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Both hindbrain roof plate epithelium (hRPe) and choroid plexus epithelium (hCPe) produce morphogens and growth factors essential for proper hindbrain development. Despite their importance, little is known about how these essential structures develop. Recent genetic fate maps indicate that hRPe and hCPe descend from the same pool of dorsal neuroectodermal progenitor cells of the rhombic lip. A linear developmental progression has been assumed, with the rhombic lip producing nonmitotic hRPe, and seemingly uniform hRPe transforming into hCPe. Here we show that hRPe is not uniform but rather is comprised of three spatiotemporal fields, differing in organization, proliferative state, order of emergence from the rhombic lip, and molecular profile of either the constituent hRPe cells themselves and/or their parental progenitors. Only two fields contribute to hCPe. We also show an hCPe contribution directly by the rhombic lip at late embryonic stages when hRPe is no longer present; indeed the production interval for hCPe by the rhombic lip is surprisingly extensive. Further, we show that the hCPe lineage appears unique among the varied rhombic lip-derived lineages in its proliferative response to constitutively active Notch1 signaling. Collectively, these findings provide a new platform for investigating hRPe and hCPe as neural organizing centers and provide support for the model that they are themselves patterned structures, likely capable of influencing neural development along multiple spatial and temporal axes.
Roof plate epithelium (RPe), through secretion of patterning signals and growth factors, induces adjacent neuroectoderm to produce specific neuron subtypes (Lee et al., 2000; 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 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 roof plate epithelium 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 critical 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 et al., 1995). An initial defining feature of RPe and its progenitor cells is 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, may have the capacity to influence neural patterning along the AP as well as DV axis.
Further distinguishing hRPe, for example from spinal cord RPe, is its proposed generation of hindbrain choroid plexus epithelium (hCPe), a ribbon-like cuboidal epithelium which 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, was found to be comprised of lineage-restricted compartments (Awatramani et al., 2003). A linear progression in development has been assumed, with the rhombic lip producing nonmitotic 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 through its secretion of morphogens and growth factors, including BMPs and fibroblast growth factors (Emerich et al., 2005).
Here we extend our understanding of how hRPe and hCPe develop. We show that hRPe is comprised of three molecularly distinct spatiotemporal fields with only two appearing to contribute to hCPe. Further, we show that hCPe receives contributions directly from the rhombic lip at late embryonic stages when hRPe seems no longer present. This supports a model of hCPe development in which transformation through an hRPe intermediate is not required and counters the view that hCPe formation is strictly a conservative process. Further, we show that the hCPe lineage appears unique among rhombic lip-derived lineages in that it proliferates in response to ligand-independent activation of the Notch1 pathway. Together, these findings offer a new platform for investigating development, function, aging, and regeneration of the hRPe and hCPe, and support the model that they are themselves patterned, segmental structures, perhaps capable of influencing neural diversity along multiple spatial and temporal axes.
Mouse lines include: Gdf7::cre (Lee et al., 2000); En1-cre (Kimmel et al., 2000); Egr2::cre (Voiculescu et al., 2000); Rse2::cre (Awatramani et al., 2003); Wnt1::cre (Chai et al., 2000); Math1::cre (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 (Awatramani, Farago and Dymecki, unpublished Flpe-responsive nβgal indicator); RC::PFttc (Farago and Dymecki, unpublished Cre-responsive nβgal indicator); RC::PFwe (Farago et al., 2006); R26::stop-Notch1-ICD::IRES-nGFP (Murtaugh et al., 2003).
For each developmental stage, pregnant females were injected intraperitoneally with 30 μg of BrdU (Roche) per gram of body weight.
Intraperitoneal TAM (Sigma) injections were given as follows: injection time point→harvest time point=dose per 40 g body weight. E7.5→E11.5=6mg; E8.5→E11.5= 7mg; E9.5→E11.5=8mg; E7.5→E16.5=5mg; E8.5→E16.5=6mg; E9.5→E16.5=7mg; E10.5→E16.5=7mg; E11.5→E16.5=8mg; E12.5→E16.5=9mg; E13.5→E16.5=10mg; E14.5→E16.5=11mg; E15.5→E16.5=12mg (Hunter et al., 2005).
