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First published online 29 August 2007
doi: 10.1242/dev.003095
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Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
* Author for correspondence (e-mail: dymecki{at}genetics.med.harvard.edu)
Accepted 20 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Roof plate, Neural patterning, Choroid plexus, Genetic fate map, Inducible recombinases, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lee and Jessell, 1999). In
addition to patterning adjacent neuroectoderm, RPe derives from neuroectoderm
(Awatramani et al., 2003;
Landsberg et al., 2005) -
specifically, from cells situated most laterally in the neural plate, which
ultimately come to reside in the dorsal midline following transformation of
the neural plate into the neural tube. In most anteroposterior (AP) regions of
the neural tube, RPe thus appears as a morphologically distinct strip of cells
at the dorsal midline (Fig.
1D). At the level of the hindbrain, however, RPe becomes an
expansive dorsolateral sheet of cells. This is because closure of the neural
plate to form a tube is transient in the hindbrain [occurring at around
embryonic day (E)9]; instead of remaining closed at the dorsal midline as a
tube, the edges of the hindbrain neural plate flare out laterally as the
hindbrain flexes (Fig. 1A,B),
with the RPe needing to tent over an expansive fourth ventricle (4v)
(Fig. 1A-C). Regardless of
whether RPe is a large sheet of cells, as in the hindbrain (hRPe,
Fig. 1A,B), or a thin midline
strip, as in other brain regions (RPe, Fig.
1D), it secretes morphogens crucial for neural tube patterning
along the dorsoventral (DV) axis; for example, members of the bone
morphogenetic protein (BMP) family
(Chizhikov and Millen,
2004b).
Differentiation of RPe from neuroectoderm requires signals from neighboring
epidermal ectoderm (Lee et al.,
2000; Lee and Jessell,
1999; Liem, Jr et al.,
1995). An initial defining feature of RPe and its progenitor cells
is the expression of Lmx1a, a LIM-homeodomain transcription factor
(Chizhikov and Millen, 2004b;
Millonig et al., 2000). In
spinal cord, RPe formation requires Lmx1a
(Chizhikov and Millen, 2004a;
Millen et al., 2004;
Millonig et al., 2000);
however, in the hindbrain, only certain AP regions of the hRPe are lost in the
absence of Lmx1a (Chizhikov et al.,
2006; Manzanares and Krumlauf,
2000; Millonig et al.,
2000), hinting at possible molecular heterogeneity within the
hRPe. Consistent with such heterogeneity, we have shown previously that hRPe
is segmented along the AP axis such that hRPe derived from different
rhombomeres (different AP levels) do not intermix
(Awatramani et al., 2003) - a
contrast to floor plate cells, the ventral counterpart of the roof plate,
where extensive mixing occurs among cells derived from different rhombomeres
(Fraser et al., 1990). The
hRPe, as a result of these AP variations, might have the capacity to influence
neural patterning along the AP as well as the DV axis.
A finding that further distinguishes hRPe, for example from spinal cord
RPe, is its proposed generation of hindbrain choroid plexus epithelium (hCPe),
a ribbon-like cuboidal epithelium that produces cerebrospinal fluid (CSF) and
serves as the blood-CSF barrier (Redzic et
al., 2005; Segal,
2000). Recent genetic fate maps suggest that the hCPe (a
late-embryonic/adult structure suspending into the 4v) and the hRPe (an
early/transient structure tenting over the 4v) descend from a common
progenitor cell pool comprising dorsal-most neuroectodermal territory referred
to as the rhombic lip (Fig.
