1: Int J Dev Biol. 2005;49(2-3):231-5. 

Isthmus organizer and regionalization of the mesencephalon and metencephalon.

Nakamura H, Watanabe Y.

Department of Molecular Neurobiology, Graduate School of Life Sciences and
Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
nakamura@idac.tohoku.ac.jp

The brain vesicles that are formed at an early stage of neural development are
the fundamentals of the brain plan. Heterotopic transplantation revealed that
the diencephalon could change its fate when juxtaposed to the isthmus
(mes-metencephalic boundary), which indicated that the isthmus functions as an
organizer for the mesencephalon and metencephalon. Fgf8 is identified as an
isthmus organizing signal. Misexpression of Fgf8a and Fgf8b indicated that a
strong Fgf8 signal organizes cerebellar development. The transcription factors
define the fate of the region. Overlapping expression of Otx2, En1 and Pax2 may
define the mesencephalic region and additional expression of Pax3/7 may instruct
the mesencephalic region to differentiate into the tectum. The di-mesencephalic
boundary is determined by repressive interaction between Pax6 and En1/Pax2 and
the mes-metencephalic boundary is defined by repressive interaction between Otx2
and Gbx2. Fgf8 is induced at the border of the Otx2 and Gbx2 expression domain,
overlapping with Gbx2 expression.

Publication Types:
    Review
    Review, Tutorial

PMID: 15906236 [PubMed - indexed for MEDLINE]


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2: J Neurosci. 2004 Jun 2;24(22):5258-68. 

Functional integration of embryonic stem cell-derived neurons in vivo.

Wernig M, Benninger F, Schmandt T, Rade M, Tucker KL, Bussow H, Beck H, Brustle
O.

Institute of Reconstructive Neurobiology, University of Bonn Medical Center and
Hertie Foundation, University of Bonn, D-53105 Bonn, Germany.

Pluripotency and the potential for continuous self-renewal make embryonic stem
(ES) cells an attractive donor source for neuronal cell replacement. Despite
recent encouraging results in this field, little is known about the functional
integration of transplanted ES cell-derived neurons on the single-cell level. To
address this issue, ES cell-derived neural precursors exhibiting neuron-specific
enhanced green fluorescent protein (EGFP) expression were introduced into the
developing brain. Donor cells implanted into the cerebral ventricles of
embryonic rats migrated as single cells into a variety of brain regions, where
they acquired complex morphologies and adopted excitatory and inhibitory
neurotransmitter phenotypes. Synaptic integration was suggested by the
expression of PSD-95 (postsynaptic density-95) on donor cell dendrites, which in
turn were approached by multiple synaptophysin-positive host axon terminals.
Ultrastructural and electrophysiological data confirmed the formation of
synapses between host and donor cells. Ten to 21 d after birth, all
EGFP-positive donor cells examined displayed active membrane properties and
received glutamatergic and GABAergic synaptic input from host neurons. These
data demonstrate that, at the single-cell level, grafted ES cell-derived neurons
undergo morphological and functional integration into the host brain circuitry.
Antibodies to the region-specific transcription factors Bf1, Dlx, En1, and Pax6
were used to explore whether functional donor cell integration depends on the
acquisition of a regional phenotype. Our data show that incorporated neurons
frequently exhibit a lacking or ectopic expression of these transcription
factors. Thus, the lack of an appropriate regional "code" does not preclude
morphological and synaptic integration of ES cell-derived neurons.

PMID: 15175396 [PubMed - indexed for MEDLINE]


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3: J Neurobiol. 2004 Apr;59(1):48-56. 

Polarity and laminar formation of the optic tectum in relation to retinal
projection.

Nakamura H, Sugiyama S.

