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The Journal of Neuroscience, June 11, 2008, 28(24):6152-6164; doi:10.1523/JNEUROSCI.5593-07.2008

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Development/Plasticity/Repair
A Role for Receptor Protein Tyrosine Phosphatase in Midbrain Development

Anja Badde and Dorothea Schulte

Department of Neuroanatomy, Max Planck Institute for Brain Research, 60528 Frankfurt, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mid-hindbrain boundary (MHB) harbors an important organizing center for the adjacent brain regions. Here, we present evidence that the receptor protein tyrosine phosphatase (RPTP) is part of the complex molecular network that maintains and shapes the MHB region. RPTP is expressed in a tight band of cells in the caudal midbrain, anterior to the transverse ring of Wnt1 expression. Forced expression of RPTP across the mid-hindbrain region repressed expression of Wnt1, whereas RNA interference-mediated knock-down of RPTP resulted in expansion and distortion of the Wnt1 domain. When ectopically expressed in the mesencephalon, RPTP specifically inhibited the induction of Wnt1 expression after subsequent stimulation with Fgf8. Reduced Wnt1 expression after RPTP transfection correlated with a decrease in Ras- mitogen-activated protein kinase activity at the MHB. We further show that in the embryonic midbrain, RPTP can bind to β-catenin, a central component of the canonical Wnt signaling pathway. Overexpression of RPTP suppressed the activity of a β-catenin responsive promoter in the midbrain and reduced progenitor cell proliferation. Cotransfection of Wnt1 or of a stabilized form of β-catenin together with RPTP partially rescued the RPTP-mediated proliferation defect. Together, these data suggest that RPTP may play a dual role in the control of midbrain development: as a negative modulator of Fgf8-induced Wnt1 expression at the MHB, which may help to confine the Wnt1 domain to it characteristic tight ring at the MHB; and as an inhibitor of canonical Wnt signaling through interaction with and presumably sequestration of β-catenin.

Key words: RPTP; Fgf8; Wnt1; β-catenin; midbrain; chick

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mid-hindbrain (MHB) (isthmic) organizer, located near the constriction between the developing mesencephalic and metencephalic vesicles, acts as a secondary organizer for the control of growth and pattern formation of the adjacent midbrain and hindbrain (Nakamura et al., 1986; Martinez and Alvarado-Mallart, 1990; Martinez et al., 1991). Its activity is regulated by an interdependent network of nuclear factors and signaling proteins, which includes the transcription factors En1/En2, Pax2/Pax5, Lmx1b, Hes1/Hes3, iro1/iro7, and members of the Fgf and Wnt families of secreted proteins (Bally-Cuif et al., 1992; Rowitch and McMahon, 1995; Araki and Nakamura, 1999; Shamim et al., 1999; Adams et al., 2000; Hirata et al., 2001; Itoh et al., 2002; Matsunaga et al., 2002; O'Hara et al., 2005).

Fibroblast growth factor 8 (Fgf8), expressed in the rostral hindbrain adjacent to the MHB, is of central importance for midbrain and hindbrain development. Beads soaked in recombinant Fgf8 and implanted in prosomeres p1, p2 of the diencephalon, in the midbrain or hindbrain can mimic the inductive capacity of isthmic transplants (Crossley et al., 1996; Martinez et al., 1999; Shamim et al., 1999; Irving and Mason, 2000). Conversely, zebrafish ace mutants, which lack functional Fgf8, and mice hypomorphic for Fgf8 fail to maintain gene expression at the MHB and display massive defects in midbrain and cerebellar development (Meyers et al., 1998; Reifers et al., 1998). An important target of Fgf8 at the MHB is the secreted glycoprotein Wnt1. After isthmic organizer formation, Wnt1 expression is restricted to a narrow band of cells in the caudal mesencephalon, anterior to the Fgf8 expression domain (Bally-Cuif et al., 1995; Hollyday et al., 1995). Wnt signaling is critically involved in mid-hindbrain development, because targeted deletion or tissue-specific overexpression of Wnt1 at the MHB lead to massive perturbation of mid-hindbrain growth (Thomas and Capecchi, 1990; McMahon et al., 1992; Panhuysen et al., 2004).

