Prdm proto-oncogene transcription factor family expression and interaction with the Notch-Hes pathway in mouse neurogenesis.

Abstract

Establishment and maintenance of a functional central nervous system (CNS) requires a highly orchestrated process of neural progenitor cell proliferation, cell cycle exit, and differentiation. An evolutionary conserved program consisting of Notch signalling mediated by basic Helix-Loop-Helix (bHLH) transcription factor activity is necessary for both the maintenance of neural progenitor cell character and the progression of neurogenesis; however, additional players in mammalian CNS neural specification remain largely unknown. In Drosophila we recently characterized Hamlet, a transcription factor that mediates Notch signalling and neural cell fate.
Hamlet is a member of the Prdm (PRDI-BF1 and RIZ homology domain containing) proto-oncogene transcription factor family, and in this study we report that multiple genes in the Prdm family (Prdm6, 8, 12, 13 and 16) are expressed in the developing mouse CNS in a spatially and temporally restricted manner. In developing spinal cord Prdm8, 12 and 13 are expressed in precise neuronal progenitor zones suggesting that they may specify discrete neuronal subtypes. In developing telencephalon Prdm12 and 16 are expressed in the ventricular zone in a lateral to medial graded manner, and Prdm8 is expressed in a complementary domain in postmitotic neurons. In postnatal brain Prdm8 additionally shows restricted expression in cortical layers 2/3 and 4, the hippocampus, and the amygdala. To further elucidate roles of Prdm8 and 16 in the developing telencephalon we analyzed the relationship between these factors and the bHLH Hes (Hairy and enhancer of split homolog) effectors of Notch signalling. In Hes null telencephalon neural differentiation is enhanced, Prdm8 expression is upregulated, and Prdm16 expression is downregulated; conversely in utero electroporation of Hes1 into the developing telencephalon upregulates Prdm16 expression.
Our data demonstrate that Prdm genes are regulated by the Notch-Hes pathway and represent strong candidates to control neural class specification and the sequential progression of mammalian CNS neurogenesis.

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Prdm Proto-Oncogene Transcription Factor Family
Expression and Interaction with the Notch-Hes Pathway
in Mouse Neurogenesis
Emi Kinameri1, Takashi Inoue2, Jun Aruga2, Itaru Imayoshi3, Ryoichiro Kageyama3, Tomomi Shimogori4*,
Adrian W. Moore1*
1Molecular Neuropathology Group, RIKEN Brain Science Institute, Wako, Saitama, Japan, 2Laboratory for Behavioral and Developmental Disorders, RIKEN Brain Science
Institute, Wako, Saitama, Japan, 3Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan, 4Shimogori Research Unit, RIKEN Brain Science Institute, Wako,
Saitama, Japan
Abstract
Background: Establishment and maintenance of a functional central nervous system (CNS) requires a highly orchestrated
process of neural progenitor cell proliferation, cell cycle exit, and differentiation. An evolutionary conserved program
consisting of Notch signalling mediated by basic Helix-Loop-Helix (bHLH) transcription factor activity is necessary for both
the maintenance of neural progenitor cell character and the progression of neurogenesis; however, additional players in
mammalian CNS neural specification remain largely unknown. In Drosophila we recently characterized Hamlet, a
transcription factor that mediates Notch signalling and neural cell fate.
Methodology/Principal Findings: Hamlet is a member of the Prdm (PRDI-BF1 and RIZ homology domain containing) proto-
oncogene transcription factor family, and in this study we report that multiple genes in the Prdm family (Prdm6, 8, 12, 13 and
16) are expressed in the developing mouse CNS in a spatially and temporally restricted manner. In developing spinal cord
Prdm8, 12 and 13 are expressed in precise neuronal progenitor zones suggesting that they may specify discrete neuronal
subtypes. In developing telencephalon Prdm12 and 16 are expressed in the ventricular zone in a lateral to medial graded
manner, and Prdm8 is expressed in a complementary domain in postmitotic neurons. In postnatal brain Prdm8 additionally
shows restricted expression in cortical layers 2/3 and 4, the hippocampus, and the amygdala. To further elucidate roles of
Prdm8 and 16 in the developing telencephalon we analyzed the relationship between these factors and the bHLH Hes (Hairy
and enhancer of split homolog) effectors of Notch signalling. In Hes null telencephalon neural differentiation is enhanced,
Prdm8 expression is upregulated, and Prdm16 expression is downregulated; conversely in utero electroporation of Hes1 into
the developing telencephalon upregulates Prdm16 expression.
Conclusions/Significance: Our data demonstrate that Prdm genes are regulated by the Notch-Hes pathway and represent
strong candidates to control neural class specification and the sequential progression of mammalian CNS neurogenesis.
Citation: Kinameri E, Inoue T, Aruga J, Imayoshi I, Kageyama R, et al. (2008) Prdm Proto-Oncogene Transcription Factor Family Expression and Interaction with the
Notch-Hes Pathway in Mouse Neurogenesis. PLoS ONE 3(12): e3859. doi:10.1371/journal.pone.0003859
Editor: Michael Hendricks, Temasek Life Sciences Laboratory, Singapore
Received August 29, 2008; Accepted November 7, 2008; Published December 3, 2008
Copyright: ? 2008 Kinameri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding provided by the RIKEN Brain Science Institute. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tshimogori@brain.riken.jp (TS); adrianm@brain.riken.jp (AWM)
Introduction
The nervous system of mammals contains a large number of
neurons in a diverse array of neuron classes. Transcription factors
play central roles in generating this complexity by controlling
neural progenitor cell proliferation, patterning, and defining
neuron fate [1,2]. For example, it is well established that an
evolutionary conserved basic Helix-Loop-Helix (bHLH) transcrip-
tion factor cascade downstream of Notch signalling is necessary for
both the maintenance of neural progenitor cell character and the
progression of neurogenesis. High Notch activity maintains neural
progenitors through an effector pathway consisting of the bHLH
Hairy and enhancer of split homologue transcription factors Hes1
and Hes5. Notch upregulates the Hes factors that then function as
DNA-binding repressors and antagonize the expression of
proneural bHLH genes [3]. Hence, low Notch activity reduces
Hes activity and leads to upregulation of proneural bHLH factors
such as Ngn2 (Neurogenin2) and Mash1 (Mammalian achaete-
scute homolog1); these factors then repress neural progenitor cell
maintenance and promote neuron differentiation [4].