Department of Molecular Neurobiology, Graduate School of Life Sciences and
Institute of Development Aging and Cancer, Tohoku University, Seiryo-machi 4-1,
Aoba-ku, Sendai 980-8575, Japan. nakamura@idac.tohoku.ac.jp

The mes-metencephalic boundary (isthmus) works as an organizer for the tectum,
and the organizing molecule may be Fgf8. The region where Otx2, En1, and Pax2
are expressed overlappingly may differentiate into the mesencephalon. The
di-mesencephalic and mes-metencephalic boundaries are determined by repressive
interaction of Pax6 and En1/Pax2 and of Otx2 and Gbx2, respectively. The optic
tectum is a visual center in lower vertebrates. The tectum and the retina should
be regionalized and be positionally specialized for the proper retinotopic
projection. Gradient of En2 plays a crucial role in rostrocaudal polarity
formation of the tectum. En2 confers caudal characteristics of the retina by
inducing ephrinA2 and A5, which are the repellant molecules for the growth cones
of temporal retinal ganglion cells. Grg4 antagonizes the isthmus-related genes,
and is involved in the formation of di-mesencephalic boundary and tectal
polarity formation at an early phase of development. Then, Grg4 plays a role in
tectal laminar formation by controlling the migration pathway. Migration pathway
of tectal postmitotic cells changes after E5. The late migratory cells split the
early migratory neurons to form laminae h-j of SGFS. Grg4 is expressed in the
ventricular layer after E5, and forces postmitotic cells to follow the late
migratory pathway, though retinal fibers terminate at laminae a-f of SGFS.
Misexpression of Grg4 disrupts the lamina g, and in such tecta retinal arbors
invade deep into the tectal layer, indicating that lamina g is a nonpermissive
lamina for the retinal arbors. Copyright 2004 Wiley Periodicals, Inc. J
Neurobiol 59: 48-56, 2004

Publication Types:
    Review
    Review, Tutorial

PMID: 15007826 [PubMed - indexed for MEDLINE]


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4: J Neurosci. 2004 Feb 4;24(5):1255-64. 

Pax6 and engrailed 1 regulate two distinct aspects of renshaw cell development.

Sapir T, Geiman EJ, Wang Z, Velasquez T, Mitsui S, Yoshihara Y, Frank E, Alvarez
FJ, Goulding M.

Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, California
92037, USA.

Many of the interneuron cell types present in the adult spinal cord contribute
to the circuits that control locomotion and posture. Little is known, however,
about the embryonic origin of these cell types or the molecular mechanisms that
control their differentiation. Here we provide evidence that V1 interneurons
(INs), an embryonic class of interneurons that transiently express the En1
transcription factor, differentiate as local circuit inhibitory interneurons and
form synapses with motor neurons. Furthermore, we show that a subset of V1 INs
differentiates as Renshaw cells, the interneuronal cell type that mediates
recurrent inhibition of motor neurons. We analyze the role that two V1
IN-related transcription factor genes play in Renshaw cell development. Pax6
(paired box gene 6) is necessary for an early step in Renshaw cell development,
whereas Engrailed 1 (En1), which is genetically downstream of Pax6, regulates
the formation of inhibitory synapses between Renshaw cells and motor neurons.
Together, these results show that Pax6 and En1 have essential roles in
establishing the recurrent inhibitory circuit between motor neurons and Renshaw
cells.

PMID: 14762144 [PubMed - indexed for MEDLINE]


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5: Development. 2002 Mar;129(6):1327-38. 

Pax6 regulates specification of ventral neurone subtypes in the hindbrain by
establishing progenitor domains.

Takahashi M, Osumi N.

Department of Developmental Neurobiology, Tohoku University Graduate School of
Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.