Reversible protein phosphorylation on tyrosine residues is a key mechanism underlying intracellular signal transduction. Protein tyrosine phosphatases (PTPs) antagonize the activity of protein tyrosine kinases (PTKs) and thereby limit the duration and intensity of the PTK signal (Stoker and Dutta, 1998; den Hertog, 1999; Paul and Lombroso, 2003; Tonks, 2006). Receptor protein tyrosine phosphatase (RPTP; also called RPTP), a member of the type IIb family of receptor tyrosine phosphatases, is expressed in a spatially and temporally dynamic manner in the embryo and has been shown recently to be necessary for somitogenesis and convergent extension during gastrulation (Aerne et al., 2003; Aerne and Ish-Horowicz, 2004). We reported previously the developmental expression of RPTP in the CNS and have shown that its expression is particularly dynamic anterior to the isthmic constriction (Badde et al., 2005). Here, we have taken a gain-of-function and loss-of-function approach to show that RPTP can modulate mesencephalic development through a dual mechanism: it inhibits the induction of Wnt1 expression by Fgf8, which may help to restrict the Wnt1 domain to its characteristic tight ring at the MHB, and binds to β-catenin and appears to thereby negatively modulate canonical Wnt signaling.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression constructs and in ovo electroporation. cDNAs encoding full-length mouse RPTP [National Center for Biotechnology Information (NCBI) accession number U55057] or RPTP-IC (corresponding to nucleotides 352–2701 of NCBI accession number U55057) were fused to a triple HA-tag and cloned into the expression vector pMES, which includes an internal ribosomal entry site (IRES)-green fluorescent protein (GFP) cassette (Swartz et al., 2001) or into the vector pMIWIII, which lacks IRES-GFP (Suemori et al., 1990). The coding sequence of chick RPTP was cloned using degenerate primers based on published sequences of the mouse, rat, and human orthologs and found to be mostly identical to NCBI accession number AY147868 (sequences are available on request). The coding sequences of chick Fgf8b, mouse Wnt1, or mouse En1 were cloned into pMIWIII. For dnRas^N17-pMES, a dominant-negative form of the Ras protein, which contains a serine to asparagine mutation at residue 17, was inserted into pMES (Clontech). Axin-pMES carried the full-length coding region of chick Axin, a generous gift from F. Costantini (Columbia University Medical Center, New York, NY) (Zeng et al., 1997). For caβ-catenin-pMIWIII, a stabilized form of β-catenin was cloned into pMIWIII (Funayama et al., 1995). For the rescue experiment with RPTP and Wnt1, the coding sequence of GFP in RPTP-pMES was replaced by a single ClaI site (RPTP-pMEC). The coding region of Wnt1 was then cloned into this ClaI site resulting in RPTP-Wnt1-pMEC. Unless otherwise noted, 1–2 µg/µl of each construct were injected into the neural tube of Hamburger–Hamilton stage 9–10 (HH9–HH10) chick embryos (White Leghorn) (Hamburger and Hamilton, 1992). Electroporation was performed as described, except that five pulses of 10 V for 50 ms were given to facilitate DNA uptake (Schulte et al., 1999). To visualize DNA uptake, 0.5–0.8 µg/µl of plasmids expressing enhanced GFP (GFP-pMIWIII), Discosoma red fluorescent protein (dsRed-pCMV), or a nuclear version of red fluorescent protein (nRFP-pCAGGS) were coelectroporated. To control for possible unspecific effects of the procedure, pMES (which carries IRES-GFP) or GFP-pMIWIII was introduced. For consecutive electroporation into the same region of the neural tube (see Fig. 3A), GFP with or without the full-length form of RPTP or the catalytically inactive allele RPTP-IC were introduced into the mesencephalic vesicle at HH9–10. Six hours after initial transfection, a time window known to be sufficient for robust transgene expression (Momose et al., 1999), an expression plasmid carrying the coding sequence of Fgf8b together with a plasmid carrying dsRed were electroporated into the same area that had received the first electroporation 6 h earlier. Only embryos that exhibited extensive coexpression of both fluorescent markers, indicating that both transgenes were targeted to the same region of the neural tube, were chosen for additional analysis. For TOP:dgfp reporter analysis, 1 µg/µl of the TOP:dgfp reporter construct, which drives expression of destabilized green fluorescent protein off the TOPFLASH promoter/enhancer (Korinek et al., 1997; Dorsky et al., 2002), were electroporated into the mesencephalic vesicle alone or in combination with 1.5 µg/µl RPTP-pMIWIII or 1.5 µg/µl RPTP-IC-pMIWIII. GFP expression was monitored and documented 24 h later.

Bead implantation. Heparin-coated acrylic beads (Sigma-Aldrich) were soaked in 0.015–0.15 mg/ml mouse recombinant Fgf8b protein (R&D Systems) overnight at 4°C, washed in sterile PBS, and split with forceps. Appropriately sized bead fragments were implanted into the lateral wall of the mesencephalic vesicle of HH11 chick embryos. The embryos were collected 24 h after bead implantation and processed for in situ hybridization.

RNA in situ hybridization. To ensure specificity of the probe, a partial sequence of chick RPTP was used for all in situ hybridization experiments (Badde et al., 2005). RNA probes for cGrg4, cSprouty2, and cSef were cloned from chick HH15–20 whole head total RNA by reverse transcriptase-PCR with gene-specific primers (primer sequences are available on request). The cDNAs used to generate in situ probes for cEn1, cFgf8, cLmx1b, cOtx2, cPax2, cPax5, or cWnt1 were gifts from C. Tabin (Harvard Medical School, Boston, MA), C. Logan (University of Calgary, Calgary, Alberta, Canada), or H. Rohrer (Max Planck Institute for Brain Research, Frankfurt, Germany). In situ hybridization on whole embryos was performed as described previously (Schulte et al., 1999). For open book preparation, the mesencephalic vesicle was opened along the dorsal midline and flat-mounted. Two-color in situ hybridization was performed as described with the exception that the first transcripts were detected using 5-bromo-4-chlor-indolyl-phosphate (BCIP) and nitroblue–tetrazolium–chloride as chromophores, which results in a dark blue/purple precipitate, whereas the second transcripts were detected with BCIP alone, which results in a bright blue/turquoise precipitate (Schulte and Cepko, 2000). Specimens were photographed with a Zeiss StemiSV11 microscope and a Canon PowerShot G5 camera or with a Leica MZ12 microscope and a Leica MPS60 camera. Brightness and contrast were adjusted in Adobe Photoshop (Adobe Systems). In Figure 1, C, D, D', I', and I'' were false colored in Adobe Photoshop for better visual contrast.