Much of our understanding of the mechanisms of Notch and
bHLH function in the mammalian central nervous system (CNS) is
derived from seminal studies examining neurogenesis in the
peripheral nervous system (PNS) of the fruit fly Drosophila
melanogaster [5]. In the fly we recently identified Hamlet, a
transcription factor as acting to instruct neuron and glial cell fate
and mediating Notch signalling in a neural lineage-specific
manner [6,7]. Hamlet is a member of the relatively uncharacter-
ized transcription factor family known as the Prdm (PRDI-BF1
and RIZ homology domain containing) family [8]. Prdm family
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members are characterized by an N-terminal PR domain, and in
addition all but one (Prdm11) contain zinc fingers (Fig. 1). The PR
domain is 20–30% identical to the SET (Su(var)3-9, Enhancer-of-
zeste, and Trithorax) domain, a histone methyltransferase catalytic
module [9].
We hypothesized that the Prdm gene family may also
significantly participate in the development of the mammalian
nervous system as this family meets the molecular criteria to be
active in neurogenesis. Prdm family members are known to control
cell proliferation both in cancer [10–15] and in normal
development [16]. Furthermore, Prdm family members are also
used to define cell fate. For example, a great deal of interest has
been generated by the abilities of Prdm16 to control the switch
between skeletal muscle and brown fat in mice [17,18], and Prdm1
to act as a switch between fast and slow twitch muscle in zebrafish
(Danio rerio) [19]. In addition, Notch signalling is an essential
control mechanism in neurogenesis, and Prdm family function in
cell fate can occur through mediation of Notch signalling. For
example, both Drosophila hamlet and its Caenorhabditis elegans
homologue EGL-43 mediate Notch-controlled cell fate decisions
[7,20,21].
Roles in nervous system development have already been
demonstrated or suggested for a few members of the Prdm family.
Both hamlet and EGL-43 are required for sensory neuron
differentiation [6,22]. Furthermore, Prdm3 (Mds1/Evi1), the mouse
homologue of hamlet, is also expressed in the PNS within the
developing cranial and dorsal root ganglia [23], and knockout of
Prdm3 in mice leads to nervous system hypoplasia [24]. prdm1
(blimp1) is expressed in sensory neuron precursors in both zebrafish
[25] and Drosophila [26]. In zebrafish prdm1 expression at the edge
of the neural plate specifies the precursor cells competent to form
primary sensory neurons [25,27]. In these cells prdm1 functions
within the context of a Notch-bHLH pathway since sensory
neurogenesis also requires downregulation of Notch signalling and
subsequent induction of ngn1 [28].
In this study we present evidence for the function of multiple
relatively uncharacterized Prdm gene family members during
mammalian neurogenesis. By employing mRNA in situ hybridiza-
tion (ISH) analysis we show that several Prdm family members are
expressed in spatially restricted and related domains of neuronal
progenitors in the developing CNS consistent with a role in neural
class specification. In addition, we find that a subset of Prdm family
members remain expressed in the postnatal brain. Furthermore,
by analyzing Hes loss- and gain-of-function embryos, we
demonstrate that Prdm family gene expression in the developing
telencephalon is controlled by the Notch-Hes pathway and
regulated during the sequential progression of neurogenesis. We
suggest that the genes of the Prdm family represent strong new
candidates to function in neural progenitor cell proliferation and
neural differentiation in the mammalian CNS.
Results
Prdm5–16 expression in midgestation mouse embryos
Fifteen mouse members of the Prdm family (Fig. 1, Table S1)
were identified from the National Center for Biology Information
(NCBI) http://www.ncbi.nlm.nih.gov/ and Mouse Genome
Informatics (MGI) http://www.informatics.jax.org/ databases.
The expression patterns of Prdm1–4 have been investigated in
detail; of these, Prdm1, 3 and 4 are expressed in the nervous system
[23,29,30] (data not shown). Prdm2 expression has not been
reported in the CNS and our preliminary studies detected no
CNS-specific expression (data not shown).
We designed two or three independent, gene-specific primer
pairs for Prdm5–16. Using these primers we carried out reverse
transcription polymerase chain reaction (RT-PCR) on total
Figure 1. The domain structure of the mouse Prdm1–16 proteins. Illustrations of the protein domain structure of the Prdm family members
1–16. These illustrations are drawn to scale based on the sequence analysis shown in Table S1. Zinc fingers are represented by blue boxes and the PR
domain by a red box. Those proteins for which a nervous system specific expression pattern is reported in this study are indicated with asterisks.
doi:10.1371/journal.pone.0003859.g001
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mRNA isolated from stage embryonic (E) day 11.5 (whole embryo)
and 13.5 (head only) tissue. In both samples we detected
expression of all Prdm5–16 genes (data not shown). Furthermore,
we confirmed that we had amplified cDNA from the correct
predicted gene by sequencing the RT-PCR-generated amplicons
from each primer pair.