Recent studies have shown that generation of different kinds of neurones is
controlled by combinatorial actions of homeodomain (HD) proteins expressed in
the neuronal progenitors. Pax6 is a HD protein that has previously been shown to
be involved in the differentiation of the hindbrain somatic (SM) motoneurones
and V1 interneurones in the hindbrain and/or spinal cord. To investigate in
greater depth the role of Pax6 in generation of the ventral neurones, we first
examined the expression patterns of HD protein genes and subtype-specific
neuronal markers in the hindbrain of the Pax6 homozygous mutant rat. We found
that Islet2 (SM neurone marker) and En1 (V1 interneurone marker) were
transiently expressed in a small number of cells, indicating that Pax6 is not
directly required for specification of these neurones. We also observed that
domains of all other HD protein genes (Nkx2.2, Nkx6.1, Irx3, Dbx2 and Dbx1) were
shifted and their boundaries became blurred. Thus, Pax6 is required for
establishment of the progenitor domains of the ventral neurones. Next, we
performed Pax6 overexpression experiments by electroporating rat embryos in
whole embryo culture. Pax6 overexpression in the wild type decreased expression
of Nkx2.2, but ectopically increased expression of Irx3, Dbx1 and Dbx2.
Moreover, electroporation of Pax6 into the Pax6 mutant hindbrain rescued the
development of Islet2-positive and En1-positive neurones. To know reasons for
perturbed progenitor domain formation in Pax6 mutant, we examined expression
patterns of Shh signalling molecules and states of cell death and cell
proliferation. Shh was similarly expressed in the floor plate of the mutant
hindbrain, while the expressions of Ptc1, Gli1 and Gli2 were altered only in the
progenitor domains for the motoneurones. The position and number of
TUNEL-positive cells were unchanged in the Pax6 mutant. Although the proportion
of cells that were BrdU-positive slightly increased in the mutant, there was no
relationship with specific progenitor domains. Taken together, we conclude that
Pax6 regulates specification of the ventral neurone subtypes by establishing the
correct progenitor domains.

PMID: 11880342 [PubMed - indexed for MEDLINE]


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6: Prog Neurobiol. 2001 Dec;65(5):473-88. 

Regionalisation and acquisition of polarity in the optic tectum.

Nakamura H.

Department of Molecular Neurobiology, Institute of Development, Aging and
Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, 980-8575, Sendai, Japan.
makamura@idac.tohoku.ac.jp

The optic tectum differentiates from the alar plate of the mesencephalon and
receives retinal fibres in a precise retinotopic manner. Here, mechanisms of
tectum polarisation and regionalisation are reviewed. Misexpression of Pax2,
Pax5 or En can change the fate of the presumptive diencephalon to that of the
tectum. Ephrin A2 and A5 are expressed in a gradient in the tectum, caudal high
and rostral low, and may play important roles in the formation of a precise
retinotectal projection map. Retinal fibres that express receptors for these
ligands, and which come from the temporal retina, are repulsed by the ligands
and do not invade the caudal tectum. Both En1 and En2 can regulate posterior
characteristics in the tectum by inducing ephrin A2 and A5. Transplantation
experiments in chick have indicated that the mes/metencephalic boundary works as
an organiser for the tectum and the cerebellum. Fgf8 is a candidate signalling
molecule in the organiser. Pax2/5, En, and Fgf8 are in a positive feedback loop
for their expression such that misexpression of one of these genes in the
diencephalon turns on the feedback loop and can result in induction of an optic
tectum. Otx2 and Gbx2 appear to repress each other's expression and contribute
to defining the posterior border of the tectum. Misexpression of Otx2 in the
metencephalon can change the fate of its alar plate to a tectum, and
misexpression of Gbx2 in the mesencephalon can cause anterior shifting of the
caudal limit of the tectum. The anterior border of the tectum may be determined
as a result of repressive interactions between Pax6 and En1/Pax2. Along the
dorsoventral axis of the mesencephalon, Shh contributes to ventralize the
tissue; that is, Shh can change the fate of the presumptive tectum to that of
the tegmentum that is the ventral structure. It is proposed that the brain
vesicle that expresses Otx2, Pax2, and En1 may differentiate into the tectum.

Publication Types:
    Review
    Review, Tutorial

PMID: 11689282 [PubMed - indexed for MEDLINE]


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7: Trends Neurosci. 2001 Jan;24(1):32-9. 

Regionalization of the optic tectum: combinations of gene expression that define
the tectum.

Nakamura H.