Embryos were prepared for cryosection (30 μm) and mRNA ISH (Hunter et al., 2005). Riboprobes directed against the following mRNAs were used: AP-2α (Genbank 3983850, Open Biosystems), Barhl1 (Genbank AI324745, Research Genetics), Bmp7 (C. Cepko), Br (C. Tabin), Gdf7 (K. Millen), 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), and Wnt1 (A. McMahon). Detection of βgal 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.
For co-detection of either βgal 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-βgal (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 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 βgal 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-βgal 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.
Recent genetic fate maps show that the mouse hRPe at ~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 βgal (nβgal) as lineage tracer (Farago et al., 2006). We coupled this new Cre-responsive nβgal indicator allele with recombinase transgene Egr2::cre (Voiculescu et al., 2000) to map hRPe emerging from rhombomeres (r) 3 and 5. As predicted (Awatramani et al., 2003), nβgal+ 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 (lat, Fig. 2B). 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 (med, Fig. 2B) (Fraser et al., 1990). Interestingly, this newly observed medial hRPe population appears to disperse anteriorly to a greater extent than posteriorly (also seen when using an r2-specific Cre driver (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 are 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, while 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 βgal indicator mice to distinguish hRPe cells (βgal+) 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) while medially-located hRPe cells (Fig. 2C,F medially-located green cells) disperse among mesenchymal cells (Fig. 2C blue cells and Fig. 2F red cells). While this dispersion of medial hRPe among mesenchyme is reminiscent of some neural crest, we detect no expression of various neural crest markers (e.g. AP-2α, Pax3, Sox9, (Chan, 2003; Zhao et al., 1997)) in these cells (Supplemental Fig. 1).
To determine whether hRPe cells in these two fields differ in their proliferative capacity, immunodetection of the nuclear antigen Ki-67 was performed: Wnt1::cre; Cre-responsive βgal indicator mice were used in order to distinguish, by βgal expression, hRPe cells from neighboring mesenchyme and epidermal ectoderm. hRPe at E9.5 showed co-localization of βgal and Ki-67 whether medially- or laterally-located (white arrowheads, Fig. 2D,E). 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 is becoming mitotically quiescent, while laterally located hRPe cells are quiescent immediately upon emerging from the rhombic lip (white arrowhead, Fig. 2G, inset).
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 nonmitotic 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 with a single dose of tamoxifen (TAM) at different stages, 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 likely emerge from the rhombic lip during a ~12 hour window, starting ~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. While 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 likely 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 may be involved in their production. Wnt1 and Wnt1::cre are expressed in dorsal hindbrain neuroepithelium but not hRPe or neighboring mesenchyme (Supplemental Fig. 2 and data not shown); cumulative fate mapping using doubly transgenic mice (Wnt1::cre; Cre-responsive βgal 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 begins between ~E9.25-9.5, with Cre activity in Gdf7::cre; Cre-responsive βgal indicator mice first detectable at ~E9.5, presenting as βgal 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 expressed Wnt1 but not yet Gdf7, while cells of the lateral hRPe field derive from antecedents that expressed both.
In addition to deriving from temporally and molecularly distinct progenitor cells of the rhombic lip, the two hRPe fields themselves differ in molecular expression. Lateral but not medial hRPe fields express genes such as Bmp7, Gdf7, Lmx1a, Kcne2 (the latter encoding a potassium voltage-gated channel protein (Lundquist et al., 2006)), and transthyretin (TTR, encoding the carrier protein for thyroid hormone (Harms et al., 1991; Herbert et al., 1986)) (Fig. 4A–C, D–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–C, D–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 βgal activity in En1-cre; Cre-responsive βgal 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 Figure 4T: a medial field (yellow), an r2-r8 caudolateral field (light blue), and an r1 rostrolateral field (dark blue).