Prdm8, 12 and 13 show restricted nervous system
expression from early embryogenesis
To examine the expression of Prdm5–16 in detail, we carried out
whole mount in situ hybridization (WISH) at E9.5 and E10.5.
Three Prdm genes (8, 12 and 13) showed spatially restricted
expression in nervous system tissue (Fig. 2). Prdm8 and 13 showed
specific expression in spinal cord at E9.5 when neurogenesis starts
(Fig. 2A, C), and maintained their expression in this tissue at E10.5
(Fig. 2D, F).
Prdm12 was also expressed in the spinal cord at E9.5. In
addition, weak expression of Prdm12 was observed in the caudal
forebrain and midbrain (Fig. 2B, blue and yellow arrowheads). At
E10.5, these two expression domains of Prdm12 in forebrain and
midbrain became stronger (Fig. 2E, red, blue and yellow
arrowheads, respectively). For better visibility, we next dissected
out the brain at E10.5 to analyze detailed expression of Prdm12
(Fig. 2G). Prdm12 was expressed in several regions of the
diencephalon, p3 (Fig. 2G, red and green arrowheads), p1
(Fig. 2G, blue arrowhead), hypothalamus (Fig. 2G, pink
arrowhead), and a small dorsal region in the midbrain (Fig. 2G,
yellow arrowhead). To obtain precise spatial information about the
Prdm12 expression domain in the diencephalon we performed two-
colour ISH to compare Prdm12 expression with Shh (sonic
hedgehog). Shh marks the Zli (zona limitans intrathalamica), the
definitive border of p2 and p3 (Fig. 2H–K) [31]. Our analysis
clearly revealed that Prdm12 was expressed in postmitotic neurons
adjacent to, but not overlapping, the Zli (Fig 2I, J). In addition,
Prdm12 was expressed in the p1 region in the diencephalic VZ but
was excluded from the dorsal midline (Fig. 2I, K).
In addition to CNS expression, we also detected Prdm12
expression in the PNS. Prdm12 was expressed in a repeated pattern
lateral to the spinal cord in the dorsal root ganglia (DRG) (Fig. 2E,
black arrowheads) and in the head region in the cranial ganglia
(Fig. 2B, E, white arrowheads).
Prdm8, 12 and 13 family members are expressed in
interrelated domains along the dorsal-ventral axis of the
spinal cord
To understand the detailed expression pattern of the Prdm gene
family members in the spinal cord we performed further ISH on
sections (Fig. 3). In the ventral neural tube distinct classes of
motor- and interneurons are derived from distinct VZ progenitor
cell populations. Each progenitor cell population is defined by the
expression of subsets of homeodomain (HD) transcription factors
[32]. We used two-colour ISH at the cervical level to map the
expression domains of Prdm8, 12 and 13 in the VZ relative to
previously described HD factors.
The dorsal limit of the Prdm8 (Fig. 3A–C) expression domain
was identical to that of Dbx1 (Developing brain homeobox 1), which
marks the neuronal progenitor domain p0 (Fig. 3B, J) [33]. The
Prdm8 expression domain extended to a ventral limit overlapping
with that of Olig2 (Oligodendrocyte transcription factor 2) (data not
shown) and was exclusive to the expression domain of Nkx2.2 (NK2
transcription factor related, locus 2) (Fig. 3C, J) [34,35]. Hence, the
Prdm8 expression domain encompassed the VZ of progenitor
regions p0, p1, p2 and pMN (Fig. 3J). Prdm12 expression (Fig. 3D–
E) had a dorsal limit at the Dbx1 expression domain (Fig. 3D) and
a ventral limit significantly dorsal to Olig2 (Fig. 3E); hence, Prdm12
was expressed only in the p1 (Fig. 3J). In addition, Prdm12 was
expressed in the DRG (Fig. 3D, E, orange arrowheads). Prdm13
was localized in the VZ of the dorsal spinal cord with a ventral
expression border at the Dbx1 dorsal limit (Fig. 3F, J).
Figure 2. Whole mount ISH analysis of the Prdm gene family at
E9.5 and E10.5. The spatial distribution of Prdm8, 12, and 13 are
shown by WISH at E9.5 (A–C) and E10.5 (D–F) in mouse. Strong
expression of Prdm8 (A, D) and Prdm13 (C, F) is observed in spinal cord
at E9.5 and E10.5. Strong expression of Prdm12 in spinal cord and in
cranial ganglia (white arrowhead) and weak expression in caudal
diencephalon and midbrain (blue and yellow arrowheads) is observed
at E9.5 (B). (E) At E10.5, additional expression of Prdm12 in the rostral
brain is observed in p3 (red arrowhead), and stronger expression in p1
(blue arrowhead) and the midbrain (yellow arrowhead). In addition
Prdm12 is expressed in the dorsal root ganglia (black arrowheads) and
cranial ganglia (white arrowhead). (G–I) WISH of the dissected brain at
E10.5 is shown, lateral is to the left. Prdm12 is expressed in the ventral
hindbrain, p3 (red and green arrowheads), p1 (blue arrowheads),
midbrain (yellow arrowhead), and hypothalamus (pink arrowheads) (G).
