Download Regionalization of the nervous system 2

Regionalization of the
nervous system 2
September 21, 2016
Yasushi Nakagawa
Department of Neuroscience
Thursday, September 22
Coffee Hour: 10:15-11:15am at Syrdyk’s Cafe in
Northrop Auditorium
A big picture for regionalization
-Early anterior-posterior patterning of vertebrate neural tissue is closely linked to
neural induction
-There are four major steps in regionalization of the brain:
1. Ectodermal cells acquire neural identity (neural induction)
BMP inhibition results in the formation of anterior neural tissue.
2. Adoption of crude positional character (anterior vs posterior)
Opposition between caudalizing factors and their inhibitors (especially Wnts and Wnt
inhibitors) establish crude AP patterning.
3. Formation of cell populations (“secondary organizers”) within the neural tissue that
secretes signaling molecules (morphogens)
4. These secondary organizers modulate and refine initial regional patterning such that
the expression domains of newly induced genes begin to subdivide the neural plate into
discrete territories that prefigure the various structures of the mature CNS.
-Same molecular pathways (Wnt, BMP, FGF, RA, Shh, etc.) play a role in more than one
steps at different times and places.
Gross AP patterning in early neural
plate results in the formation of
a sharp border
Conclusions
P
A
Wnt
BMP
Figure 6 | Double-gradient model of embryonic axis
formation. The model shows how perpendicular activity
gradients of Wnts and bone morphogenetic proteins (BMPs)
regulate head-to-tail and dorsal–ventral patterning. The colour
scales of the arrows indicate the signalling gradients; arrows
indicate the spreading of the signals. Patterning begins at
Growth-factor antagonists that are secreted by the
Spemann–Mangold organizer are at the heart of a
three-dimensional coordinate system of positional
information that functions during axial patterning in
the vertebrate gastrula.
gradient(s)
Otx2 The Nodal signalling
Gbx2
is crucial to set this process in motion by inducing both
Wnt and BMP growth factors as well as their antagonists at different doses (FIG. 5). A modernized doublegradient model can therefore be proposed78, in which
orthogonal BMP and Wnt gradients pattern the DV and
AP axis (FIG. 6). In the classical models that were put forward for amphibian embryos, the AP-graded factor was
proposed to be both mesoderm-inducing as well as
posteriorizing2. The two processes were indistinguishable because mesoderm induction is accompanied by
induction of posteriorizing factors. Today, mesoderm
induction and posteriorization can be uncoupled,
with Nodals inducing mesoderm, and Wnts, FGF and
retinoic acid acting as posteriorizing agents. Gastrulation elaborates the DV–AV Nodal gradient into two
orthogonal gradients of BMP and Wnt. BMP antagonists
A
P
Vieira et al., 2010
Secondary organizers are formed
within the neural tissue
Otx2
Gbx2
Vieira et al., 2010
ANR: anterior neural ridge
ZLI: zona limitans intrathalamica
IsO: isthmic organizer
Anterior neural ridge (ANR)
-formed at the junction between most anterior
neural tissue (anterior commissure later forms
here) and non-neural ectoderm
-requires AVE for its formation
-expresses Fgf8.
-ectopic Fgf8 induces rostral forebrain phenotypes
in more caudal tissue
-Fgf8 is required for the telencephalic identity
-Fgf8 also regulate the AP polarity of the cerebral
cortex (discussed later).
Vieira et al., 2010
ANR polarizes the cerebral cortex
later in development
Sanes Fig.2.29
Fgf8 and Fgf15 are
expressed in the ANR
region, which is
located at the anterior
pole of the future
cerebral cortex in
mammals.
