glial cells
(other than radial glia). This is consistent with classical
[3H]thymidine birthdating studies in the diencephalon of many
other species (Altman and Bayer, 1979a-c, 1988b,c, 1989a-c;
Tay and Straznicky, 1982). The fact that there were any single
cell glial clones is of some interest. The siblings of these cells
may have been missed due to the inefficiency of the PCR
and/or sequencing, or death. Alternatively, the single cell glial
clones may have arisen from radial glia. Several lines of
evidence have indicated that radial glia transform into astrocytes later in development (Schmechel and Rakic, 1979; Levitt
and Rakic, 1980; Pixley and De Vellis, 1984; Voigt, 1989;
Cullican et al., 1990; Gray and Sanes, 1992). A large percentage of radial glial clones were in fact single cell clones, indicating that they were born near the time of the retroviral
infection, consistent with birthdating of radial glia in other
studies (Levitt and Rakic, 1980; Misson et al., 1988).
Unusual clone types
Several unusual clone types were identified in this study. One
type was bilateral, with sibling cells on either side of the third
ventricle, but unlike the bilateral clones observed in the diencephalon from another study (Arnold-Aldea and Cepko, 1995),
the bilateral clones in this study were not symmetric. At least
three possible migration pathways could result in bilateral
clones. A cell could cross through one of the major commissures (see Fig. 6), which include the anterior, posterior and
supraoptic decussation dorsalis. Analysis at E18 has led to
identification of cells within each of these commissures.
However, as yet, clonally related cells on each side of the diencephalon and within one of these commissures have not been
observed within the infected brains in the current data set. A
second pathway to the generation of bilateral clones is for cells
to migrate around the anterior, inferior or posterior limits of
the diencephalon (Fig. 6B, cell A). We have not seen this type
of migration in the material that we have examined, but a more
extensive examination of younger brains would be required to
exclude these routes. A third possibility is that a population of
progenitor cells exists along the midline that is capable of generating siblings that can migrate to populate both sides of the
diencephalon (Fig. 6B, cell B). Similar midline cells are
present in invertebrates (Crews et al., 1988; Nambu et al.,
1990, 1991; Crews et al., 1992), and in vertebrates such as the
zebrafish (Hatta et al., 1991), and have been proposed as the
source of bilaterally symmetrical clones in the chick diencephalon seen at E8 (Arnold-Aldea and Cepko, 1995). We
have no direct evidence from the current data set for this
mechanism of generating bilateral clones.
One rare subset of clones had their origins in the lateral
ventricle and descendants in the diencephalon. Radial glia with
their cell bodies in the wall of the lateral ventricle and
processes radiating medially into the diencephalon were
observed to have sibling cells, both glial and neuronal, within
the body of the diencephalon. Several other studies have
suggested that progenitors from the ventricular zone of the
lateral ventricle could provide cells to specific nuclei in the
diencephalon (Rakic and Sidman, 1969; Altman and Bayer,
1978b) Although no specific nuclei were populated by the
clones originating in the lateral ventricles, the few clones
observed in this study preclude a definitive analysis.
Surprisingly, clonally related cells in the diencephalon and
the mesencephalon were observed. Since the first cerebral
vesicles that form are the prosencephalon, the mesencephalon
and the rhombencephalon, the presence of clonally related cells
in the diencephalon and mesencephalon indicates that even
these very early segments of the nervous system do not form
absolute lineage boundaries. Such clones are undoubtably
more frequent than reported here as AP+ cells in all regions of
the brain were not systematically analyzed. However, if they
are rare, it is not clear what meaning one can ascribe to them.
They could simply be due to progenitor cells that are situated
exactly on the border between the two areas. Alternatively,
they could play a more meaningful role in patterning or connections between brain areas. AP+ cells were not sampled from
the telencephalon and therefore no clones would have been
identified that crossed from the diencephalon into the telencephalon. However, clones were found in the hypothalamus
and thalamus. In the model where prosomeres form the
segments of the brain, the thalamus and hypothalamus are in
distinct prosomeres, including the hypothalamus arising from
the ventral telencephalon (Puelles and Rubenstein, 1993;
Rubenstein et al., 1994; Rubenstein and Puelles 1994). Clones
that occupy both the thalamus and hypothalamus thus would
cross the diencephalon-telencephalon boundary if the model
proposed for prosomeres held for the chick.
In summary, lineage analysis has been conducted in the
diencephalon of the chick using a complex retroviral library.