Two-colour WISH for Prdm12 (blue) and Shh (brown) in Zli (zona limitans
intrathalamica) shows the spatial relationships of the expression
domains of these genes (red and green arrowheads) (H). A section
taken at the plane illustrated with a dashed line in panel H is shown in
panel I. In addition, magnified regions from panel I, highlighted by
dashed boxes, are shown in panel J and K. Bar in C, 500 mm (A–C),
700 mm (D–F), 400 mm (G–H), 200 mm (I), and 100 mm (J, K).
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In the spinal cord expression of Hes5 marks the proliferating
neural precursors in the VZ and classIII ß-tubulin marks postmitotic
neurons in the mantle zone (Fig. 3H, I, K, N). In the ventral spinal
cord Prdm6 was expressed not in the VZ, but solely in a small
subset of postmitotic neurons (Fig. 3G). However, both Prdm12
and 8 had more complex expression patterns encompassing both
Hes5-positive proliferating VZ cells and adjacent postmitotic cells
in the mantle zone (Fig. 3K–M). Furthermore, the localization of
Prdm8 in the VZ was not consistent along the dorso-ventral axis. At
the dorsal limit of Prdm8 expression, where Dbx1 was also
expressed, Prdm8 was present only weakly in the VZ but much
more strongly in the mantle zone distal to the region expressing
Dbx1 (Fig. 3B, C, L, yellow arrowheads). In the dorsal spinal cord
Prdm13 was expressed throughout the VZ but was highest in the
cells at the VZ lateral margin (Fig. 3N–O).
Prdm8, 12 and 16 label interrelated domains in the
developing forebrain
WISH on embryos isolated at E9.5, E10.5 and E11.5
demonstrated that Prdm12 was expressed in the brain from E9.5
Figure 3. Spatially restricted expression of Prdm family members in the spinal cord. Spatial expression of Prdm gene family members is
shown by ISH in transverse spinal cord cervical sections at E11.5. (A) Prdm8 has strong expression in the ventral VZ (blue arrowhead) and some
postmitotic neurons (red arrowheads). (B, C) Two-colour ISH for Prdm8 (blue) and Dbx1 or Nkx2.2 (brown) in spinal cord progenitor regions shows the
dorsal and ventral expression limits of Prdm8. (D, E) Two-colour ISH for Prdm12 (blue) and Dbx1 or Olig2 (brown) demonstrate that the Prdm12
expression domain is restricted to p1. Strong expression in the DRG is also observed (orange arrowhead). (F) Expression of Prdm13 (blue) is restricted
to the dorsal half of spinal cord and its ventral limit is the Dbx1 (brown) expressing p0 progenitor domain. (G) Expression of Prdm6 is observed in
ventrally located postmitotic neurons (red arrowheads) and also is in putative sclerotome (blue arrowhead). (H) Hes5 marks the ventricular zone and a
small population of precursor cells in the DRG (orange arrowhead). (I) class III ß-tubulin marks postmitotic differentiating neurons in the mantle zone
(inside the red dashed lines) and DRG (orange arrowhead). (J) A summary cartoon illustrating which progenitor domains express the transcription
factors shown in panels A–F. Bar in A 100 mm (A–I). (K–O) A comparison of Hes5 and Prdm family gene expression; each panel shows a magnified
region of the spinal cord as boxed in red (K–M) and blue (N, O) in panel H. In the ventral spinal cord Hes5 (K) expression marks the VZ (red arrowhead)
and is not expressed in postmitotic neurons (blue arrowhead); on the other hand, Prdm8 (L) and Prdm12 (M) are expressed in both the VZ (red
arrowhead) and postmitotic region (blue arrowhead). Prdm8 expression in the Dbx1 zone is low in the ventricular zone where it overlaps Dbx1 and
stronger in the mantle zone a where it expands beyond the extent of Dbx1 expression (yellow arrowheads in B, C, L). In the dorsal spinal cord Hes5
expression (N) and Prdm13 expression (O) overlap. Prdm13 expression is weak in the proximal VZ (red arrowhead) and strong (purple arrowhead) at
the interface between the VZ and the mantle zone (blue arrowhead).
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(Fig. 2) and that Prdm8 and 16 began to be expressed in the
forebrain at E11.5 (data not shown). To analyze brain-specific
expression of these factors in more detail we performed section
ISH at E12.5 (Fig. 4A–C, F–H). We compared the expression
pattern of the Prdm genes to those of Hes5, which marks
proliferating VZ cells, and classIII ß-tubulin, which marks
postmitotic neurons (Fig. 4D, E, I, J).
At E12.5 Prdm16 was expressed in the VZ throughout the rostral
to caudal telencephalon (Fig. 4A, F). The expression was strongest
at the pallium/subpallium boundary (psb) (Fig. 4A, F black
arrowhead) and formed a lateral/strong to medial/weak gradient
(Fig. 4A, F). Prdm16 was additionally expressed in the septum
(Fig. 4A, white arrowhead), choroid plexus (Fig. 4F, red
arrowhead), and pretectum in the diencephalon (Fig. 4F, purple
arrowhead). Prdm12 was expressed in the dorsal telencephalic VZ
with a steep lateral/strong to medial/weak gradient from the psb
(Fig. 4B, G black arrowhead). Prdm12 was also expressed in
postmitotic neurons in the septum, striatum (Fig. 4B, white and
blue arrowheads), and lateral region of the prethalamus (Fig. 4G,
pink arrowhead). Prdm8 was solely expressed in postmitotic
neurons in the dorsal telencephalon (Fig. 4C, H, orange
arrowheads). In the rostral cortex the lateral edge of the Prdm8
expression domain was complementary to that of the Prdm12
expression domain in the pallium (Fig. 4B, C, blue arrowheads).