Sanes Fig.2.28
Areas in the mature cortex
M: motor area
S: somatosensory area
A: auditory area
V: visual area
Primary somatosensory area of
rodents can be identified
histologically
Li and Crair (2011)
Ectopic expression of Fgf8 changes areal
organization of the cerebral cortex
rostrally
Wp1
Wp2
anterior
medial
lateral
posterior
Sanes Fig.2.29
Wp1: original somatosensory map
Wp2: new somatosensory map
C: Increasing Fgf8 expression in the anterior cortex by in vivo electroporation results in
the expansion of anterior cortical areas, such as the motor area (red)
D: Ectopic Fgf8 expression by in vivo electroporation in the caudal cortex causes the
formation of duplicated, mirror image of the somatosensory map.
Fgf8 controls differential gene
expression in cortical progenitor cells
Fgf8 suppresses the
expression of Emx2 and
Coup-TF1, two transcription
factors expressed in anteriorlow, posterior-high gradients
in the immature cortex in
mice.
Decreasing the expression of
Fgf8 results in increased
expression of Emx2 and
Coup-TF1
O’Leary et al. (2007) Neuron 56: 252-269
Fgf8 regulates cortical patterning by
controlling gene expression network
Analysis of knockout and
over-expression mice show
that Emx2 and Coup-TF1
impart the posterior cortical
area identity.
Fgf8 suppresses the
posterior cortical area identity
by suppressing the
expression of Emx2 and
Coup-TF1 in anterior cortical
progenitor cells
Other transcription factors
such as Pax6 and Sp8 are
also involved in this gene
regulation network.
O’Leary et al. (2007) Neuron 56: 252-269
Isthmic organizer (IsO)
-formed at the junction between midbrain and
hindbrain
-controls the regionalization of the midbrain and
anterior hindbrain
-requires repressive interaction between
transcription factors Otx2 and Gbx2 for its formation
-IsO expresses Fgf8.
Vieira et al., 2010
Grafted IsO or Fgf8-soaked beads
induced midbrain and cerebellum in
the host forebrain
-reminiscent of Spemann’s organizer transplant
experiment
(re-patterning of the surrounding neuroepithelium
upon transplantation)
-Fgf8-soaked beads mimic the effects of sO
transplantation.
-The induced ectopic midbrain is correctly
polarized, suggesting that Fgf8 has a role in
patterning the midbrain, as it has a patterning
role in the neocortex
Eschevaria et al. 2003
Zona limitans intrathalamica (ZLI)
ZLI is located within the diencephalon, immediately
anterior to the thalamus.
ZLI is critical for AP patterning of the thalamus.
Vieira et al., 2010
Hindbrain is divided into seven
rhombomeres
Hindbrain is segmented in a
progressive process and is
divided into seven discrete units
called rhombomeres.
Cell sorting mechanisms create
segregated groups of cells that
adopt distinct characteristics (e.g.,
cranial nerves)
Sanes, Fig.2.6
Segmental expression patterns of
mammalian Hox genes in the
hindbrain and spinal cord
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b4
b5
b6
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a10
a11
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Hoxb5
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Hoxb4
Hoxa4
Hoxb3
Hoxb4
Hoxb2
Hoxd4
Hoxb1
Forelimb
r1
r2
r3
r4
r5
Rhombomeric hindbrain
r6
r7
1
2
3
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Occipital
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Cervical
Thoracic
PSM
Lumbar
Alexander et al. (2009)
Functional support for the role of Hox genes
in regulating the segmental identity of rhombomeres has come through analyses of phenotypes arising from loss- and gain-of-function
experiments in several species (Lumsden 2004,
Maconochie et al. 1996, Rijli et al. 1998, Moens
& Prince 2002). The requirement for Hox proteins in many different tissues often results in
complex defects in Hox mutants. Moreover,
ties, genetic s
the role of H
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Access provided by University of Minnesota - Twin Cities - Law Library on 09/17/15. For personal use only.
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Expression of Hox genes are
Revie
regulated by signals from the somite
-Retinoic acid (RA) level is highest near the caudal end of
the hindbrain.
a
RA
Hoxb1
←−−−−−−−−−
Krox20
Figure 2
Kreisler
vhnf1
iro7
pr1
pr2
pr3
pr4
pr5
pr6
pr7
Pre-rhombomeric hindbrain
b
Krox20
?