Dept of Molecular Neurobiology, Institute of Development, Aging and Cancer,
Tohoku University, Seiryo-machi 4-1, Aoba-ku, 980-8575, Sendai, Japan.
nakamura@idac.tohoku.ac.jp

The optic tectum differentiates from the alar plate of the mesencephalon. Here,
the molecular mechanisms for differentiation of the tectum are reviewed.
Mis-expression of Pax2, Pax5 or En can change the fate of the presumptive
diencephalon to become the tectum. En, Fgf8, Pax2 and Pax5, exist in a positive
feedback loop for their expression so that mis-expression of any of these genes
acts on the feedback loop resulting in induction of the optic tectum in the
diencephalon. Otx2 and Gbx2 can repress the expression of each other and
contribute to the formation of the posterior border of the tectum.
Mis-expression of Otx2 in the metencephalon changed the fate of its alar plate
to the tectum. The anterior border of the tectum might be determined as a result
of repressive interaction of Pax6 with En1 and Pax2. Along the dorsoventral axis
of the mesencephalon, Shh contributes to the ventralization of the tissue, that
is, the area affected by Shh differentiates into the tegmentum. It is proposed
that the brain vesicle that expresses Otx2, Pax2 and En1 might differentiate
into the tectum.

Publication Types:
    Review

PMID: 11163885 [PubMed - indexed for MEDLINE]


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8: Development. 2001 Jan;128(2):181-91. 

EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse
mid/hindbrain region.

Liu A, Joyner AL.

Howard Hughes Medical Institute and Developmental Genetics Program, Skirball
Institute of Biomolecular Medicine, Department of Cell Biology, New York
University School of Medicine, New York, NY 10016, USA.

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required
for and sufficient to induce the formation of midbrain and cerebellar
structures. To address through what genetic pathways FGF8 acts, we examined the
epistatic relationships of mid/hindbrain genes that respond to FGF8, using a
novel mouse brain explant culture system. We found that En2 and Gbx2 are the
first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain
explants treated with FGF8-soaked beads. By examining gene expression in En1/2
double mutant mouse embryos, we found that Fgf8, Wnt1 and Pax5 do not require
the En genes for initiation of expression, but do for their maintenance, and
Pax6 expression is expanded caudally into the midbrain in the absence of EN
function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region,
forebrain mutant explants were treated with FGF8 and, significantly, the EN
transcription factors were found to be required for induction of Pax5. Thus,
FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor
other than FGF8 could be involved in initiating normal Pax5 expression in the
mesencephalon/metencephalon. The En genes also play an important, but not
absolute, role in repression of Pax6 in forebrain explants by FGF8. Previous
Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the
midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8
can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1
is initially broadly induced in Gbx2 mutant explants, as in wild-type explants,
but not subsequently repressed in cells near FGF8 that normally express Gbx2.
Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts
downstream of FGF8 in repression of Wnt1. This is the first such epistatic study
performed in mouse that combines gain-of-function and loss-of-function
approaches to reveal aspects of mouse gene regulation in the
mesencephalon/metencephalon that have been difficult to address using either
approach alone.

PMID: 11124114 [PubMed - indexed for MEDLINE]


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9: Development. 2000 Jun;127(11):2357-65. 

Pax6 defines the di-mesencephalic boundary by repressing En1 and Pax2.

Matsunaga E, Araki I, Nakamura H.

Department of Molecular Neurobiology, Institute of Development, Aging and
Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan.

Transcriptional factors and signaling molecules are responsible for
regionalization of the central nervous system. In the early stage of neural
development, Pax6 is expressed in the prosencephalon, while En1 and Pax2 are
expressed in the mesencephalon. Here, we misexpressed Pax6 in the mesencephalon
to elucidate the mechanism of the di-mesencephalic boundary formation.
Histological analysis, expression patterns of diencephalic marker genes, and
fiber trajectory of the posterior commissure indicated that Pax6 misexpression
caused a caudal shift of the di-mesencephalic boundary. Pax6 repressed En1, Pax2
and other tectum (mesencephalon)-related genes such as En2, Pax5, Pax7, but
induced Tcf4, a diencephalon marker gene. To know how Pax6 represses En1 and
Pax2, we ectopically expressed a dominant-active or negative form of Pax6. The
dominant-active form of Pax6 showed a similar but more severe phenotype than
Pax6, while the dominant-negative form showed an opposite phenotype, suggesting
that Pax6 acts as a transcriptional activator. Thus Pax6 may repress
tectum-related genes by activating an intervening repressor. The results of
misexpression experiments, together with normal expression patterns of Pax6, En1
and Pax2, suggest that repressive interaction between Pax6 and En1/Pax2 defines
the di-mesencephalic boundary.