Immunohistochemical staining. Localization of ectopically expressed RPTP-HA or RPTP-IC-HA was visualized on cryosections using a polyclonal anti-HA antibody (1:1000; Roche Diagnostics). GFP expression was confirmed using a polyclonal antibody (1:1000; Invitrogen). The mitotic index was determined with a polyclonal anti-phosphorylated Histone H3 (pH3) antibody (1:1000; Upstate Biotechnology). All antibodies were diluted in PBS containing 5% Chemiblocker (Millipore Bioscience Research Reagents) and 0.5% Triton X-100 before use. Microscope fields with a magnification of 40x were documented either with an Axioplan2 (Zeiss), a FluoView 1000 confocal microscope (Olympus), or a LSM5 Pascal confocal microscope (Zeiss). Immunohistochemical detection of diphosphorylated Erk1/2 with the monoclonal anti-dpErk antibody (clone M-8159 Sigma-Aldrich) was performed as described by Corson et al. (2003).

RNA interference. For RNA interference (RNAi)-mediated knock-down of RPTP or Wnt1 expression, the psiSTRIKE-U6 Hairpin Cloning System was used (Promega). The short hairpin RNA target sequences directed against nonoverlapping regions of the cRPTP cytoplasmic domain were GTCAACATGACCAAAGCAA (RPTP_si-a) and GCTTCAAGCAGGAGTATGA (RPTP_si-b). The efficiency and specificity of the RNAi targeting constructs were assessed by luciferase reporter assay. Human embryonic kidney 293T (HEK293T) cells were cotransfected with the RNAi targeting constructs RPTP_si-a, RPTP_si-b, targeting constructs containing randomized sequences of RPTP_si-a or RPTP_si-b, respectively (GAACTACGTAAACCAGACA or GCATAGACGAATCGGTATG), or an unrelated targeting construct (GAAGGTACACGAATTATGT) together with a psiCHECK-2 reporter construct, which contained the intracellular portion of cRPTP fused to Renilla luciferase (1.5 µg of each construct/six-well plate; Promega). After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter 1000 Assay System (Promega) in a GloMax96 plate Luminometer (Promega). For in vivo analysis, RPTP_si-a or RPTP_si-b was electroporated into the neural tube at a concentration of 1.5 µg/µl together with GFP-pMIWIII (0.8–0.5 µg/µl) at the developmental ages indicated. Short hairpin RNA target sequences against Wnt1 were GGTCATCTACGGCAACAAA and GCGCCTCGAGGGTCATCTA. Simultaneous transfection of both RNA targeting constructs (1.5 µg/six-well dish) repressed the activity of a psiCHECK-2 reporter construct, which contained the coding region of chick Wnt1 fused to Renilla luciferase, to 34% compared with a targeting construct carrying the randomized sequence CGAATCAGTCAGTCCAGAA (data not shown).

Immunoprecipitation. RPTP-HA-pMES or RPTP-IC-HA-pMES was electroporated into the midbrain at HH10. After 24 h, the midbrain was dissected in ice-cold PBS and lysed in 150 mM NaCl, 10 mM Na-Phosphate buffer, pH 7.2, 1% NP-40, 1% Desoxycholate, 0.1% SDS, 50 mM Na-F, 0.2 mM Na-orthovanadate, and protease inhibitors (Complete tablets; Roche Diagnostics). Precipitation of the transgenes was achieved using a monoclonal, agarose-coupled anti-HA antibody (Sigma-Aldrich). Immunoprecipitation and Western blot analysis were performed as described previously (Mühleisen et al., 2006). Primary antibodies used for Western blot analysis were rat anti-HA high-affinity antibody (1:1000; Roche Diagnostics) or polyclonal anti-β-catenin (1:1000; Upstate Biotechnology). For stripping off the first set of antibodies, the blots were incubated in 0.1 M glycine, pH 2.5, for 30 min at 37°C.

Analysis of cell proliferation and programmed cell death. RPTP-HA-pMES or pMES were electroporated into the midbrain of HH9 chick embryos. After 24 h, bromodeoxyuridine (BrdU) was injected into the midbrain vesicle; labeling duration was 2 h. Detection of BrdU-positive cells was performed on 15-µm-thick cryosections using the BrdU Labeling and Detection Kit I (Roche Diagnostics) and colabeling with a GFP-specific antibody to visualize transfected cells. For analysis of programmed cell death, RPTP-pMIWIII together with nRFP-pCAGGS, or nRFP-pCAGGS alone, was electroporated into the midbrain vesicle. Detection of apoptotic cells was performed on cryosections using the Fluorescein In Situ Cell Death Detection Kit (Roche Diagnostics).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
RPTP is expressed in a domain tightly associated with the ring of Wnt1 expression at the MHB and is regulated by Fgf8
We reported previously that between the 19 and 40 somite stages (HH13–HH19), RPTP is expressed in a tight, transverse ring anterior to the isthmic constriction and to the transverse domain of Wnt1 expression (Fig. 1A,B) (Badde et al., 2005). To correlate the precise location of this ring to that of Wnt1 at the organizer, RPTP- and Wnt1-specific transcripts were visualized together in the same specimen by two-color in situ hybridization. Expression of the two molecules was found in tightly associated domains at HH14 (Fig. 1C) and HH19 (Fig. 1D,D'). Although both expression domains appear to slightly overlap, in all specimens analyzed, RPTP-specific transcripts were found rostral to the ring of Wnt1 expression that abuts the MHB. Therefore, during this developmental period, the Wnt1 domain at the MHB is bordered by a ring of RPTP-expressing cells at its anterior side. Based on these expression data, the RPTP expression domain at the MHB may lie within the future preisthmic domain, the caudal-most aspect of the mesencephalic alar plate, which is characterized by coexpression of Otx2, Pax2, and possibly Wnt1 (Hidalgo-Sánchez et al., 2005).