Krox20
Hoxb1
Hoxb2
-RA binds to receptors in the cell body, which translocates to
the nucleus and acts as a transcription factor. Many Hox
genes are directly regulated by the RA receptor.
-Adding RA during early embryogenesis results in expansion
of posterior hindbrain at the expense of the anterior
hindbrain. Hox gene expression patterns change
accordingly.
RA
Hoxa1
Hoxa2
vhnf1
Kreisler
Hoxa3
Hoxb3
RA
Hoxa4
Hoxb4
Hoxd4
r1
r2
r3
r4
r5
r6
Rhombomeric hindbrain
r7
Gene patternin
hindbrain. (a) N
the rhombome
represents the
by the Raldh2 (
the somites flan
response to RA
(both in green)
retinoic acid re
their 3′ flankin
expression of K
territory. In the
between iro7 (
the hindbrain i
further subdivi
paralogs, Krox2
(b) Network at
The borders of
hindbrain coin
earlier expressi
become localiz
this time Hoxa1
the hindbrain.
mediated by cr
group 2 paralog
upstream regul
in response to R
green)]. Throug
the Cyp26 fam
( yellow backgrou
the caudal end
display higher
rhombomeres,
shading for Ho
the Hox genes a
expression of H
Alexander et al. (2009)
Hox genes regulate the
segmental identity of the
hindbrain
Hoxa1 knockout mice:
-r5 is lost
-r4 and r6 are fused.
fundamental DV landmarks, ends rostrally at the molecularly distinct midline that separates the mamillary bodies
(the latter are currently assigned to the basal plate; see
Figures 10.1–10.3). The floor plate is primarily coextensive
with its inducer, the notochord, a relationship known as
being ‘epichordal.’ This viewpoint, recently incorporated
into the prosomeric model (Puelles et al., 2012a), importantly implies that the entire forebrain including the
hypothalamus and the telencephalon is fundamentally
since they are continuous from left to right across the terminal wall, both at neural plate stages and in the neural
tube (Puelles, 1995, 2001; Shimamura et al., 1995). This
portion of the midline is thus best understood as a singular transversal landmark at the terminal midline, extending topologically from ventral (floor) to dorsal (roof). Its
diverse prospective subregions within the hypothalamus therefore can be interpreted conveniently, even if
paradoxically, as being all equally rostralmost, akin to
Is the rostral part of the brain
segmented like the hindbrain?
191
10.2 NEURAL PLATE, CLONAL STRUCTURE, AND TOPOLOGIC SPATIAL DIMENSIONS
AHP
ac Poa
Eye
Pall
FIGURE 10.2
Secondary
prosencephalon
TG
THy
hp2
PHy
ch
Tel (evaginated)
hp1
p2
Alar–basal
boundary
RP
Isthmic
organizer
AP
BP
Th
St
Ep
m1
PThE
Pal
p1 PT
FP
PTh
Dg
Midbrain
m2
Cb
Prepontine
Pontine
Hindbrain choroidal roof
Hindbrain
Septal
roof plate
POA
p3
ZI
ub
hp2
ac
Midbrain
m1
m2
Cb
ntia nigra
sta
RM
PRM
M
hp1 RTu
PM
THy
Pa
SPa
Tu
Eye
Pontomedullary
PT
p1
Di
zli
PIsth
pc
n
phalo
ce
n
e
Th
p2
zli
Diencephalon
BIC
E
hbc
Hb
PTh
p3
SC
IC
Forebrain choroidal
roof
hp
m
rm sth
Model of neural plate topology of
the fundamental AP and DV subdivisions. Some parts
are colored for contrast. The spinal cord is shortened
for simplicity.