Prdm8 and 12 are expressed in specific populations of
neurons in the postnatal brain
To examine later stages of Prdm expression we extended our
ISH analysis to E16.5 and early postnatal day (P) 6 brains. At
E16.5 Prdm8 continued to be str7ongly expressed in postmitotic
neurons in the developing forebrain (data not shown). Further-
more, at E16.5 Prdm16 was still weakly expressed in the
telencephalic VZ; however, Prdm12 was no longer expressed in
this structure (data not shown). At E16.5 both Prdm16 and Prdm12
continued to be expressed in the septum (data not shown).
At P6Prdm8expressionwasina sharplydefinedlamina patternin
the neocortex (Fig. 5A). Comparison of the Prdm8 expression
domain with Nissl staining and Tbr1 (T-box brain gene 1) [36]
expression domains (Fig. 5B, white arrowheads, C) showed that
Prdm8 was expressed in layers 2/3 and 4 (Fig. 5A). Prdm8-expressing
cells were also scattered in dentate gyrus (DG) and in CA2 and CA3
regions of the pyramidal cell layer in the hippocampus (Fig. 5D). In
addition, Prdm8 was expressed in the nucleus of the lateral olfactory
tract (nLOT) (Fig. 5E, arrow) as confirmed by the identical
expression of Tbr1 (Fig. 5F, arrow) and by Nissl staining (Fig. 5G,
arrow). The nLOT is connected to the main olfactory bulb and the
piriform cortex, and influences nonpheromonal olfactory-guided
behaviours, especially feeding [37].
By P6 Prdm16 was no longer expressed. Prdm12, on the other
hand, was expressed in specific populations of postmitotic neurons
in the hypothalamus where it was restricted to the dorsomedial
nucleus (Fig. 5H). Prdm12 was also expressed in the dorsal half of
the zona incerta of the thalamus (Fig. 5I, arrows). Taken together,
these results suggest additional roles of Prdm8 and 12 in
differentiated neurons besides their roles in progenitors in the
developing CNS.
Prdm16 is positively regulated and Prdm8 negatively
regulated by Hes activity during telencephalic
neurogenesis
Notch signalling is an essential control mechanism to regulate
mammalian neurogenesis. We have previously shown that the
Drosophila Prdm gene hamlet is a modifier of Notch signalling during
Drosophila peripheral neurogenesis [7]. C. elegans EGL-43 was also
demonstrated to be downstream of Notch signalling and to
mediate the effect of Notch during vulva formation [20,21].
Finally, zebrafish prdm1 is required to enable Notch signalling
pathway-mediated specification of sensory neurons [25,28].
In mammalian cortical neurogenesis high levels of Notch
signalling maintain neural progenitors by upregulating the bHLH
Figure 4. Expression of Prdm family members in the developing forebrain. Each panel shows ISH carried out on one hemisphere of either
rostral or caudal E12.5 forebrain. (A) Prdm16 is expressed in the VZ of the dorsal and ventral telencephalon in a lateral/strong to medial/weak
gradient. (F) Sections from the caudal part of the brain show expression of Prdm16 in choroid plexus epithelium (CPe) (red arrowhead) and pretectum
(purple arrowhead). The boundary of the pallium and subpallium (psb) is marked by a black arrowhead. (B) Prdm12 is expressed in lateral
telencephalic VZ with a steep lateral/strong to medial/weak gradient from the psb (black arrowhead). It also has expression in postmitotic neurons in
the septum (white arrowhead) and pallium (blue arrowhead). (G) Beside the expression of Prdm12 in the lateral VZ, a small expression domain in the
prethalamus (pink arrowhead) is detected. (C, H) Prdm8 is expressed in the postmitotic neurons of the lateral and dorsal regions of the cortex (orange
arrowheads). (D, E, I, J) The progenitor region and the postmitotic region is indicated by Hes5 (D, I) and classIII ß-tubulin (E, J) respectively. Scale bar in
A, 250mm (A–E); scale bar in F, 500mm (F–J). Abbreviations: lv, lateral ventricle; psb, pallium-subpallium boundary; sep, septum; cge, caudal ganglionic
eminence; th, thalamus; CPe, choroid plexus epithelium.
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effectors Hes1 and Hes5 [3]. In particular, Hes1 maintains neural
progenitor cell identity and represses proneural genes such as
Ngn2. These proneural factors drive the progenitor to exit cell cycle
and begin neural differentiation [38]; hence, loss of Hes activity
leads to upregulation of proneural genes and consequent
premature neurogenesis. Prdm16 expression in the telencephalic
ventricular zone overlaps with that of the Hes genes (Fig. 4A, D, F,
I). Furthermore, the expression of Prdm8 in early telencephalic
development is complementary to that of the Hes genes (Fig. 4C,
D, H, I). Hence, we examined if the Hes effectors of Notch
signalling regulate Prdm family member gene expression during
vertebrate telencephalic neurogenesis.
Three Hes genes (Hes1, 3 and 5) can potentially substitute for
each other in the ventricular zone [39]; hence we chose to analyze
the expression of Prdm8 and 16 in Hes triple-null forebrain. To
obtain Hes triple-null forebrains we crossed Hes1 (floxed/floxed);
Hes32/2; Hes52/2mice with Emx1-Cre; Hes1+/2; Hes32/2;
Hes52/2mice. We hereafter refer to the triple-null forebrains of
the embryos derived from this cross as Hes cTKO forebrains. We
examined E14.5 Hes cTKO forebrains for both Prdm8 and Prdm16
expression as well as the expression of the proneural factor Ngn2. It
has previously been shown that in Hes cTKO mutant embryos
cortical neurogenesis occurs prematurely [39]. Indeed, in Hes
cTKO mutant telencephalon, compared to wildtype, there was a
large increase in the number of cells expressing Prdm8 (Fig. 6A, A9,
B, B9, n=4) and Ngn2 (Fig. 6C, D, bracket, n=4). These data
show that, like Ngn2, Prdm8 expression is repressed by Hes activity,
and they further imply that Prdm8 is activated during the temporal
progression of neurogenesis in a fashion similar to Ngn2.