1A,B) (Awatramani et al.,
2003; Landsberg et al.,
2005). Furthermore, hCPe, like hRPe, appears to comprise
lineage-restricted compartments (Awatramani
et al., 2003). A linear progression in development has been
assumed, with the rhombic lip producing non-mitotic hRPe, and hRPe
transforming into the more expansive hCPe
(Thomas and Dziadek, 1993;
Wilting and Christ, 1989)
without cell gain (Dziegielewska et al.,
2001; Kappers,
1955; Sturrock,
1979). The enormous increase in surface area of hCPe versus hRPe
has been viewed as arising solely through changes in cell shape - densely
packed pseudostratified hRPe spreads out to form the simple cuboidal hCPe
(Dohrmann, 1970). In addition
to producing CSF, the hCPe is thought to play a patterning role during late
embryogenesis via its secretion of morphogens and growth factors, including
BMPs and fibroblast growth factors
(Emerich et al., 2005).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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);
En1::cre (En1-cre)
(Kimmel et al., 2000);
Egr2::cre (Voiculescu et al.,
2000); Rse2::cre (Rse2 refers to a rhombomere
2-specific enhancer) (Awatramani et al.,
2003); Wnt1::cre (Chai
et al., 2000); Math1::cre (Math1 is also known
as Atoh1 - Mouse Genome Informatics)
(Matei et al., 2005);
Wnt1::Flpe (Awatramani et al.,
2003); Wnt1::FlpeERT2
(Hunter et al., 2005);
R26R (Soriano, 1999);
R26::FRAP (Awatramani et al.,
2001); RC::Fa (R. Awatramani, A. F. Farago and S.M.D.,
unpublished; Flp-responsive nßgal indicator); RC::PFttc (A. F.
Farago and S.M.D., unpublished; Cre-responsive nßgal indicator);
RC::PFwe (Farago et al.,
2006); R26::stop-Notch1-ICD::IRES-nGFP
(Murtaugh et al., 2003).
Bromodeoxyuridine (BrdU) injections
For each developmental stage, pregnant females were injected
intraperitoneally with 30 µg of BrdU (Roche) per gram of body weight.
Tamoxifen (TAM) administration
Intraperitoneal TAM (Sigma) injections were given as follows: injection
time point to harvest time point = dose per 40 g body weight. E7.5 to E11.5 =
6 mg; E8.5 to E11.5 = 7 mg; E9.5 to E11.5 = 8 mg; E7.5 to E16.5 = 5 mg; E8.5
to E16.5 = 6 mg; E9.5 to E16.5 = 7 mg; E10.5 to E16.5 = 7 mg; E11.5 to E16.5 =
8 mg; E12.5 to E16.5 = 9 mg; E13.5 to E16.5 = 10 mg; E14.5 to E16.5 = 11 mg;
E15.5 to E16.5 = 12 mg (Hunter et al.,
2005).
In situ hybridization (ISH), X-gal and alkaline phosphatase (PLAP) histochemistry
Embryos were prepared for cryosection (30 µm) and mRNA ISH
(Hunter et al., 2005).
Riboprobes directed against the following mRNAs were used:
AP-2
(GenBank 3983850, Open Biosystems), Barhl1
(GenBank AI324745, Research Genetics), Bmp7 (C. Cepko, Harvard
Medical School, MA), Br (C. Tabin, Harvard Medical School, MA),
Gdf7 (K. Millen, University of Chicago, IL),
FlpeERT2 (Hunter et
al., 2005), Kcne2 (C. Cepko), Lmx1a (K. Millen),
Math1 (mAtoh1) (GenBank BC010820, Open Biosystems),
Notch1 (C. Cepko), Pax3 (C. Tabin), Sox9 (C.
Tabin), Ttr (C. Walsh, Harvard Medical School, MA), and Wnt1
(A. McMahon, Harvard University, MA). Detection of ßgal on whole tissue
and sections was performed as described
(Farago et al., 2006).
Detection of PLAP followed established protocols
(Hunter et al., 2005), except:
slides were post-fixed in 4% PFA, dehydrated in methanol, cleared in benzyl
alcohol:benzyl benzoate (1:2, Sigma) and rehydrated.