We have characterized the patterns of dispersion of clones and
found several novel distributions. Clones were found to spread
Clones in the chick diencephalon
in all directions. Dispersion of clones from medial to lateral
and from dorsal to ventral was found to parallel the pattern of
radial glial fibers, suggesting that migration along radial glial
fibers is one mechanism for clonal dispersion. Furthermore,
siblings were found to occupy multiple nuclei, including nuclei
derived from more than one diencephalic neuromere. Characterization of the cell types within clones demonstrated a high
frequency of clones containing neurons and glia, supporting
the hypothesis that progenitor cells in the chick diencephalon
are multipotential. In addition to the common clones, unusual
clones showed bilateral dispersion, origins in the lateral
ventricle and/or siblings in both the diencephalon and mesencephalon. The significance of these rare patterns of dispersion
remains unknown.
We would like to thank Suzanne Bruhn, John Lin and Francis Szele
for critically reviewing the manuscript and for making many helpful
suggestions. We are grateful to Julie Zitz for technical assistance.
Supported by grants from the NIH (NS-01664 to J. A. G.; NS-23021
to C. L. C.) and the Howard Hughes Medical Institute.
REFERENCES
Altman, J. and Bayer, S. A. (1978a). Development of the diencephalon in the
rat. I. Autoradiographic study of the time of origin and settling patterns of
neurons of the hypothalamus. J. Comp. Neurol. 182, 945-971.
Altman, J. and Bayer, S. A. (1978b). Development of the diencephalon in the
rat. II. Correlation of the embryonic development of the hypothalamus with
the time of origin of its neurons. J. Comp. Neurol. 182, 973-993.
Altman, J. and Bayer, S. A. (1978c). Development of the diencephalon in the
rat. III. Ontogeny of the specialized ventricular linings of the hypothalamic
third ventricle. J. Comp. Neurol. 182, 995-1015.
Altman, J. and Bayer, S. A. (1979a). Development of the diencephalon in the
rat. IV. Quantitative study of the time of origin of neurons and the
internuclear chronological gradients in the thalamus. J. Comp. Neurol. 188,
455-471.
Altman, J. and Bayer, S. A. (1979b). Development of the diencephalon
in the rat. V. Thymidine-radiographic observations on internuclear
and intranuclear gradients in the thalamus. J. Comp. Neurol. 188, 473499.
Altman, J. and Bayer, S. A. (1979c). Development of the diencephalon in
the rat. VI. Re-evaluation of the embryonic development of the thalamus on
the basis of thymidine-radiographic datings. J. Comp. Neurol. 188, 501524.
Altman, J. and Bayer, S. A. (1988a). Development of the rat thalamus: I.
Mosaic organization of the thalamic Neuroepithelium. J. Comp. Neurol. 275,
346-377.
Altman, J. and Bayer, S. A. (1988b). Development of the rat thalamus: II.
Time and site of origin and settling pattern of neurons derived from the
anterior lobule of the thalamic neuroepithelium. J. Comp. Neurol. 275, 378405.
Altman, J. and Bayer, S. A. (1988c). Development of the rat thalamus: III.
Time and site of origin and settling pattern of neurons of the reticular nucleus.
J. Comp. Neurol. 275, 406-428.
Altman, J. and Bayer, S. A. (1989a). Development of the rat thalamus: IV. The
intermediate lobule of the thalamic neuroepithelium, and the time and site of
origin and settling pattern of neurons of the ventral nuclear complex. J.
Comp. Neurol. 284, 534-566.
Altman, J. and Bayer, S. A. (1989b). Development of the rat thalamus: V. The
posterior lobule of the thalamic neuroepithelium and the time and site of
origin and settling pattern of neurons of the medial geniculate body. J. Comp.
Neurol. 284, 567-580.
Altman, J. and Bayer, S. A. (1989c). Development of the rat thalamus: VI. The
posterior lobule of the thalamic origin and settling pattern of neurons of
the lateral geniculate and lateral posterior nuclei. J. Comp. Neurol. 284, 581601.
Angevine, J. B. (1970). Time of neruon origin in the diencephalon of the
mouse: an autoradiographic study. J. Comp. Neurol. 139, 129-188.
Arnold-Aldea, S. and Cepko, C. L. (1995) Dispersion patterns of clonally
77
related cells during development of the ventral forebrain. Dev. Biol. in
press.