S
Sbpall
OP
Septal roof
isth
Cephalic
flexure
cbc
r1
r2
ch
r4
PHy
NH
Hindbrain
r3
Pons
r5
r6
r7
r8
r9 r10
r11
Medullary
Spinal cord
Spinal cord
Telencephalon
Alar hypothalamus
Basal hypothalamus
Puelles (2013)
FIGURE 10.3
Model of further subdivisions
(including
neuromeric ones) in
the neural
tube. Roof and floorcan
plate areas
are infurther
gray (AC, anterior
Based on morphology and gene
expression
pattern,
forebrain
and
midbrain
be
commissure), and choroidal roof tissue in black. The colored areas are the same marked in Figure 10.2, adding the pontine hindbrain region in blue,
that (Puelles
separates the prepontine
hindbrain
(isth, r1, r2) from the pontomedullary
(r5, r6), leaving caudally the medullary hindbrain (r7–r11).
subdivided into “prosomeres”
and
Rubenstein,
1993, hindbrain
2003).
Dotted lines indicate shared alar DV subdivisions within the forebrain–midbrain tagma as well as main basal DV subdivisions in the hypothalthe terminal wall of the forebrain has to be regarded as a
dorsoventrally organized part of the neural wall, like the
lateral walls, though it is singular in occupying the midline (Puelles, 1995, 2001; Puelles et al., 2012a,b), whereas
the floor plate is a longitudinally organized brain zone.
Kingsbury (1922) was the first author who proposed
that the neural floor plate does not reach the anterior
neural ridge (Figure 10.2; Puelles, 1995; Shimamura
et al., 1995). On the basis of the peculiar histologic
appearance of the hindbrain floor, which displays a
median astroglial raphe that seemed to end rostrally at
the isthmic fossa, he held that the floor plate ends at
the prospective isthmus (at the midbrain–hindbrain border; Figure 10.1). However, Johnston (1923) corrected
analogous floor plate glial specialization found along
the ventral midline of midbrain and diencephalon,
which ends roughly at the mamillary pouch (see also
Kuhlenbeck, 1973; Puelles, 1995; Puelles et al., 1987a).
alar midbrain
region by
has
been reproduced as an inset to show its major subregions.
Johnston’samus.
(1923) The
description
was corroborated
observation of an early epichordal strip of midbrain and diencephalic median floor cells that differentially express
acetylcholinesterase (AChE; Puelles et al., 1987a). A
handful of floor plate gene markers (e.g., Shh, Ntn1,
Lmx1b, Nr4a2) have become known subsequently that I. INDUCTION AND PATTERNING OF THE CNS AND PNS
clearly stop rostrally at mamillary level, jointly with
the primary rostral end of the notochord (Puelles et al.,
2012a; see the Allen Developing Mouse Brain Atlas).
Note that a direct contact of the notochord with the neu-
Complex morphology makes it difficult to define the true A-P axis and divide the forebrain
into domains, but expression of Shh and its target genes (e.g., Nkx2.2) helps the definition
of A-P axis. This concept is crucial to study molecular mechanisms of regionalization.
being ‘epichordal.’ This viewpoint, recently incorporated
into the prosomeric model (Puelles et al., 2012a), importantly implies that the entire forebrain including the
hypothalamus and the telencephalon is fundamentally
ing topologically from ventral (floor) to dorsal (roof).
diverse prospective subregions within the hypotha
mus therefore can be interpreted conveniently, even
paradoxically, as being all equally rostralmost, akin
DV patterning
TG
SC
IC
ch
Tel (evaginated)
alon
eph
c
en
Th
p2
zli
PT
p1
Pal
10.2 NEURAL PLATE, CLONAL STRUCTURE, AND TOPOLOGIC SPATIAL DIMENSIONS
AHP
Sbpall
OP
Septal roof
ac Poa
Eye
Pall
FIGURE 10.2
Secondary
prosencephalon
Septal
roof plate
PHy
m
rm sth
hp1
zli
p2
RP
Isthmic
organizer
AP
BP
Th
hp2
ac
Eye
Midbrain
m1
m2
Cb
ntia nigra
isth
Cephalic
flexure
cbc
r1
r2
ch
r4
PHy
NH
Hindbrain
r3
Pons
r5
Ep
r6
r7
r8
r9 r10
r11
p1 PT
FP
m1
Midbrain
m2
Cb
POA
u
ta
bs
RM
PRM
M
hp1 RTu
PM
THy
Pa
SPa
Tu
PTh
p3
Diencephalon
Alar–basal
boundary
Dg
Forebrain choroidal
roof
hp
ZI
Model of neural plate topology of
the fundamental AP and DV subdivisions. Some parts
are colored for contrast. The spinal cord is shortened
for simplicity.