Immunodetection
For co-detection of either ßgal and BrdU or GFP and BrdU, embryos were
soaked in 30% sucrose/PBS for 3 hours at 4°C, embedded in OCT
(Tissue-Tek), sectioned (30 µm), fixed in 4% PFA/PBS for 10 minutes,
blocked in 5% goat serum and 0.1% Triton X-100/PBS (BB), incubated overnight
with rabbit anti-ßgal (MP Biomedicals, 1:5000) or rabbit anti-GFP
(Molecular Probes, 1:5000) in BB at 4°C, incubated with goat anti-rabbit
Cy2 (Jackson ImmunoResearch, 1:500) in BB for 3 hours at room temperature
(RT), incubated overnight at 4°C with rat anti-BrdU (Serotec, 1:500) in
BB, incubated with goat anti-rat Cy3 (Jackson ImmunoResearch, 1:500) in BB for
3 hours at RT, exposed to 1 µg/ml DAPI (Sigma), and mounted. For
co-detection of ßgal and Ki-67 or GFP and Ki-67, the above protocol was
used except: embryos were fixed in 0.2% PFA for 3 hours prior to sucrose;
combined primary antibodies rabbit anti-ßgal and mouse anti-Ki-67 (BD
PharMingen, 1:200) or rabbit anti-GFP and mouse anti-Ki-67 were incubated at
4°C; combined secondary antibodies anti-rabbit Cy2 and anti-mouse Cy3
(Jackson ImmunoResearch) were incubated at RT.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E11 is comprised
of lineage-restricted compartments along the AP dimension
(Awatramani et al., 2003) - a
contrast to the AP cell dispersion characterizing floor plate
(Fraser et al., 1990). Here,
we re-examined our earlier finding of hRPe segmentation using a genetic fate
mapping indicator allele with improved sensitivity due to encoding a
nuclear-localized ßgal (nßgal) as lineage tracer
(Farago et al., 2006). We
coupled this new Cre-responsive nßgal indicator allele with recombinase
transgene Egr2::cre (Voiculescu
et al., 2000) to map hRPe emerging from rhombomeres 3 and 5 (r3
and r5). As predicted (Awatramani et al.,
2003), nßgal-positive (nßgal+) cells were found in
stripes in the lateral hRPe at E11.5, supporting the model that these
cells develop in a segmental, lineage-restricted fashion
(Fig. 2B, lat). Not predicted
was our detection of a midline hRPe field harboring an admixture of cells
derived from different rhombomeres - reminiscent of the cell dispersion
reported for floor plate (Fig.
2B, med) (Fraser et al.,
1990). Interestingly, this newly observed medial hRPe population
appeared to disperse anteriorly to a greater extent than posteriorly [also
seen when using an r2-specific Cre driver (Rse2::cre)
(Awatramani et al., 2003), data
not shown]; the basis for such differential cell movement is unknown.
Next, we examined whether compartments versus admixtures of hRPe cells were
present earlier. At E9.5, we found AP mixing of nearly all hRPe cells
(Fig. 2A). Together with the
finding that hRPe arises from dorsal-most neuroectoderm within the rhombic lip
(Awatramani et al., 2003;
Landsberg et al., 2005), these
results suggest a model whereby hRPe cells emerging from the rhombic lip at
E9.5 mix along the AP axis, whereas hRPe cells emerging later constrain to AP
compartments and reside laterally.
These two hRPe fields, defined by either compartmentalization or dispersion
along the AP dimension, were also found to differ dorsoventrally. Using doubly
transgenic Wnt1::cre; Cre-responsive ßgal indicator
mice to distinguish hRPe cells (ßgal+) from neighboring mesenchyme and
overlying epidermal ectoderm, we found that laterally located hRPe cells
(Fig. 2C,G, laterally located
green cells) segregate from overlying mesenchyme and epidermal ectoderm
(Fig. 2C,G, and cartooned in
Fig. 1C), whereas medially
located hRPe cells (Fig. 2C,F,
medially located green cells) disperse among mesenchymal cells
(Fig. 2C, blue cells and
Fig. 2F, red cells). Although
this dispersion of medial hRPe among mesenchyme is reminiscent of some neural
crest cells, we detected no expression of various neural crest markers [e.g.
AP-2, Pax3 and Sox9 (Chan, 2003;
Zhao et al., 1997)] in these
cells (see Fig. S1 in the supplementary material).
To determine whether hRPe cells in these two fields differed in their proliferative capacity, immunodetection of the nuclear antigen Ki-67 was performed: Wnt1::cre; Cre-responsive ßgal indicator mice were used in order to distinguish, by ßgal expression, hRPe cells from neighboring mesenchyme and epidermal ectoderm. hRPe at E9.5 showed co-localization of ßgal and Ki-67 whether medially or laterally located (Fig. 2D,E, white arrowheads). By contrast, at E11.5, little co-labeling was observed, with an occasional cycling hRPe cell detected medially but only quiescent hRPe cells laterally (Fig. 2F,G). Thus, by E11.5, midline hRPe was becoming mitotically quiescent, whereas laterally located hRPe cells were quiescent immediately upon emerging from the rhombic lip (Fig. 2G, inset, white arrowhead).