View larger version (73K): [in this window] [in a new window]   Figure 1. Expression and regulation of RPTP at the MHB. A, Expression of RPTP in a WT chick embryo at HH15. B, Expression of Wnt1 at HH17. C, Two-color in situ hybridization showing expression of RPTP in dark blue and Wnt1 in turquoise at HH14. The ring of Wnt1 expression at the MHB is bordered by RPTP-expressing cells at its rostral side. D, D', Open book preparation of a HH19 (E3.5) chick midbrain showing RPTP expression in dark blue and Wnt1 expression in turquoise. RPTP and Wnt1 expression occurs in tightly associated, partially overlapping domains at the MHB; anterior is to the top, and posterior is to the bottom. D', Higher magnification of the boxed area in D. E–I'', Whole-mount in situ hybridizations on HH15–HH16 chick embryos, electroporated with different expression constructs. In all panels, detected transcripts are indicated in the bottom left corner, and misexpressed transgenes are indicated in brackets in the bottom right corner. E, Expression of RPTP (dark blue) was not altered after misexpression of GFP (turquoise; 1.5 µg/µl). F, Transfection of 1 µg/µl of the expression plasmid Fgf8b-pMIWIII together with GFP-pMIWIII effectively repressed RPTP expression at the MHB. F', Two-color in situ hybridization on the embryo shown in F for RPTP (dark blue) and GFP (turquoise). G, G', Transfection of 0.05 µg/µl of Fgf8b-pMIWIII together with GFP-pMIWIII (turquoise) induced RPTP expression (dark blue) in the dorsal midbrain. G' is a higher magnification of the boxed area shown in G. H, RPTP expression at the MHB was lost after misexpression of the dominant-negative RasN17 together with GFP. H', Two-color in situ hybridization on the embryo shown in H (RPTP, dark blue; GFP, turquoise). I, Forced expression of Fgf8b (together with GFP) in the MHB region shifted the ring of RPTP expression rostrally around the transfected area. The image is an overlay of the GFP fluorescence photographed immediately after harvesting of the embryo and the RPTP expression domain as detected subsequently by in situ hybridization (dark blue). I', I'', Spatial relationship of RPTP (purple) and Wnt1 (turquoise) expression in the embryo shown in I. I'' is a higher magnification of the boxed area in I'. In all panels, upregulation of Wnt1 is indicated by arrowheads, and expression of RPTP (or lack thereof) is indicated by arrows. The asterisks in D mark upregulation of RPTP expression in the mesencephalic alar plate after HH18. C, D, D', I', and I'' are false colored for better visual contrast. fp, Floor plate; mes, mesencephalon; met, metencephalon. Scale bars: A, B, E–G, H–I, 500 µm; C, 250 µm.

 
As a first step to understanding RPTP function at the MHB, we investigated whether its expression was regulated by the secreted protein Fgf8. Fgf8 is expressed in rhombomere 1 adjacent to the isthmic organizer and can induce organizer characteristic gene expression and cell fate changes when ectopically applied in the caudal diencephalon or mid-hindbrain region (Crossley et al., 1996; Martinez et al., 1999). Transfection of 1 µg/µl to 0.1 µg/µl of an expression plasmid carrying the splicing isoform Fgf8b into the mid-hindbrain region of chick embryos has been shown previously to induce ectopic cerebellar development, whereas transfection of 0.01 µg/µl promoted tectal growth (Sato et al., 2001). We introduced an Fgf8b expression plasmid at different concentrations into the mid-hindbrain region together with an expression plasmid carrying GFP for visualization (Fig. 1F–G'). Expression of Wnt1 and RPTP at the MHB were unaltered after transfection with the GFP expressing plasmid alone compared with wild-type (WT) control embryos, demonstrating that in ovo electroporation per se does not affect gene expression at the MHB (Fig. 1E) (data not shown) (n = 36 of 37 for RPTP; n = 12 of 12 for Wnt1). Consistent with our observation that RPTP is not expressed in the hindbrain region during normal development, we found that widespread electroporation across the mid-hindbrain territory of 1 µg/µl of the Fgf8b-pMIWIII expression plasmid, a concentration known to stimulate cerebellar development, effectively repressed RPTP expression at the MHB (n = 9 of 10) (Fig. 1F,F'). Electroporation of 0.01 µg/µl of the Fgf8b expression plasmid, a concentration that enhanced midbrain growth in a previous study (Sato et al., 2001), did not alter RPTP expression (n = 7 of 7) (data not shown). However, RPTP-specific transcripts were upregulated in the midbrain vesicle at an intermediate concentration of 0.05 µg/µl Fgf8b-pMIWIII (n = 3 of 5) (Fig. 1G,G'). Fgf8b misexpression at either concentration was unable to stimulate RPTP expression in the hindbrain, supporting the idea that metencephalic tissue is not permissive for RPTP expression (n = 18 of 18) (Fig. 1F–G') (data not shown). Together, these results suggest that RPTP expression at the isthmic organizer requires a distinct Fgf8 signaling level, which is below the level required for metencephalic development but above the level that stimulates tectal growth. Fgf8 signal transduction at the MHB appears to be mediated primarily through the Ras-mitogen-activated protein (MAP) kinase pathway (Corson et al., 2003; Sato and Nakamura, 2004). To confirm the dependency of RPTP expression on Fgf8 signaling at the MHB, we misexpressed a dominant-negative form of Ras (dnRas^N17), which was shown previously to effectively block Fgf8 signal transduction at the isthmic organizer (Sato and Nakamura, 2004). RPTP expression was consistently lost in cells forced to express dnRas^N17, demonstrating that RPTP expression at the MHB indeed requires Fgf8/Ras-MAP kinase pathway activity (Fig. 1H,H') (n = 8 of 9).