Furthermore, in Hes cTKO telencephalon the progenitor cell
population is reduced but not fully depleted [39]. Indeed, in Hes
cTKO forebrains there was also a thinning of the Prdm16 expressing
VZ region in the telencephalon (Fig. 6E, F, purple arrowheads)
and a stronger reduction of Prdm16 in the VZ of the striatum
(Fig. 6E, F, blue arrowheads, n=4).
Hes1 promotes neural progenitor cell identity by repressing
genes that promote cell cycle exit and neural differentiation [38].
Targets of Hes1 activity can be determined by Hes1 electroporation
into the telencephalon followed by examination of changes in
putative target gene expression via ISH [38]. Our Hes loss of
function (Hes cTKO) data implied that Prdm16 may be positively
regulated by Hes1, and hence be part of a suite of genes expressed
in neural progenitor cells. To examine if Prdm16 is positively
Figure 5. Expression of Prdm family genes in the early
postnatal brain. Coronal sections through P6 brains, processed to
show Prdm8 expression (A, D, E), Prdm12 expression (H, I), Tbr1
expression (C, F) or Nissl (B, G). Prdm8 is expressed in layer 2/3 and 4 of
the neocortex (A). Nissl stain clearly reveals the cortical layers, especially
the barrel hollows in the somatosensory cortex in layer 4 (B,
arrowheads). Layer 2/3, 5, and 6 are specifically labelled by Tbr1 (C).
Cells expressing Prdm8 in the hippocampus are concentrated in the
dentate gyrus (DG) and the CA2/3 regions (D). At P6, Prdm8 is expressed
specifically in nucleus of lateral olfactory tract (nLOT) (E), which is also
revealed by Tbr1 (F) and Nissl staining (G). Prdm12 expression in the
hypothalamus is restricted in dorsomedial nucleus (H). In the thalamus,
expression of Prdm12 is specific to the dorsal half of the zona incerta (I,
arrows). Scale bar in C is 500 mm for A–H and 1.3 mm for I.
doi:10.1371/journal.pone.0003859.g005
Figure 6. Prdm8 is upregulated, and Prdm16 downregulated, in
Hes-null forebrain. Coronal sections of wildtype (A, C, E) and Hes cTKO
(B, D, F) E14.5 telencephalon with ISH to detect Prdm8 (A, B), Ngn2 (C,
D), and Prdm16 (E, F). Prdm8 and Ngn2 are upregulated in developing
Hes-null brains. This upregulation is due to more cells expressing Prdm8
(compare A9 and B9, which are magnified regions of the boxed areas in
A and B respectively) and Ngn2 (compare the brackets in C and D). On
the other hand, Prdm16 is strongly downregulated in the subpallium
(blue arrowheads) and slightly downregulated in the pallium (purple
arrowheads). Scale bar in A, 500 mm.
doi:10.1371/journal.pone.0003859.g006
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regulated by Hes we performed in utero electroporation of Hes1
cDNA along with EGFP cDNA (pEF-Hes1 and pEF-EGFP) into
the telencephalon at E13.5 (Fig. 7A–D); we also electroporated
EGFP cDNA alone as a control (Fig.7E–H, bracket, n=5,
respectively). Eighteen hours later we sacrificed the embryos and
carried out ISH to determine Prdm16 gene expression. Prdm16 was
strongly upregulated by Hes1 overexpression (Fig. 7A, B, bracket
and arrowheads, n=5) implying that Prdm16 is positively regulated
by Hes1 during neurogenesis and expressed in the neural
progenitor cell population. As Hes1 protein is believed to act as
a transcriptional repressor [3] positive regulation of Prdm16 by
Hes1 may not be direct; it is possible that Hes1 acts by repressing a
repressor of Prdm16 expression. At the same time we examined
Ngn2 expression, and as previously reported Ngn2 was repressed by
Hes1 overexpression (Fig. 7C, D, arrow, n=4) [38]. Electropora-
tion of EGFP alone did not cause any change in Prdm16 or Ngn2
gene expression (Fig. 7E–H).
Discussion
Prdm family-mediated neural class specification of the
developing spinal cord
Our data suggest that several members of the Prdm family could
play a role in neuronal specification. During spinal cord
development distinct classes of neurons are generated from
progenitor cells located at different dorso-ventral positions within
the VZ. These domains are spatially defined by the restricted
expression of members of the HD transcription factor family and
the bHLH factor Olig2. The individual code of transcription
factors expressed in each region of VZ defines the fate of the
neurons that are generated in that specific region [32]. We can
now add the Prdm transcription factors as another family in which
multiple members delineate specific progenitor regions. The Prdm
family is hence an interesting candidate for involvement in
controlling neuron class identity in the spinal cord.