Our data thus far suggest that, at E11.5, there are two distinct hRPe
fields differing in adhesion properties and proliferative capacity: a lateral
field of non-mitotic cells organized into lineage-restricted compartments
versus a medial field of an admixture of cells from different axial levels of
which a few are mitotic. Similarities between the medial hRPe field at E11.5
and earlier hRPe cells at E9.5 suggest a production sequence whereby medially
located hRPe cells emerge from the rhombic lip prior to those situated
laterally, with both populations having migrated dorsomedially from the
rhombic lip. To address this suggested production sequence, we generated a
temporal fate map of hRPe progenitors. We partnered with a Flp-responsive
alkaline phosphatase (PLAP) indicator allele
(Awatramani et al., 2001), an
inducible version of Flpe recombinase [FlpeERT2
(Hunter et al., 2005)]
expressed under the control of Wnt1 regulatory elements
(Fig. 1E). By activating
FlpeERT2 at different stages with a single dose of tamoxifen (TAM),
we were able to activate PLAP as a lineage tracer in temporally sequential
cohorts of Wnt1-expressing progenitor cells in the rhombic lip and
determine their fate within the E11.5 hRPe. In these experiments, PLAP
activation occurred within the rhombic lip, as opposed to within the
descendant cells themselves, because the latter do not express Wnt1
or FlpeERT2 (Hunter et
al., 2005), and because the half-life of FlpeERT2 is
relatively short and thus does not perdure into descendant cells. Further,
hRPe cells marked by PLAP probably emerge from the rhombic lip during an
approximate 12 hour window, starting at around 12 hours post-TAM
administration (Hunter et al.,
2005).
Following E9.5 TAM administration, and therefore E10.0-E10.5 lineage
tracer activation, we observed at E11.5 an abundance of PLAP+ cells throughout
the lateral but not medial hRPe field (Fig.
2I). Following E7.5 TAM administration, and therefore
E8.0-E8.5 lineage tracer activation, we detected at E11.5 scattered PLAP+
cells in both medial and lateral hRPe fields
(Fig. 2H) - albeit few in
number because the Wnt1::FlpeERT2 transgene (like
endogenous Wnt1) is just beginning to be expressed at this early time
point with little FlpeERT2 protein yet present. Although limited by
both low Wnt1 expression and dose of tamoxifen that can be given at
this early time point, these findings nonetheless support a model whereby TAM
administration at E7.5 triggers recombination events within rhombic lip cells
at E8, with these cells subsequently undergoing asymmetric divisions -
one set of daughter cells emerging dorsomedially from the rhombic lip to
populate the medial hRPe field while the other set remains cycling in the
rhombic lip to later give rise to progeny hRPe cells that situate laterally.
In this model, generation of the medial hRPe field is largely complete by
E10, after which progenitor cells of the rhombic lip give rise to lateral
fields. Thus, medial-to-lateral position within the E11.5 hRPe probably
corresponds to a temporal axis of cell production from the rhombic lip.
Having uncovered two hRPe fields differing in position, tissue
organization, proliferation and time of emergence from the rhombic lip, we
asked whether the respective parental progenitor cells for each field differ
molecularly, reflecting that different genetic programs might be involved in
their production. Wnt1 and Wnt1::cre were expressed in
dorsal hindbrain neuroepithelium but not in hRPe or neighboring mesenchyme
(see Fig. S2 in the supplementary material, and data not shown); cumulative
fate mapping using doubly transgenic mice (Wnt1::cre; Cre-responsive
ßgal indicator) showed extensive cell marking throughout the
entire hRPe at E11.5 (Fig. 3A-D
and Fig. 2C-G). By contrast,
Gdf7 and Gdf7::cre expression in the rhombic lip began
between E9.25 and E9.5, with Cre activity in Gdf7::cre;
Cre-responsive ßgal indicator mice first detectable at
E9.5, presenting as ßgal activity in the rhombic lip and lateral but
not medial hRPe fields (Fig.
3E-H). Thus, cells constituting the medial hRPe field derive from
progenitor cells that express Wnt1 but not yet Gdf7, whereas
cells of the lateral hRPe field derive from antecedents that expressed
both.