THy
hp2
PTh 191
p3
S
Di
PThE
PIsth
pc
Hb
St
BIC
E
hbc
Prepontine
Pontine
Hindbrain choroidal roof
Hindbrain
Pontomedullary
Medullary
Spinal cord
Telencephalon
Alar hypothalamus
Basal hypothalamus
FIGURE 10.3
Four domains along DV axis
-roof plate (most lateral/dorsal)
-alar plate
-basal plate
-floor plate (most medial/ventral)
Spinal cord
Model of further subdivisions (including neuromeric ones) in the neural tube. Roof and floor Puelles
plate areas
are in gray (AC, ante
(2013)
commissure), and choroidal roof tissue in black. The colored areas are the same marked in Figure 10.2, adding the pontine hindbrain region in b
The notochord controls the dorsalventral polarity of the neural tube
B. Removal of the notochord
results in the loss of the floor
plate and motor neurons.
C. Ectopic transplantation of
the notochord near the dorsal
neural tube induces a second
floor plate and ectopic motor
neurons.
Sanes, Fig.2.22
Sonic hedgehog (Shh) controls the
ventral identity of the neural tube
Shh is a homologue of the Drosophila segment
polarity gene, Hedgehog.
Notochord-derived Shh induces the expression
of Shh in the floor plate.
Shh is secreted and form a concentration
gradient along the DV axis.
Sanes, Fig.2.26
Shh and BMP signals antagonize each
other in DV patterning
Non-neural ectoderm produces BMPs, which
induces the expression of BMPs in the roof
plate.
Shh and BMP signals antagonize each other to
define the identity of neural progenitor cells
along the dorsal-ventral axis
Neural crest is derived from the lateral border of
the neural plate (green), which contribute to
many cell types including neurons of the
peripheral nervous system (e.g., the sensory
neurons and autonomic neurons, Schwann
cells).
Sanes, Fig.2.26
2.1 GENERAL PRINCIPLES
OF MORPHOGEN GRADIENTS
the 1980s t
was define
1988a,b). T
soon therea
Anderson,
have been d
teins; exam
scription fa
derivative
Sonic hedgehog (Shh) controls the
ventral identity of the neural tube
2.1.1 History of the Morphogen
and Morphogenetic Field
The concept of a morphogen can be traced to the turn
of the twentieth century, when Morgan postulated the
presence of ‘formative substances’ as the basis for different regeneration rates in worms (Morgan, 1901). Very
soon thereafter, Boveri entertained this idea for normal
development (Boveri, 1901). A seminal event for this
field was the discovery of a localized source for morphogens known as the Spemann organizer (Spemann and
Mangold, 1924). The term ‘morphogen’ was coined by
Turing, who described how uniformly distributed signals made by cells can spread, self-organize, and generate pattern (Turing, 1952). The Turing process remains
highly relevant, but for this chapter, the more relevant
concept is that of nonuniform graded distributions of
morphogens, an idea formalized in the famous ‘French
Flag’ model of Wolpert (Figure 2.1; Wolpert, 1969).