HD transcription factor proteins expressed in the spinal cord
VZ are divided into two groups, classes I and II. A single class I
and a single class II factor are paired in such a way that there is a
sharp boundary between the domains that express each member
of the pair. HD transcription factors are repressors, and the sharp
boundaries of expression between each pair of class I and II factors
are achieved by mutual cross-repression [32]. Interestingly, Prdm
proteins associate with a wide range of chromatin-remodelling
enzymes and also act predominantly as transcription repressors
[16,40–44]. The expression domains of Prdm13 and 8 have a sharp
mutual border, raising the interesting possibility that these factors
may repress each other. In addition, Prdm13, 8 and 12 all have
sharp borders with domains that express specific HD factors,
raising the possibility of repression between Prdm and HD factors.
In addition to progenitor cell regions, Prdm8 and 12 are
expressed in adjacent cells in the mantle zone. Furthermore, both
Prdm8 and 13 are expressed at a high level by cells at the margin of
the VZ. Therefore a second point in spinal cord neurogenesis that
Prdm genes may be active is as the cells exit the proliferative zone
and begin to differentiate as neurons. In this context, we note that
in the Drosophila PNS hamlet specifies neuron class fate by acting
only transiently at the point where intermediate precursor cells
undergo a final division and the immature neuron is formed [6].
prdm1 also acts in a transient fashion to specify slow twitch muscle
in zebrafish [19]. It is tempting to speculate that such transient
Prdm protein activity could involve establishing a new stable
chromatin state in the differentiating cells, either by direct PR
domain-mediated remodelling or indirectly by recruiting remod-
elling enzymes [16,40–44].
Prdm family-mediated patterning of the developing
brain
In the developing telencephalon patterning is controlled by
transcription factors expressed not in discrete domains but rather
in a graded fashion [45]. In the telencephalon both Prdm12 and 16
are expressed in lateral/strong to medial/weak gradients (Fig. 4).
Furthermore, initial domains of Prdm12 expression are adjacent to
the Zli and isthmus, both of which act as signalling centres
regionally patterning the developing brain. Hence, Prdm16 and
especially Prdm12 are candidates that merit further examination
for roles in brain patterning.
In the early postnatal brain Prdm8 is expressed in cortical layers
2/3 and 4, and Prdm12 in the hippocampus, part of the
hypothalamus, and the thalamus (Fig. 5). Furthermore, outside
the brain, Prdm12 is strongly expressed in both dorsal root and
cranial ganglia. These expression domains imply that, in addition
to a possible involvement in patterning, these factors may also play
a role in the differentiation and function of specific neuron classes.
Probable evolutionary conservation of individual Prdm
family member functions during vertebrate neurogenesis
A recent survey in zebrafish described homologues of the entire
mouse Prdm gene family [46]. Zebrafish Prdm family members that
have CNS specific expression during neurogenesis (prdm8a, 8b, 12,
13 and 16) are the homologues of those mouse Prdm family
Figure 7. Prdm16 is upregulated in response to Hes1 overex-
pression. pEF-EGFP and pEF-Hes1 together (A–D) or a control
consisting of pEF-EGFP alone (E–H) were electroporated into telence-
phalic neural progenitors at E13.5. Eighteen hours later, the brains were
harvested, sectioned coronally, and then ISH was carried out to detect
GFP (A, C, E, G), Prdm16 (B, F), and Ngn2 (D, H). Hes1 electroporation into
the telencephalon caused upregulation of Prdm16 and a concomitant
downregulation of Ngn2. Scale bar in A, 500 mm.
doi:10.1371/journal.pone.0003859.g007
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Page 8

members we describe in this study [46]. Furthermore, there is
considerable conservation in the domains of expression for some of
these homologues, suggesting probable evolutionary conservation
of function. For example, zebrafish prdm8a and 13 are expressed in
the spinal cord [46] similar to Prdm8 and 13 in mouse and it will be
interesting to ascertain if they have analogous expression domains
along the dorso-ventral axis. In the developing telencephalon,
similar to mouse, zebrafish prdm16 is expressed during early
neurogenesis (18 hours postfertilization) and downregulated later
(by 24 hours postfertilization) [46]. These data suggest an early
role for prdm16 in telencephalic neurogenesis and raise the
question of whether prdm16 also marks neural precursors in
zebrafish as it does in mouse. prdm3 is also expressed in early
telencephalic development in zebrafish [46] but not mouse ([23]
and data not shown); this overlapping telencephalic expression of
prdm3 and 16 in zebrafish is interesting because they encode very
similar proteins that are likely to share conserved mechanisms of
action [40–42].
Putative conserved roles of Prdm family members in
neuronal, smooth muscle, germ cell, and haemopoietic
development, and in leukaemogenesis
Prdm16, originally named Mel1, was first identified as being
expressed at highly elevated levels in leukaemia. Expression of high
levels of a truncated form of Prdm16 that does not encode the PR
domain (called sPrdm16 or Mel1s) is associated with acute myeloid
leukaemia (AML) in humans [11,47] and causative of AML in mouse
[48,49]. In this study we demonstrated that Prdm16 expression in the
developing CNS is mediated by the Notch-Hes pathway. Interest-
ingly,haemopoiesisalsoutilizesNotchsignallingtomaintainstemcell
identity and to diversify cell types during lineage elaboration [50].
Moreover, certain mutations that constitutively activate Notch1
protein cause leukaemia; although this is usually T-cell acute
lymphoblastic leukaemia rather than the AML associated with
Prdm16 [51]. Hence, the relationship of Prdm16 to Notch signalling
and a conserved role for Prdm16 in the maintenance of progenitor cell
fate are very interesting prospects for further investigation in
haemopoiesis and leukaemogenesis, as well as neurogenesis.