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)] and transthyretin [Ttr, encoding the carrier
protein for thyroid hormone (Harms et al.,
1991; Herbert et al.,
1986)] (Fig. 4A-F,
and data not shown). The expression profiles of Kcne2 and
Ttr not only distinguish lateral from medial hRPe but also identify
an unexpected AP subdivision within the lateral field
(Fig. 4A-F). At E11.5,
Kcne2 and Ttr transcripts were detectable in lateral hRPe
spanning axial levels r2-r8, but not in lateral hRPe territory situated at the
level of r1 [the latter territory demarcated by ßgal activity in
En1::cre (En1-cre); Cre-responsive ßgal
indicator mice (Machold and Fishell,
2005; Zervas et al.,
2004)]. Thus, the hRPe at E11.5 appears divisible into at least
three domains, shown in Fig.
4T: a medial field (yellow), an r2-r8 caudolateral field (light
blue) and an r1 rostrolateral field (dark blue).
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E12.5 (Fig. 4L), 3
days later than that observed for field 2
(Fig. 4B,E). Thus, although
emerging simultaneously from the rhombic lip, lateral roof plate derivatives
from r1 show a temporal lag in molecular fate as compared with those from
r2-r8.
hRPe fields contribute differentially to choroid plexus epithelium
hRPe cells are thought to undergo shape changes beginning at E12 in
order to form directly the entire hCPe
(Lindeman et al., 1998).
Having established that the hRPe is comprised of at least three fields
distinguished in their spatial, temporal and molecular development, we sought
to determine whether these differences correlate with cell fate with respect
to hCPe. Previously, we have shown that the hCPe is a compartmentalized
structure, with little mixing of hCPe cells that arise from different
rhombomeres (Awatramani et al.,
2003). Indeed, we observed this further in the present r1
(En1::cre) fate map of the hCPe
(Fig. 4M,Q). Because areas of
hCPe harboring an admixture of marked and unmarked cells were not observed, it
seems unlikely that the medial hRPe (Fig.
4T, field 1) contributes substantially to hCPe. Because medial
hRPe cells (field 1) appeared to emerge from the rhombic lip earlier than
lateral hRPe cells (fields 2 and 3), we can further address the contribution
by field 1 cells to the hCPe by extending our earlier presented temporal fate
map of the Wnt1::FlpeERT2+ rhombic lip
(Fig. 2H,I) to late embryonic
stages. Such studies would also define, for the first time, the production
interval for hCPe.
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E10-E14.
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E9.5-E13.5,
consistent with that determined by genetic inducible fate mapping. Together,
these findings are consistent with a model whereby only lateral hRPe cells
(fields 2 and 3) contribute to the hCPe. Interestingly, by E12.5, the
epithelium tenting over the 4v appears morphologically to be hCPe and not hRPe
(cuboidal versus pseudostratified, respectively). Therefore, at these later
stages of development (E12.5-E14), the rhombic lip might generate cells
that differentiate directly into hCPe.
Differential response among rhombic lip lineages to ligand-independent activation of the Notch1 pathway
In many CNS regions, activation of the Notch signaling pathway in a cell
sustains its capacity to proliferate
(Louvi and Artavanis-Tsakonas,
2006) and thus imposes a progenitor cell-like identity. Multiple
Notch genes are expressed in the ventricular zone of the hindbrain, including
in the rhombic lip territory (Lindsell et
al., 1996) (Fig.
6B). Using a R26::stop-Notch1-ICD::IRES-nGFP allele
(Murtaugh et al., 2003), we
expressed the intracellular domain of Notch1 (Notch1-ICD) - the constitutively
active fragment - and a nuclear-localized green fluorescent protein (nGFP) in
two different progenitor cell populations of the rhombic lip and their
descendant lineages: (1) the dorsal-most progenitor cells in the rhombic lip
(Gdf7+) and their progeny cells - lateral hRPe and hCPe; or (2)
Math1 (Atoh1)-expressing progenitor cells of the
rhombic lip and their progeny cells - including precerebellar mossy fiber
neurons, cerebellar and cochlear nuclei granule cells
(Farago et al., 2006;
Landsberg et al., 2005;
Wang et al., 2005).