Given that a region of the hCPe appears to derive from r1 (En1-cre fate map in Fig. 4I,M,Q), we asked when r1-derived hRPe (field 3, Fig. 4T) or r1-derived hCPe become TTR+ and Kcne2+. This occurs after ~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 to those from r2-r8.
hRPe cells are thought to undergo shape changes beginning at ~E12 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 as relates to hCPe. Previously, we have shown that the hCPe is a compartmentalized structure, with little mixing of hCPe cells arising 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 (field 1, Fig. 4T) contributes substantially to hCPe. Because medial hRPe cells (field 1) appear to emerge from the rhombic lip earlier than lateral hRPe cells (fields 2 and 3), we can further address contribution by field 1 cells to the hCPe by extending to late embryonic stages our earlier presented temporal fate map of the Wnt1::FlpeERT2+ rhombic lip (Fig. 2H,I). Such studies would also define, for the first time, the production interval for hCPe.
Doubly transgenic embryos (Wnt1::FlpeERT2; Flp-responsive PLAP indicator) were given single doses of TAM at different embryonic stages, and the fate of rhombic lip descendant cells (PLAP+) determined as relates to hCPe at E16.5 (Fig. 5). Because neither hRPe nor hCPe cells express FlpeERT2, rather just cells in the rhombic lip (Fig. 5A-F), and because no recombinase activity is detected in the absence of TAM (Fig. 5G-L, insets), then PLAP activity in E16.5 hCPe cells should reflect rhombic lip as the spatial site of origin, while the time of TAM administration should define a temporal window of emergence from the rhombic lip. Only following TAM administration at time points spanning E9.5-E13.5 were PLAP+ cells observed in the E16.5 hCPe (Fig. 5G-K). Earlier (E7.5 and E8.5) (Supplemental Fig. 3) and later (E14.5, Fig. 5L) administrations failed to result in detectable hCPe cell marking. These results suggest an hCPe production interval by the rhombic lip that spans ~E10–14.
Corroborating this hCPe production interval are our results obtained following injection of the S-phase tracer BrdU. We performed analyses in doubly transgenic embryos (Wnt1::cre; Cre-responsive βgal indicator) to distinguish hCPe cells (nβgal+) from overlying mesenchymal and ectodermal cells. Co-localization of BrdU and nβgal was detectable following single BrdU injections between E10.5-E13.5 (Fig. 5N-Q), with double-labeled hCPe cells most abundant following injection at E11.5-E12.5 (Fig. 5O,P). Few double-labeled cells were observed following BrdU injection at E9.5 (Fig. 5M) and none following injection at E14.5 (Fig. 5R). These findings predict a production interval spanning ~E9.5-13.5, 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 may generate cells that differentiate directly into hCPe.
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 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 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-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).
While these lineages demonstrated nGFP activity in doubly transgenic animals (Fig. 6L,P,T and data not shown), indicating expression of the Notch1-ICD transgene, only within the hRPe and hCPe lineages did we detect a proliferation response. On post natal day (P) 7, doubly transgenic Gdf7::cre; R26::stop-Notch1-ICD::IRES-nGFP mice showed substantial expansion of the hCPe (Fig. 6E,G singly versus F,H doubly transgenic); indeed, the cerebellum and midbrain appear pushed forward as a consequence (Fig. 6H). Moreover, brain ventricles were expanded and CSF was in excess, reminiscent of hydrocephalus (Lindeman et al., 1998). 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 two hours later hCPe were co-immunostained for GFP (marker for hCPe cells expressing Notch1-ICD) and BrdU. ~20% of hCPe cells were co-labeled (Fig. 6L), suggesting that when Notch1-ICD is expressed, normally nonmitotic 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 cochlear nucleus and 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).