Austin, C. P. and Cepko, C. L. (1990). Cellular migration patterns in the
developing mouse cerebral cortex. Development 110, 713-732.
Bergquist, H. (1952). Studies on the cerebral tube in vertebrates. The
neuromeres. Acta Zool. 33, 117-187.
Birgbauer, E. and Fraser, S. (1994). Violation of cell lineage restriction
compartments in the chick hindbrain. Development 120, 1347-1356.
Bulfone, A., Kim, H. J., Puelles, L., Porteus, M. H., Grippo, J. F. and
Rubenstein, J. L. (1993a). The mouse Dlx-2 (Tes-1) gene is expressed in
spatially restricted domains of the forebrain, face and limbs in midgestation
mouse embryos [published erratum appears in Mech Dev 1993
Aug;42(3):187]. Mech. Dev. 40, 129-140.
Bulfone, A., Puelles, L., Porteus, M., Frohman, M., Martin, G. and
Rubenstein, J. (1993b). Spatially restricted expression of Dlx-1, Dlx-2 (Tes1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines
potential transvere and longitudinal segmentation boundaries. J
Neuroscience 13, 3156-3172.
Crews, S., Franks, R., Hu, S., Matthews, B. and Nambu, J. (1992).
Drosophila single-minded gene and the molecular genetics of CNS midline
development. J. Exp. Zool. 261, 234-244.
Crews, S. T., Thomas, J. B. and Goodman, C. S. (1988). The drosophila
single-minded gene encodes a nuclear protein with sequence similarity to the
per gene product. Cell 52, 143-151.
Cullican, S., Baumrind, N., Yamamoto, M. and Pearlman, A. (1990).
Cortical radial glia: identification in tissue culture and evidence for their
transformation into astrocytes. J Neurosci. 10, 684-692.
Fekete, D. and Cepko, C. (1993). Replication-competent retroviral vectors
encoding alkaline phosphatase reveal spatial restriction of viral gene
expression/transduction in the chick embryo. Mol Cell Biol. 13, 26042613.
Fekete, D., Perez-Miguelsanz, J., Ryder, E. and Cepko, C. (1995). Clonal
analysis in the chicken retina reveals tangential dispersion of clonally related
cells. Dev. Biol. In press.
Figdor, M. and Stern, C. (1993). Segmental organization of embryonic
diencephalon. Nature 363, 630-634.
Frank, E. and Sanes, J. R. (1991) Lineage of neurons and glia in chick dorsal
root ganglia: analysis in vivo with a recombinant retrovirus. Development
111, 895-908.
Frantz, G., Bohner, A., Akers, R. and McConnell, S. (1994a). Regulation of
the POU domain gene SCIP during cerebral cortical development. J.
Neurosci. 14, 472-485.
Frantz, G., Weimann, J., Levin, M. and McConnell, S. (1994b). Otx1 and
Otx2 define layers and regions in developing cerebral cortex and cerebellum.
J. Neurosci. 14, 5725-5740.
Fraser, S., Keynes, R. and Lumsden, A. (1990). Segmentation in the chick
embryo hindbrain is defined by cell lineage restrictions. Nature 344, 431435.
Galileo, D., Gray, G., Owens, G., Majors, J. and Sanes, J. (1990). Neurons
and glia arise from a common progenitor in chicken optic tectum:
demonstration with two retroviruses and cell type-specific antibodies. Proc.
Natl Acad. Sci. USA 87, 458-462.
Golden, J. and Cepko, C. (1995). Lineage analysis using retroviral vectors.
American Zoologist In press.
Golden, J., Fields-Berry, S. and Cepko, C. (1995). Construction and
charaterization of a highly complex retroviral library for lineage analysis.
Proc. Natl Acad. Sci. USA 92, 5704-5708.
Gray, G. and Sanes, J. (1992). Lineage of radial glia in the chicken optic
tectum. Development 114, 271-283.
Guthrie, S. (1995). The status of the neural segment. Trends in Neurosciences
18, 74-79.
Guthrie, S. and Lumsden, A. (1991). Formation and regeneration of
rhombomere boundaries in the developing chick hindbrain. Development
112, 221-230.
Guthrie, S. and Lumsden, A. (1992). Motor neuron pathfinding following
rhombomere reversals in the chick embryo hindbrain. Development 114,
663-673.