In this model, Wolpert described smoothly declining
gradients of morphogen concentration within a ‘morphogenetic field’ of cells. These gradients were imagined
to arise via diffusion from a localized source toward a
sink, thus giving cells within the morphogenetic field
different positional values based on morphogen concentration. The positional values then determined the fates
adopted by cells in the field (Figure 2.1). It was not until
2.1.2 Ho
The conc
derstandin
is erroneou
nants of cel
toire of mo
ontogeny a
of cell type
phogens co
on their ow
different pr
petencies in
provided b
ple, this cha
netic prot
fibroblast g
formation
neural pote
The defi
specify tw
dependent
essary to e
(Freeman a
between th
Briscoe, 20
boundaries
boundaries
the express
specify cel
1975). Und
mation is co
tive) chang
central pro
as will be
Different dorsal-ventral domains of
neural progenitor cells are defined
by differential expression of
transcription factors.
Ribes and Briscoe, (2009) Cold Spring
Harbor Perspectives in Biology
Morphogen
The differential expression patterns
of transcription factors are
established by graded Shh
signaling.
2
1
Distance from source
Sonic hedgehog (Shh) controls the
ventral identity of the neural tube
Establishment of spatial organization of
neurons from ventral progenitor cells in the
spinal cord is regulated by Shh signaling.
Distinct subtypes of interneurons (V0-V3) and
motor neurons (MN) are generated from each
of the six progenitor domains.
Ribes and Briscoe, (2009) Cold Spring
Harbor Perspectives in Biology
Regionalization
-summary
Anterior-posterior difference in the future nervous system is formed early in development,
and the early mechanism is linked tightly to neural induction.
During and after gastrulation, organizer region and its mesodermal derivatives
(notochord, prechordal plate) send signals for neural induction (e.g., BMP inhibition) and
the anterior fate (e.g., Wnt inhibition). Anterior mesoendodermal tissue (e.g., AVE) also
antagonizes caudalizing factors (Fgf, RA, Wnt, etc.) produced by caudal mesoendoderm
(e.g., primitive streak). This initial mechanism roughly divides the nervous system into
anterior and posterior halves.
”Secondary organizers” are formed in the neural tissue and produce signaling
molecules. These signals further refine the grossly patterned nervous system into smaller
domains of neural progenitor cells (both along AP and DV axes). Establishment of these
distinct progenitor pools is crucial for the generation of different types of neurons. This
process is mediated by a network of transcription factors.
AP patterning of the Drosophila embryos is a prototype for the research on vertebrate
nervous system, and has provided a number of important genes whose vertebrate
homologues play crucial roles.
How can we apply the knowledge to
ES cell-mediated neurogenesis?
Gaspard and Vanderhaeghen (2010)
From fibroblasts to neurons
-via iPS cells
(induced pluripotent stem cells)
o be determined before they
ransplantation in patients.
mising, there are still many
t make it difficult to translate
rmalities, at the genetic and
SCs are a major hurdle that
SC-derived neurons can be
g. These aberrations include
Mol Neurobiol (2012) 45:586–595
-Similar strategies are used to
generate specific types of neurons
from ES cells and iPS cells.
-There are methods that bypass the
formation of iPS cells, either directly
generating neural stem cells or even
neurons.
cytokine leukemia inhibitory factor and cell signaling molecules such as Wnts [48, 51–53]. Chemicals, such as histone
deacetylase (HDAC) inhibitors and DNA methyltransferase
inhibitors, have also been used by several groups to enhance
fibroblast reprogramming [54, 55]. The promising technological development of such chemicals provides great hope
that patient-specific iNs can be generated and implanted
without the risk of patients developing tumors due to onco-
Use of iPSCs in translational
neuroscience
Therapeutic use: transplantation of iPSCs-derived neurons or glial cells into damaged
brain
replacement
chaperone effects
Modeling of human diseases
generate iPSCs from patients with various neurological or psychiatric disorders
differentiate iPSCs in vitro into neurons or glia and identify defects of patient-derived
cells
3D culture system has allowed the formation of “organoids” that mimic the eye or the
cerebral cortex
screen for drugs that ameliorate the defects