Next, we targeted the Fgf8b expression plasmid (at a concentration of 1 µg/µl) to a narrow region surrounding the isthmic organizer and analyzed the expression of RPTP and Wnt1 together in the same specimen 24 h later (Fig. 1I–I''). The domain of RPTP expression was shifted rostrally around the region targeted for Fgf8 misexpression (recognizable by the fluorescence of the coelectroporated GFP; n = 9 of 10) (Fig. 1I, arrow), consistent with the idea that RPTP expression is inhibited by strong Fgf8b signals but induced at a distance from the Fgf8b source, where Fgf8b protein levels are expected to be lower. When Wnt1- and RPTP-specific transcripts were visualized together, both expression domains were arranged in concentric circles, with the RPTP domain (arrow) always located anterior to the Wnt1 domain (Fig. 1I',I'', arrowhead) (n = 6 of 6). Fgf8-induced upregulation of Wnt1 and RPTP thus mimics their normal spatial relationship at the MHB, where the Wnt1 domain separates the Fgf8 and RPTP expression domains (Badde et al., 2005) (Fig. 2G). This raises the possibility that the threshold of Fgf8 signaling differs from the induction of Wnt1 and RPTP expression at the MHB, and that Wnt1 expression requires higher levels of Fgf8 signaling than expression of RPTP. To investigate this in more detail, we narrowed down the concentrations of Fgf8 necessary for inducing Wnt1 and RPTP expression, respectively, by implanting fragments of beads soaked in different concentrations of Fgf8b. As reported previously, Wnt1 expression was induced around a bead fragment soaked in 0.15 mg/ml Fgf8b (Fig. 2A,A', arrowheads) (n = 12 of 12) (Crossley et al., 1996). Induction of RPTP expression was also consistently observed, yet its expression domain was located at a distance to the Fgf8b soaked bead and must therefore lie distal of the ring of Wnt1 expression, which generally surrounded the Fgf8 source (Fig. 2B,B', arrows) (n = 15 of 15). When the Fgf8b concentration was lowered to 0.05 mg/ml, some ectopic Wnt1 expression could still be observed surrounding the Fgf8 soaked bead, and the ring of RPTP-specific transcripts was located closer to the bead (Fig. 2C,D) (n = 5 of 8 for Wnt1; n = 7 of 7 for RPTP). Fgf8b at 0.015 mg/ml was not sufficient to induce Wnt1 expression (Fig. 2E) (n = 1 of 3) but could still elicit robust ectopic RPTP expression (Fig. 2F,F') (n = 4 of 4). Although additional work is clearly needed to fully understand Fgf receptor signaling dynamics at the MHB, these results strongly suggest that transcriptional activation of Wnt1 and RPTP requires different levels of Fgf8 signaling.

View larger version (106K): [in this window] [in a new window]   Figure 2. Fgf8-induced upregulation of Wnt1 and RPTP mimics their normal spatial relationship at the MHB. A'–F', Expression of Wnt1 (A, A', C, E) and RPTP (B, B', D, F, F') after implantation of beads soaked in different concentrations of recombinant Fgf8b. Wnt1 expression (A, A') is ectopically upregulated in the mesencephalon closely around a partial bead soaked in 0.15 mg/ml Fgf8b, RPTP expression only at a distance to the bead (B, B'). A' is a back view of the embryo shown in A. C, D, Ectopic expression surrounding beads soaked in 0.05 mg/ml Fgf8b. E, F, F', Expression surrounding beads soaked in 0.015 mg/ml Fgf8b. In F', the Fgf8b-releasing bead was removed for better visual clarity. G, Schematic drawing of the gene expression patterns of Fgf8, Wnt1, and RPTP at the MHB of a normal chick embryo (HH15). H, Schematic drawing of Wnt1 and RPTP expression around a Fgf8-releasing bead fragment (pink) implanted into the mesencephalic alar plate, which acts as ectopic Fgf8 source in the dorsal midbrain. The asterisks in all panels indicate the location of Fgf8b-releasing beads. Scale bar, 100 µm.