PMID: 10804178 [PubMed - indexed for MEDLINE]


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10: Mech Dev. 1998 Dec;79(1-2):185-97. 

Neurons derived in vitro from ES cells express homeoproteins characteristic of
motoneurons and interneurons.

Renoncourt Y, Carroll P, Filippi P, Arce V, Alonso S.

INSERM U382, Developmental Biology Institute of Marseille (IBDM),
CNRS/INSERM/Universite de la Mediterranee/AP de Marseille, France.
renoncou@ibdm.univ-mrs

We have characterized different neuronal subpopulations derived from in vitro
differentiation of embryonic stem (ES) cells using as markers the expression of
several homeodomain transcription factors. Following treatment of embryo-like
aggregates with retinoic acid (RA), Pax-6, a protein expressed by ventral
central nervous system (CNS) progenitors is induced. In contrast, Pax-7
expressed in vivo by dorsal CNS progenitors, and erbB3, a gene expressed by
neural crest cells and its derivatives, are almost undetectable. CNS neuronal
subpopulations generated expressed combinations of markers characteristic of
somatic motoneurons (Islet-1/2, Lim-3, and HB-9), cranial motoneurons (Islet-1/2
and Phox2b) and interneurons (Lim-1/2 or EN1). Molecular characterization of
neuron subtypes generated from ES cells should considerably facilitate the
identification of new genes expressed by restricted neuronal cell lineages.

PMID: 10349632 [PubMed - indexed for MEDLINE]


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11: Development. 1997 Nov;124(22):4493-503. 

PAX2 is expressed in multiple spinal cord interneurons, including a population
of EN1+ interneurons that require PAX6 for their development.

Burrill JD, Moran L, Goulding MD, Saueressig H.

Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, CA 92037, USA.

Members of the PAX family of transcription factors are candidates for
controlling cell identity in the spinal cord. We have morphologically analyzed
cells that express one of these transcription factors, PAX2, demonstrating
multiple interneuron cell types express PAX2. Two ventral populations of
PAX2-expressing interneurons in the spinal cord are marked by coexpression of
the transcription factors, EN1 and EVX1. Interestingly, the expression domains
of PAX2, EN1 and EVX1 in postmitotic neurons correlate closely with those of
Pax6 and Pax7 in the ventricular zone, implicating these patterning genes in the
regulation of PAX2, EN1 and EVX1. We show that one of these patterning genes,
Pax6, is required for the correct specification of ventral PAX2+ interneurons
that coexpress EN1. These results demonstrate that the early activity of
patterning genes in the ventricular zone determines interneuron identity in the
spinal cord.

PMID: 9409667 [PubMed - indexed for MEDLINE]


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12: Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5709-13. 

Deregulated expression of PAX5 in medulloblastoma.

Kozmik Z, Sure U, Ruedi D, Busslinger M, Aguzzi A.

Research Institute of Molecular Pathology, Vienna, Austria.

Medulloblatoma is a pediatric brain tumor originating in the human cerebellum. A
collection of 23 medulloblastomas was analyzed for expression of the
developmental control genes of the PAX and EN gene families by RNase protection
and in situ hybridization. Of all nine PAX genes investigated, only PAX5 and
PAX6 were consistently expressed in most medulloblastomas (70 and 78% of all
cases, respectively), as were the genes EN1 (57%) and EN2 (78%). EN1, EN2, and
PAX6 genes were also expressed in normal cerebellar tissue, and their expression
in medulloblastoma is consistent with the hypothesis that this tumor originates
in the external granular layer of the developing cerebellum. PAX5 transcripts
were, however, not detected in the neonatal cerebellum, indicating that this
gene is deregulated in medulloblastoma. In the desmoplastic variant of
medulloblastoma, PAX5 expression was restricted to the reticulin-producing
proliferating tumor areas containing undifferentiated cells; PAX5 was not
expressed in the reticulin-free nonproliferating islands undergoing neuronal
differentiation. These data suggest that deregulated expression of PAX5
correlates positively with cell proliferation and inversely with neuronal
differentiation in desmoplastic medulloblastoma.

PMID: 7777574 [PubMed - indexed for MEDLINE]


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