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). In addition, each hCPe cell normally attaches to a basement
membrane (Thomas et al.,
1988); by contrast, hCPe cells in doubly transgenic Gdf7::cre;
R26::stop-Notch1-ICD::IRES-nGFP animals were found in disorganized piles
(Fig. 6I,J), suggestive of a
compromised blood-CSF barrier.
Given the association between Notch signaling and proliferation
(Louvi and Artavanis-Tsakonas,
2006) together with the enlarged hCPe in doubly transgenic
Gdf7::cre; R26::stop-Notch1-ICD::IRES-nGFP animals, we asked whether
these hCPe cells had become mitotic. P0 doubly transgenic Gdf7::cre;
R26::stop-Notch1-ICD::IRES-nGFP animals were pulsed with BrdU and 2 hours
later hCPe cells were co-immunostained for GFP (marker for hCPe cells
expressing Notch1-ICD) and BrdU. Approximately 20% of hCPe cells were
co-labeled for GFP and BrdU following hCPe misexpression of Notch1-ICD and GFP
(Fig. 6L); by contrast, no hCPe
cells were co-labeled (Fig. 6K)
in wild-type animals (doubly transgenic Wnt1::Flpe; Flp-responsive
ßgal indicator animals; ßgal identifies the hCPe and BrdU
identifies cycling cells). These results (compare
Fig. 6L and 6K) suggest that
when Notch1-ICD is expressed, normally non-mitotic hCPe cells
(Kappers et al., 1958;
Knudsen, 1964;
Li et al., 2002;
Sturrock, 1979)
proliferate.
To assay for Notch1-ICD effects in Math1+ rhombic
lip-descendant cells, neurons in the pontine gray nucleus and granule cell
precursors in the cerebellum of doubly transgenic Math1::cre;
R26::stop-Notch1-ICD::IRES-nGFP animals were analyzed
(Farago et al., 2006;
Li et al., 2004). No gross
changes were observed in either the area occupied by or the density of
Barhl1+ cells - a marker for Math1-descendants
(Fig. 6M,N and Q,R).
Furthermore, neurons on route to or residing in the pontine gray nucleus
showed no proliferation in response to Notch1-ICD (yet were robustly GFP+,
indicating good expression of the Notch1-ICD transgene)
(Fig. 6O,P). Cerebellar granule
cell precursors expressing Notch1-ICD (GFP+) appeared to proliferate to the
same extent as seen in littermate controls
(Fig. 6S-V).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E10-E14. Further, we show that the
hCPe lineage appears unique among rhombic lip-derived lineages in that hCPe
cells, which are normally non-mitotic, proliferate exuberantly in response to
constitutive Notch1 signaling, whereas neuronal lineages arising from the
Math1+ rhombic lip do not. These results are summarized schematically
in Fig. 7.
Identification of distinct fields within the hindbrain roof plate
hRPe has been considered to serve two major roles: as a dorsal organizing
center (Lee et al., 2000;
Lee and Jessell, 1999;
Liem, Jr et al., 1997;
Millonig et al., 2000) and as
an intermediate epithelium that transforms into CSF-producing hCPe
(Thomas and Dziadek, 1993;
Wilting and Christ, 1989). As
an organizing center, the hRPe has been shown to influence dorsal
neuroectoderm to express distinct sets of transcription factors at different
DV positions, thereby establishing unique progenitor populations productive of
different neuron subtypes. Evidence has emerged to support the possibility
that the hRPe itself might differ along the AP axis
(Awatramani et al., 2003;
Chizhikov et al., 2006). Here,
we show that the hRPe is heterogeneous along multiple dimensions, suggesting
that, in addition to regulating hindbrain cell type production along the DV
dimension, the hRPe might have the capacity to differentially influence
hindbrain cell fate along the AP and temporal axes.
We show that the E11.5 hRPe comprises at least three fields distinguished
by spatial, temporal, molecular and architectonic parameters (schematized in
Fig. 2J,
Fig. 3I,
Fig. 4T). Field 1 cells occupy
the dorsal midline. They emerge early from the rhombic lip (E8-E9.5),
arising from a Wnt1+ but Gdf7-negative (Gdf7-)
progenitor cell population, and disperse extensively along the AP and DV axes,
mixing among hRPe cells derived from different rhombomeric levels as well as
among mesenchymal cells. Field 1 cells appear mitotic until E10.5.