 
Overexpression of RPTP suppresses Wnt1 expression at the MHB
To examine the function of RPTP at the MHB, we misexpressed the murine homolog of RPTP in the area surrounding the isthmic constriction (Fig. 3A). By fusing the coding region of RPTP to a triple HA-tag, the ectopically expressed RPTP protein could be distinguished from its endogenous form by the use of an HA-specific antibody. As expected for a transmembrane protein, RPTP-HA staining was concentrated to cellular plasma membranes, suggesting that the recombinant protein was processed normally (Fig. 3B). When GFP alone was ectopically expressed in the MHB region, Wnt1 expression was unaltered (Fig. 3C) (n = 15 of 16). In contrast, after ectopic expression of RPTP-HA, the normal Wnt1 expression pattern was markedly disturbed (Fig. 3D,E) (n = 17 of 20). In embryos in which RPTP-HA was misexpressed in a broad region surrounding the isthmic constriction, Wnt1 expression was reduced or lost from the electroporated side of the embryo, suggesting that strong RPTP expression inhibits Wnt1 expression at the MHB (Fig. 3D, arrowhead) (n = 12 of 20). In some embryos, particularly in those where RPTP-HA expression was directly targeted to the organizer (and just to the region where Wnt1 would normally be expressed), a rostral displacement or split of the Wnt1 expression domain around the RPTP-HA expressing cells was observed (Fig. 3E, arrowheads) (n = 5 of 20). Identical results were obtained when an untagged form of the protein was misexpressed, indicating that the presence of the triple-HA tag was not critical for RPTP function (data not shown). To test for the possible contribution of the cytoplasmic part of RPTP, the domain most likely to be essential for its phosphatase activity and the interaction with components of intracellular signal transduction pathways, we constructed truncated forms of RPTP in which a triple HA-tag replaced the juxtamembrane and phosphatase domains (RPTP-IC-HA). When overexpressed in the mesencephalic vesicle, RPTP-IC-HA staining was concentrated to cellular plasma membranes, indicating correct targeting of the truncated protein (Fig. 3F). In contrast to the full-length form of RPTP, electroporation of RPTP-IC-HA did not perturb Wnt1 expression (Fig. 3G) (n = 21 of 22), indicating that the cytoplasmic domain was required for RPTP function at the MHB.

View larger version (87K): [in this window] [in a new window]   Figure 3. Altered Wnt1 expression after RPTP misexpression at the MHB. A, Schematic representation of the experimental protocol. B, Double immunohistochemical labeling using antibodies against GFP (green) and the HA-epitope (red) on a frozen section through the MHB region of an embryo electroporated with RPTP-HA-pMIWIII and GFP-pMIWIII. HA-specific staining is concentrated to cellular plasma membranes. C, Expression of Wnt1 after misexpression of GFP. D, E, Different example of Wnt1 expression after misexpression of RPTP-HA together with GFP. F, Immunohistochemical analysis of RPTP-IC-HA expression as detected by expression of the HA-fusion epitope. Expression is concentrated to cellular plasma membranes. G, Wnt1 expression after transfection with RPTP-IC-HA. In C–E and G, Wnt1 expression is shown in dark blue, and GFP is shown in turquoise. The arrowheads in C–E and G mark the transverse Wnt1 domain at the MHB. Scale bars: B, F, 20 µm; (in C) C, D, E, G, 100 µm. IC, Isthmic constriction.

 
To further characterize the potential role of RPTP in modulating gene expression at the MHB, we analyzed the expression of other MHB marker genes after electroporation of RPTP-HA. The expression domain of Fgf8 was normal 24 h after misexpression of RPTP (n = 6 of 6) (supplemental Fig. 1A,B, available at www.jneurosci.org as supplemental material). The altered Wnt1 expression that we observed 24 h after RPTP transfection can therefore not be a secondary effect of an altered Fgf8 expression profile at the MHB. In addition, forced expression of RPTP did not affect expression of Lmx1b (n = 16 of 17), En1 (n = 8 of 8), Pax2 (n = 9 of 11), Pax5 (n = 9 of 10), Otx2 (n = 4 of 4), Sef1 (n = 7 of 8), sprouty2 (n = 7 of 7), or Grg4 (n = 5 of 5) within 24 h after transfection (supplemental Fig. 1C–K, available at www.jneurosci.org as supplemental material) (data not shown).

RPTP interferes with Fgf8-mediated Wnt1 induction
Next, we took advantage of the fact that Fgf8 can mimic the organizer's activity when ectopically expressed in adjacent brain regions and performed two consecutive rounds of in ovo electroporation within the mesencephalic alar plate (Fig. 4A,A'). Electroporation of the Fgf8b expression plasmid into an embryo without previous manipulation reproducibly led to ectopic Wnt1 expression (Fig. 4B,B') (n = 5 of 5). Mock-transfection with GFP or dsRed did not alter Wnt1 expression (Fig. 4G). Overexpression of Fgf8b in embryos already experiencing 6 h of GFP or RPTP-IC expression also resulted in robust upregulation of Wnt1 expression (Fig. 4C,D) (n = 5 of 5 for GFP; n = 7 of 8 for RPTP-IC). In contrast, when full-length RPTP was misexpressed in the mesencephalic vesicle (Fig. 4E), only 22% of the transfected embryos exhibited robust upregulation of Wnt1 after Fgf8b electroporation (Fig. 4F,F') (n = 4 of 18). Because overexpression of RPTP at the MHB had specifically perturbed the expression of Wnt1 but not that of MHB marker genes like En1, Pax2, or Pax5 (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), we analyzed expression of these molecules after sequential RPTP/Fgf8b electroporation. In agreement with our previous results, expression of all three molecules was strongly induced by Fgf8, regardless of previous overexpression of RPTP (supplemental Fig. 1L–O, available at www.jneurosci.org as supplemental material) (n = 6 of 7 for En1; n = 5 of 5 for Pax2; n = 5 of 5 for Pax5). Together, these results suggest that strong RPTP expression specifically interferes with the ability of cells in the embryonic mesencephalon to initiate Wnt1 expression in response to Fgf8 signals. Considering that, during normal development (or in response to local Fgf8 application), RPTP expression always extended over the Wnt1 domain at its rostral side, our data suggest that RPTP may function to rostrally restrict the domain of Wnt1 expression at the MHB.