Intriguingly, Prdm3, originally named Mds1/Evi1, is the Prdm
family member most closely related to Prdm16 [8]. Prdm3 also causes
AML when a truncated form (called Evi1) that does not encode the
PR domain is ectopically expressed [10,52]. Although Prdm3 is not
expressed in developing mouse telencephalon ([23] and data not
shown), it is expressed in embryonic and adult haemopoietic
progenitors in which it regulates proliferation [53]. The domain
structure of the Prdm16 and Prdm3 proteins are very similar (Fig. 1)
and both regulate transcription through binding to the same co-
factors, in particular the co-repressor C-terminal Binding Protein
(CtBP) [40–42]. These results suggest that Prdm16 and Prdm3
proteins could function in neural progenitor cells, haemopoietic
progenitors, and oncogenic haemopoietic progenitors via closely
related mechanisms. Hence, previous studies of Prdm3 function in
haemopoiesis and leukaemogenesis may be relevant to Prdm16
function in progenitor cells in the CNS.
Other Prdm family members also play roles in maintaining
precursor cell proliferation and pluripotency. Prdm6 is expressed in
a varietyofsmooth muscle-containing tissues where it actstosuppress
differentiation and maintain the proliferative potential of vascular
smooth muscle precursors [16]. Pluripotency is an essential feature of
germcells,andarecentreportbySaitouandcolleagueshasdescribed
a two-step process in germ cell specification that involves the
sequential activity of two Prdm family members [54]. First Prdm1 acts
to repress the somatic gene expression program; second Prdm14 acts
to promote the reacquisition of pluripotency and genome-wide
epigenetic reprogramming. Notably, Prdm14 is also upregulated in
human ES cells where it suppresses differentiation [55]. An
interesting potential link between Prdm14 function and Prdm16 is
that Prdm14 mediates the acquisition of germ cellpluripotency in part
by upregulating Sox2 [54]. Sox2 also has a crucial role in neural
precursor cell proliferation and maintenance [56]; hence in addition
to the Hes factors, Sox2 is a very good candidate for interaction with
Prdm16 during neurogenesis.
Conclusions
In this study we have shown that several members of the Prdm
gene family (Prdm6, 8, 12, 13 and 16) have interrelated expression
patterns during mouse CNS neurogenesis, which suggest roles in
neuronal class specification and differentiation. Within the
telencephalon we find that Prdm16 marks neuronal progenitor
cells and Prdm8 postmitotic neurons. In this brain region Prdm16
expression is maintained by Notch-Hes signalling and transition to
Prdm8 expression follows the down regulation of Notch. This
relationship between Prdm genes and the Notch-Hes pathway will
be interesting to investigate in wider developmental and oncogenic
contexts. Interestingly, our study and a very recent study both
show conservation of Prdm16 interaction with bHLH factors;
Prdm16 interacts with Hes1 in this study and with Myf5 in the
skeletal muscle to brown fat fate switch [18]. It is now important to
ascertain if there are common mechanisms of Prdm16 (and Prdm3)
interaction with the Notch pathway during brown fat determina-
tion, neurogenesis, haemopoiesis, and leukaemogenesis. Certainly,
the data we present in this study show that the Prdm family
interacts with the Notch-Hes pathway during neurogenesis, may
control nervous system patterning, and may modulate neuronal
progenitor cell proliferation and differentiation. Hence, the Prdm
family is an excellent candidate for further investigation relating to
the generation of nervous system complexity.
Materials and Methods
Sequence analysis
The nucleotide and peptide sequences used for primer design to
generate ISH probes from Prdm gene family members were
obtained from the NCBI and MGI databases. The accession
numbers of the cDNAs or predicted gene sequences used for the
design of the primers used in this study were as follows: Prdm5,
NP_081823; Prdm6, NP_001028453; Prdm8, NP_084223; Prdm9,
XP_619431.3; Prdm10, NP_001074286; Prdm11, CAM14371;
Prdm12, XM_355325.5;
Prdm13,
NP_001074678; Prdm15, XP_622716; Prdm16, NP_081780. All
plasmids used to generate probes for ISH are freely available upon
request from A.W.M. Protein domains for Figure 1 were identified
by utilizing PFAM [57] and BLAST.
NP_001074240;
Prdm14,
Molecular biology
Total RNA was prepared from ICR mouse embryos at E11.5
(whole embryos) and E13.5 (head) using an RNeasy Mini Kit
(QIAGEN). RT-PCR was performed using a OneStep RT-PCR
kit (QIAGEN) as per manufacturer’s instructions. The individual
PCR products were cloned into the plasmid vector pGEM-T Easy
(Promega). Additional probes used were: Hes5, classIII ß-tubulin,
Ngn2, Dbx1, Olig2, Nkx2.2, Tbr1, and GFP. Both whole mount and
section mRNA ISH were carried out using previously published
one- or two-colour methods [58].
Mouse breeding
Hes mutant mice were generated as described previously [39]. In
utero electroporation experiments were designed and executed as
Prdm Genes in Neurogenesis
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Page 9

described in recently published studies [38,59]. All animal
research and husbandry was completed in accordance with the
guidelines of the RIKEN Brain Science Institute and Kyoto
University.
Supporting Information
Table S1
domains for Prdm1-16.
Found at: doi:10.1371/journal.pone.0003859.s001 (0.04 MB
DOC)
Sequence analysis that indicates the position of protein
Acknowledgments
The authors thank J. Motoyama for constructive criticism of the
manuscript.
Author Contributions
Conceived and designed the experiments: EK AWM. Performed the
experiments: EK TI TS. Analyzed the data: EK TS AWM. Contributed
reagents/materials/analysis tools: EK TI II RK TS AWM. Wrote the
paper: EK JA TS AWM.
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