By contrast, cells in fields 2 and 3 situate laterally and emerge from
double-positive Wnt1+/Gdf7+ progenitor cells in the rhombic
lip beginning at E9.5. Cells of fields 2 and 3 are non-mitotic, and those
arising from different rhombomeric levels neither intermix nor do they mix
with overlying mesenchyme. Thus, the adhesion properties of cells in fields 2
and 3 result in compartmentalization along both the AP and DV axes. Because
r1-derived cells of field 2 can be distinguished molecularly from the rest of
field 2 (derived from r2-r8), we have designated them as field 3. Transcripts
detectable in field 2 starting at E9.5 and onward, such as Ttr
and Kcne2, are detectable in field 3 after E12.5 only.
In the formation of hCPe - a non-mitotic structure - a linear developmental
progression has been assumed, with the pseudostratified hRPe spreading out to
become the monolayer cuboidal epithelium of the hCPe. This is thought to occur
entirely through the conservative process of cell shape change. It appears
that epithelial transformation is indeed an aspect of hCPe genesis, but
involving only fields 2 and 3. Further, rhombic lip at later time points
(E12.5-E14) appeared to contribute directly to hCPe when hRPe seemed no
longer present, suggesting that some hCPe cells probably arise without
transforming through an hRPe intermediate or that the intermediate is
short-lived.
Contribution to hCPe by field 1 is unlikely, because we failed to detect
areas of hCPe that: (1) emerged from the rhombic lip between E8-E9.5 (as
assayed by temporal genetic fate mapping as well as by BrdU birth-dating); and
(2) were comprised of an admixture of cells derived from different
rhombomeres. Rather, the hCPe appeared to be built from the rhombomere-based
lineage-restricted compartments of fields 2 and 3
(Fig. 4M,Q)
(Awatramani et al., 2003).
Field 1 cells might be eliminated, although we did not detect apoptotic
activity as assayed by TUNEL or Caspase 3 immunodetection (data not
shown).
Although the fate of field 1 hRPe cells remains unknown, during embryogenesis they show similarities with roof plate cells of the spinal neural tube: field 1 hRPe cells emerge temporally from the rhombic lip coincident with the production of spinal roof plate cells (sRPe), both hRPe and sRPe situate along the dorsal midline, both cell types exhibit some degree of mitotic activity, and neither express Ttr or Kcne2. Additionally, cells either in field 1 hRPe or sRPe (but not hRPe fields 2 and 3) exhibit mixing with neighboring RPe cells. Thus, it is possible that field 1 hRPe cells conform better to what is thought of as roof plate, and that the hRPe cells in fields 2 and 3 might be better classified as postmitotic hCPe precursor cells. We propose that, during embryogenesis, organizer function shifts from field 1 hRPe (perhaps `true' roof plate) to fields 2 and 3 hRPe (incipient hCPe), and ultimately to the hCPe itself at late stages. It will be important to determine what regulates the production of these different fields. Regulators probably include differential signals emanating from mesenchyme overlying the hRPe versus incipient hCPe, as well as intrinsic molecular differences in rhombic lip progenitor cells over the course of development.
|
E9.5, remaining detectable into adulthood, whereas these
mRNAs are detectable in field 3 after E12.5 only. What is the
significance of this temporal lag in molecular expression between cells in
field 3 versus 2? The hindbrain roof plate has been shown to be a source of
signaling molecules that are important for regulating progenitor cells in the
upper or cerebellar rhombic lip (Alder et
al., 1999; Chizhikov et al.,
2006), and this function is most likely served by immediately
adjacent roof plate territory (i.e. r1-derived field 3). We studied whether
the timing of molecular changes in field 3 might correspond with an event
occurring in the cerebellar rhombic lip; for example, a shift in cell type
production. Between E12.5 and E13.5, the cerebellar rhombic lip stops
generating neurons destined for cerebellar nuclei and starts producing
precursors of cerebellar granule cells
(Machold and Fishell, 2005).
Perhaps cells in field 3 remain less differentiated molecularly, less
`hCPe-like', in order to permit the cerebellar rhombic lip to first produce
neurons destined for the cerebellar nuclei, and later become more `hCPe-like'
(reflected in the expression of Ttr and Kcne2) in order to
induce production of cerebellar granule neurons by the cerebellar rhombic lip.