Guthrie, S., Muchamore, I., Kuroiwa, A., Marshall, H., Krumlauf, R. and
Lumsden, A. (1992). Neuroectodermal autonomy of Hox-2.9 expression
revealed by rhombomere transpositions. Nature 356, 157-159.
Halliday, A. L. and Cepko, C. L. (1992). Generation and migration of cells in
the developing striatum. Neuron 9, 15-26.
Hamburger, V. and Hamilton, H. (1951). A series of normal stages in the
development of the chick embryo. J Morphol. 88, 49-91.
78
J. A. Golden and C. L. Cepko
Hatta, K., Kimmel, C., Ho, R. and Walker, C. (1991). The cyclops mutation
blocks specification of the floor plate of the zebrafish central nervous system.
Nature 350, 339-341.
Herrick, C. (1910). The morphology of the forebrain in amphibia and reptilia.
J. Comp. Neurol. 20, 215-348.
His, W. (1893). Vorschlage zur einteilung des gehirms. Arch. Anat.
Entwicklungsgesch. 17, 172-179.
Hunt, P., Gulisano, M., Cook, M., Sham, M.-H., Faiella, A., Wilkinson, D.,
Boncinelli, E. and Krumlauf, R. (1991). A distinct Hox code for the
branchial region of the vertebrate head. Nature 353, 861-864.
Jones, E. G. (1985). The Thalamus. New York: Plenum Press.
Kappers, C. U. A., Huber, G. C. and Crosby, E. C. (1960). The Comparitive
Anatomy of the Nervous System in Vertebrates. New York: MacMillan Co.
Keynes, R. and Lumsden, A. (1990). Segmentation and the origin of regional
diversity in the vertebrate central nervous system. Neuron 4, 1-9.
Keyser, A. (1972). The development of the diencephalon of the Chinese
hamster. Acta Morphologica Neerlando-Scandinavica 9, 379.
Khulenbeck, H. (1973). Overall morphologic pattern. In The Central Nervous
System of Vertebrates. Basel: Karger.
Kuenzel, W. and Masson, M. (1988). A stereotaxic atlas of the brain of the
chick (Gallus Domesticus). Baltimore, MD: The Johns Hopkins University
Press.
Leifer, D., Golden, J. and Kowall, N. (1994). Myocyte-specific enhancer
binding factor 2C expression in human brain development. Neuroscience 63,
1067-1079.
Levison, S., Chuang, C., Abramson, B. and Goldman, J. (1993). The
migrational patterns and developmental fates of glial precursors in the rat
subventricular zone are temporally regulated. Development 119, 611-622.
Levitt, P. and Rakic, P. (1980). Immunoperoxidase localization of glial
fibrillary acidic protein in radial glial cells and astrocytes of the developing
rhesus monkey brain. J. Comp. Neurol. 193, 815-840.
Lumsden, A. (1990). The cellular basis of segmentation in the developing
hindbrain. Trends in Neurosciences 13, 329-335.
Lumsden, A., Clarke, J., Keynes, R. and Fraser, S. (1994). Early phenotypic
choices by neuronal precursors, revealed by clonal analysis of the chick
embryo hindbrain. Development 120, 1581-1589.
Lumsden, A. and Keynes, R. (1989). Segmental patterns of neuronal
development in the chick hindbrain. Nature 337, 424-428.
Martinez, S. and Alvarado-Mallart, R-M. (1989). Transplanted
mesencephalic quail cells colonize selectively all primary visual nuclei of
chick diencephalon: a study using heterotopic transplants. Dev. Brain
Research 47, 263-274.
Misson, J. P., Edwards, M. A., Yamamoto, M. and Caviness, V. S. J. (1988).
Mitotic cycling of radial glial cells of the fetal murine cerebral wall: a
combined autoradiographic and immunohistochemical study. Brain
Research 466, 183-190.
Nambu, J. R., Franks, R. G., Hu, S. and Crews, S. T. (1990). The singleminded gene of Drosophila is required for the expressionof genes important
for the development of CNS midline cells. Cell 63, 63-75.
Nambu, J. R., Lewis, J. O., Wharton, K. A. and Crews, S. T. (1991). The
Drosophilia single-minded gene encodes a helix-loop-helix protein that acts
as a master regulator of CNS midline development. Cell 67, 1157-1167.
O’Leary, D. and Koester, S. (1993). Development of projection neuron types,
axon pathways, and patterned connections of the mammalian cortex. Neuron
10, 991-1006.