View larger version (108K): [in this window] [in a new window]   Figure 4. Misexpression of RPTP interferes with transcriptional Wnt1 activation after ectopic Fgf8 expression. A, Schematic drawing of the experimental procedure. At HH10, RPTP plus GFP, RPTP-IC plus GFP, or GFP alone were introduced into the right side of the midbrain vesicle. After 6 h (at HH12+), two expression plasmids carrying Fgf8 and dsRed, respectively, were electroporated into the same side of the midbrain vesicle. The embryos were allowed to develop for an additional 24 h. A', Example of an embryo chosen for additional analysis, based on the massive coexpression of the red and green fluorescent proteins in the midbrain vesicle. B, B', Ectopic expression of Fgf8 in the midbrain without previous manipulation leads to widespread upregulation of Wnt1. C, D, Fgf8 induced Wnt1 expression is not affected by previous introduction of GFP (C) or RPTP-IC (D). E, Example of an embryo electroporated with RPTP-pMES indicating mosaic expression of the transgene. F, F', Transcriptional upregulation of Wnt1 is mostly prevented after ectopic expression of RPTP 6 h before Fgf8 transgene introduction. B', F', Higher magnification of the boxed areas shown in B and F. G, Expression of Wnt1 in an embryo electroporated with GFP followed by misexpression of dsRed 6 h later demonstrating normal expression in the mesencephalic roof plate under these experimental conditions. H, Quantification of the results: (1), Percentage of embryos exhibiting strong ectopic expression of Wnt1 24 h after transfection with GFP or dsRed alone; (2), percentage of embryos exhibiting strong ectopic expression of Wnt1 24 h after transfection with Fgf8b (together with dsRed) without previous manipulation; (3), percentage of embryos exhibiting strong ectopic expression of Wnt1 24 h after transfection with GFP followed by Fgf8b (+dsRed); (4), percentage of embryos exhibiting strong ectopic expression of Wnt1 24 h after transfection with RPTP-IC-HA (+GFP) followed by Fgf8b (+dsRed); (5), percentage of embryos exhibiting strong ectopic expression of Wnt1 24 h after transfection with RPTP-HA (+GFP) followed by Fgf8b (+dsRed). Scale bar, 500 µm.

 
If RPTP expression at the MHB indeed serves to restrict Wnt1 expression, one would expect that reducing RPTP activity at the MHB should allow Wnt1 expression to expand anteriorly. To test this idea, two RNAi constructs targeted to nonoverlapping sequences in the intracellular domain of RPTP were designed. The activity and specificity of the constructs were confirmed by luciferase reporter assays compared with the effects of an RNAi construct targeted to an unrelated protein or with that of RNAi constructs containing randomized targeting sequences (Fig. 5A). Repression by unrelated or randomized sequences was negligible (Fig. 5A). Electroporation of either RPTP_si-a (n = 8 of 9) (Fig. 5D) or RPTP_si-b (n = 6 of 9) (data not shown) but not of the randomized control targeting construct random_a (n = 5 of 5) (Fig. 5C) into the MHB region at HH10 markedly reduced the endogenous expression of RPTP. Knock-down of RPTP also led to an anterior expansion of Wnt1-positive cells into the domain where RPTP is normally expressed (Fig. 5G–G'',I,J) (n = 9 of 14). This effect was more pronounced near the dorsal midline than in the ventral midbrain, presumably because the domain of RPTP expression is broader and any effect of knock-down of its expression may therefore be more profound in the dorsal midbrain (Fig. 5E,K). Wnt1 expression at the MHB, however, was not only expanded rostrally, but expression was also scattered within the rostral half of its normal domain in some embryos (Fig. 5F–J). We do not know the molecular basis for this effect at present. A possible explanation may come from the fact that RPTP and Wnt1 expression at the MHB appear to slightly overlap during normal development (Figs. 1C-D', 5E). It is therefore possible that RPTP stabilizes the Wnt1 expression border in the small region where both proteins are normally coexpressed. In addition, because the extracellular part of RPTP contains multiple cell adhesion-like motives, reduced RPTP expression at the MHB may alter cell adhesive properties critical for the maintenance of the coherent cellular organization within its normal expression domain. It is worth pointing out that disorganization of the Wnt1 expression domain was also observed after targeted deletion of Fgf receptor 1 from the MHB territory (Trokovic et al., 2003).