It will be important to establish whether such regulation of cell type
production is influenced by molecular changes occurring in field 3 and what
actual field 3-derived factors might be responsible. Field 3 cells did not emerge later from the rhombic lip than field 2 cells; therefore, events that control this delay in field 3 gene expression probably occur after constituent cells have emerged from the rhombic lip. The molecular boundary between fields 2 and 3 was sharp, coinciding with the r1-r2 boundary and latitude of hindbrain flexure. Future studies will involve assessing possible explanations for this exacting difference in molecular expression between fields 2 and 3, and whether, for example, the closely overlying mesenchyme situated above field 3 (rostral to hindbrain flexure) is different in its profile of cell-cell signaling molecules as compared to the mesenchyme situated above field 2 (caudal to hindbrain flexure).
Formation of the hindbrain choroid plexus epithelium is not simply a conservative transformation from pseudostratified to cuboidal cells
We present evidence that the interval for hCPe production spans from
E10 to E14, with contributions arising via transformation of hRPe cells
in fields 2 and 3, but not in field 1. Further, our data suggest that the
rhombic lip makes a direct contribution to the hCPe at later stages. This
model differs from the previously accepted view of a linear progression in
development, in which the rhombic lip is the source of non-mitotic roof plate
cells that transform to form hCPe in its entirety
(Dohrmann, 1970;
Dziegielewska et al., 2001;
Kappers, 1955;
Sturrock, 1979;
Thomas and Dziadek, 1993;
Wilting and Christ, 1989). In
this conservative model, the increased epithelial surface area crucial to hCPe
function arises solely from changes in cell shape - a densely packed
pseudostratified RPe spreads out into a simple monolayered cuboidal
epithelium. Here, we show that the addition of new cells to the hCPe by the
rhombic lip contributes to its expansion.
Selective ability of hindbrain Gdf7-derived cells to respond to constitutively active Notch1 signaling
We show that the normally non-mitotic hCPe lineage proliferates exuberantly
in response to constitutive expression of the intracellular domain of Notch1
(Notch1-ICD), and thus presumably in response to constitutive Notch1
signaling. By contrast, the rhombic lip-derived lineages characterized by
early Math1 expression did not proliferate abnormally in response to
Notch1-ICD. Are hCPe cells somehow poised to re-enter the cell cycle?
In adult rats, quiescent choroid plexus epithelium (CPe) cells in all three
ventricles gain BrdU reactivity by 2 hours post an ischemic event, with a
subset of these cells expressing neuronal nuclear antigen (NeuN) and glial
fibrillary acidic protein (GFAP); this has led to the conclusion that the
choroid plexus might harbor neural stem cells
(Li et al., 2002).
Transplantation studies in which 4v choroid plexus cells were grafted into
damaged rat spinal cord showed a gain in GFAP immunoreactivity and that at
least some of the choroid plexus cells differentiated into astrocytes
(Kitada et al., 2001). These
stem cell-like features might be further reflected in our observation of hCPe
proliferation in response to Notch1-ICD. Interestingly,
Notch3-ICD introduced into periventricular cells via retroviral
injection caused what were described as hindbrain choroid plexus tumors
(Dang et al., 2006).
In summary, we have shown that the hRPe is not a homogeneous cell population. Rather, it can be subdivided into at least three unique fields - cells in only two of these three fields contribute to the hCPe. We identify the temporal interval for hindbrain choroid plexus epithelium production and propose a novel process for its development. Our finding of spatial (mediolateral and anteroposterior), temporal and molecular differences among hRPe fields supports the model whereby the hindbrain roof plate and choroid plexus epithelium are complex structures that might exert patterning influences along AP and temporal, in addition to DV, axes. Our studies also highlight a unique property of rhombic lip-descendant hCPe cells, as compared with rhombic lip-descendant (Math1-descendant) neurons, for example, in their ability to proliferate in response to ligand-independent Notch1 signaling. This finding might have important implications for understanding choroid plexus tumor biology as well as the potential application of hCPe cells in CNS therapeutics and drug delivery.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/19/3449/DC1
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ACKNOWLEDGMENTS |
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