Orr, H. (1887). Contributions to the embryology of the lizard. J. Morphol. 1,
311-372.
Parnavelas, J. G., Barfield, J. A., Franke, E. and Luskin, M. B. (1991).
Separate progenitor cells give rise to pyramidal and nonpyramidal neurons in
the rat telencephalon. Cerebral Cortex 1, 463-468.
Pixley, S. and De Vellis, J. (1984). Transition between immature radial glia
and mature astrocytes studied with a monoclonal antibody to vimentin. Dev.
Brain Research 15, 201-209.
Price, J. and Thurlow, L. (1988). Cell lineage in the rat cerebral cortex:
a study using retroviral-mediated gene transfer. Development 104, 473482.
Price, J., Turner, D. and Cepko, C. (1987). Lineage analysis in the vertebrate
nervous system by retrovirus-mediated gene transfer. Proc. Natl Acad. Sci.
USA 84, 156-160.
Puelles, L., Amat, J. and Martinez-de-la-Torre, M. (1987). Segment-related,
mosaic neurogenetic pattern in the forebrain and mesencephalon of early
chick embryos: I. Topography of AChE-positive neuroblasts up to stage
HH18. J. Comp. Neurology 266, 247-268.
Puelles, L. and Rubenstein, J. L. (1993). Expression patterns of homeobox
and other putative regulatory genes in the embryonic mouse forebrain
suggest a neuromeric organization. Trends in Neurosciences 16, 472-479.
Puelles, L. and Zabala, C. (1982). Evidence for a concealed neuromeric
pattern in the disposition of retinorecipient grisea in the avain diencephalon.
Neurosci. Lett. Suppl. 10, 396-397.
Rakic, P. and Sidman, R. (1969). Telencephalic origin of pulvinar neurons in
the fetal human brain. Zeitschrift fur Anatomie und Entwicklungsgeschichte
129, 53-82.
Reid, C. B., Liang, I., and Walsh, C. (1995) Systematic widespread clonal
organization in cerebral cortex. Neuron 15, 299-310.
Roe, T., Reynolds, T., Yu, G. and Brown, P. (1993). Integration of murine
leukemia virus DNA depends on mitosis. EMBO J. 12, 2099-2108.
Rubenstein, J. L., Martinez, S., Shimamura, K. and Puelles, L. (1994). The
embryonic vertebrate forebrain: the prosomeric model. Science 266, 578580.
Rubenstein, J. L. and Puelles, L. (1994). Homeobox gene expression during
development of the vertebrate brain. Current Topics in Developmental
Biology 29, 1-63.
Ryder, E. F. and Cepko, C. L. (1994). Migration patterns of clonally related
granule cells and their progenitors in the developing chick cerebellum.
Neuron 12, 1011-28.
Schmechel, D. and Rakic, P. (1979). A Golgi study of radial glial cells in
developing monkey telencephalon: morphogenesis and transformation into
astrocytes. Anat. Embryol. 156, 115-152.
Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A. and Boncinelli,
E. (1992). Nested expression domains of four homeobox genes in developing
rostral brain. Nature 358, 687-690.
Tay, D. and Straznicky, C. (1982). The development of the diencephalon
in Xenopus. An autoradiographic study. Anatomy & Embryology 163, 371388.
Turner, D. L. and Cepko, C. L. (1987). A common progenitor for neurons and
glia persists in rat retina late in development. Nature 328, 131-136.
Turner, D. L., Snyder, E. Y. and Cepko, C. L. (1990). Lineage-independent
determination of cell type in the embryonic mouse retina. Neuron 4, 833-845.
Voigt, T. (1989). Development of glial cells in the cerebral wall of ferrets:
direct tracing of their transformation from radial glia into astrocytes. J.
Comp. Neurol. 289, 74-88.
Walsh, C. and Cepko, C. L. (1988). Clonally related cortical cells show
several migration patterns. Science 241, 1342-1345.
Walsh, C. and Cepko, C. L. (1992). Widespread dispersion of neuronal clones
across fuctional regions of the cerebral cortex. Science 255, 434-440.
Walsh, C. and Cepko, C. L. (1993). Clonal dispersion in proliferative layers of
developing cerebral cortex. Nature 362, 632-635.
Walsh, C., Cepko, C. L., Ryder, E. F., Church, G. M. and Tabin, C. (1992).
The dispersion of neuronal clones across the cerebral cortex (letter). Science
258, 317-320.
(Accepted 23 October 1995)