Here we present evidence for a model in which the hRPe is comprised of at least three spatiotemporal fields, differing in organization, proliferative state, order of emergence from the rhombic lip, and molecular profile of either the constituent cells and/or their parental cells. Only two of these hRPe fields appear to contribute to hCPe. We show that hCPe also receives contributions directly from the rhombic lip at late embryonic stages without seeming to transition through a morphologically hRPe-type intermediate. We define for the first time the temporal interval for hCPe production, revealing a surprisingly long period spanning ~E10–14. Further, we show that the hCPe lineage appears unique among rhombic lip-derived lineages in that hCPe cells, normally nonmitotic, proliferate exuberantly in response to constitutive Notch1 signaling, while neuronal lineages arising from the Math1+ rhombic lip do not.
hRPe has been considered to serve two major roles: as a dorsal organizing center (Lee et al., 2000; Lee and Jessell, 1999; Liem 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 may 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 may have the capacity to differentially influence hindbrain cell fate along AP and temporal axes.
We show that the E11.5 hRPe is comprised of at least three fields distinguished by spatial, temporal, molecular, and architectonic parameters (schematized in Figs. 2J, ,3I,3I, ,4T).4T). Field 1 cells occupy the dorsal midline. They emerge early from the rhombic lip (~E8-E9.5), arising from a Wnt1+ but Gdf7- progenitor cell population, and disperse extensively along 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 nonmitotic, and those arising from different rhombomeric levels do not intermix nor do they mix with overlying mesenchyme. Thus, the adhesion properties of cells in fields 2 and 3 result in compartmentalization along both 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 only after E12.5.
In the formation of hCPe, a nonmitotic 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 (~E12.5–14) appears to contribute directly to hCPe when hRPe seems no longer present, suggesting that some hCPe cells likely arise without transforming through an hRPe intermediate or that the intermediate is short-lived.
Contribution to hCPe by field 1 is unlikely because we fail to detect areas of hCPe that: (1) have emerged from the rhombic lip between E8-E9.5 (as assayed by temporal genetic fate mapping as well as by BrdU birthdating) and (2) are comprised of an admixture of cells derived from different rhombomeres. Rather, the hCPe appears built from the rhombomere-based lineage-restricted compartments of fields 2 and 3 (Fig. 4M,Q and (Awatramani et al., 2003)). Field 1 cells may 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 in either 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 production of these different fields. Regulators likely 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.
While hRPe fields 2 and 3 give rise to hCPe, they differ in onset of hCPe markers: TTR and Kcne2 mRNA are detectable in field 2 cells upon their emergence from the rhombic lip beginning at ~E9.5, remaining detectable into adulthood, while these mRNAs are detectable in field 3 only after ~E12.5. 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 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 sought 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 ~E12.5–13.5, the cerebellar rhombic lip stops generating neurons destined for cerebellar nuclei and starts producing precursors for 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 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 if 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 do not emerge later from the rhombic lip than field 2 cells; therefore, events that control this delay in field 3 gene expression likely occur after constituent cells have emerged from the rhombic lip. The molecular boundary between fields 2 and 3 is 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).
We present evidence that the interval for hCPe production spans from ~E10–14, with contributions arising via transformation of hRPe cells in fields 2 and 3, but not field 1. Further, our data suggests 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 nonmitotic roof plate cells which 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 critical to hCPe function arises solely through changes in cell shape – a densely packed pseudostratified roof plate epithelium 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.
We show that the normally nonmitotic 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 do not proliferate abnormally in response to Notch1-ICD. Are hCPe cells somehow poised to re-enter the cell cycle? In adult rats, quiescent 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 may 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 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 may 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 to rhombic lip-descendant (Math1-descendent) neurons for example, in their ability to proliferate in response to ligand-independent Notch1 signaling. This finding may have important implications for understanding choroid plexus tumor biology as well as the potential application of hCPe cells in CNS therapeutics and drug delivery.
We acknowledge R. Awatramani for helpful discussions; A. Farago, H. DiPietrantonio, C. Nielsen, and J. Mai for technical assistance; T. Jessell, A. Joyner, D. Melton, D. Rowitch, and S. Schneider-Maunoury for mouse strains; C. Cepko, A. McMahon, K. Millen, C. Tabin, and C. Walsh for riboprobe templates. Support for SMD and NLH include NIH/R21/HD044915; P01/HD0363379, and R01/NS047750.