View larger version (96K): [in this window] [in a new window]   Figure 5. RNAi-mediated knock-down of RPTP and Ras-MAP kinase pathway activation after RPTP overexpression. A, Efficiency and specificity of the RNAi targeting constructs as assessed by luciferase reporter assays. Experimental details are described in Material and Methods. Renilla luciferase activity of the reporter construct psiCHECK2-RPTP (Promega) without cotransfection of an RNAi targeting construct served as reference and was set at 100%. Cotransfection of either RPTP_si-a or RPTP_si-b together with psiCHECK2-RPTP reduced Renilla luciferase activity to <10%. Cotransfection of randomized targeting sequences or of an unrelated targeting construct did not significantly affect Renilla activity. Error bars represent SD. **p < 0.005, Student's t test. B, Representative example of GFP fluorescence 24 h after transfection of RPTP_si-a together with GFP across the MHB. C, D, F–J, Analysis of gene expression after RNAi-mediated knock-down of RPTP expression. C, RPTP expression after transfection with the randomized RNAi targeting construct random_a. D, Expression of RPTP after transfection with RPTP_si-a. E, Two-color in situ hybridization on a wild-type embryo showing expression of RPTP (dark blue) and Wnt1 (turquoise). Note that the RPTP expression domain is broader in the dorsal than in the ventral midbrain. F, H, Expression of Wnt1 in embryos transfected with the control targeting construct random_a. G, I, J, Expression of Wnt1 after transfection with RPTP_si-a (G, I) or RPTP_si-b (J). F' and G' are higher magnifications of the boxed areas in F and G. K, Schematic drawing of the normal gene expression patterns of Wnt1 (turquoise) and RPTP (purple) at the MHB. L, Schematic drawing of Wnt1 expression after RPTP_si transfection. M, Quantification of the results. Gray bars indicate unchanged expression, and black bars indicate expanded and/or scattered expression of Wnt1. N–P, DpErk1/2 staining at the MHB 6 h after electroporation of GFP (N, N') or RPTP (O–P'). N', O', and P' are higher magnifications of the boxed areas in N, O, and P. Q–T, Wnt1 expression 6 h (R) and 8 h (S, S') after RPTP transfection and in age-matched controls (Q, T). U, V, En1 expression 8 h after RPTP transfection (U, U') and in an age-matched control (V). Scale bars: C, 500 µm; F, F', 100 µm. IC, Isthmic constriction; mes, mesencephalon; met, metencephalon.

 
At least three signaling pathways, the Ras-MAP kinase, phosphatidyl inositol 3 kinase, and phospholipase C pathway, can be activated after Fgf receptor stimulation, and of the three, the Ras-MAP kinase pathway has been demonstrated to be important for mid-hindbrain development (Corson et al., 2003; Sato and Nakamura, 2004; Dailey et al., 2005). Because signal transduction in each of these pathways requires protein phosphorylation on tyrosine residues, it is possible that RPTP, because of its tyrosine phosphatase activity, interrupts signal transduction through dephosphorylation of intermediate signaling components. To test whether RPTP impinges on Ras-MAP kinase signaling, we visualized phosphorylation of the pathway intermediate Erk1/2 with the phosphorylation-specific antibody dpErk1/2 (Corson et al., 2003). Six hours after transfection of 2 µg/µl RPTP-pMES but not after transfection of pMES alone, dpErk1/2 labeling intensity was slightly reduced in the right electroporated side of the electroporated embryos (Fig. 5N–P) (n = 6 of 9). Repression of Wnt1 expression by RPTP could also be detected 6 and 8 h after RPTP transfection (Fig. 5Q–T) (n = 10 of 18). In contrast to Wnt1 but in agreement with our previous results (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), we did not observe any major disturbance of En1 expression 6 or 8 h after RPTP transfection (Fig. 5U,V) (data not shown) (n = 11 of 11). These findings indicate that the influence of RPTP on Wnt1 expression at the MHB may, at least in part, result from modulation of Ras-MAP kinase signaling intensity by RPTP.

RPTP interacts with β-catenin in the embryonic midbrain and inhibits the activity of a β-catenin responsive promoter
RPTP expression in the developing midbrain is highly dynamic during the first 4 d of chick development (Figs. 1, 6A–C) (Badde et al., 2005). Before HH12 (16 somites), RPTP is expressed throughout the mesencephalic vesicle (Fig. 6A) (Badde et al., 2005). Between HH12 and HH19, RPTP expression is lost from the midbrain alar plate but is maintained in a tight transverse domain rostral to the Wnt1 domain (Figs. 1A,C, 6B) (Badde et al., 2005). Thereafter, RPTP expression is induced once again in the mesencephalic vesicle and covers the entire mesencephalic alar plate from late E4 onwards, without crossing into the isthmic organizer region (Figs. 1D, 6C, arrowhead) (Badde et al., 2005). Because of this prominent "on-off" expression in the midbrain alar plate, we speculated that RPTP may contribute to midbrain development by mechanisms in addition to restricting Wnt1 expression at the MHB organizer.

View larger version (58K): [in this window] [in a new window]   Figure 6. RPTP binds to β-catenin in the mesencephalon and inhibits the activity of a β-catenin responsive promoter in vivo. A–C, On-off-on expression of RPTP in the mesencephalic vesicle between HH9+ and HH20 as described by Badde et al. (2005). The dashed line in B marks the isthmic constriction, the arrow in B and C marks the location of the transverse ring of RPTP expression at the MHB, and the arrowhead in C marks the rostral-to-caudal upregulation of RPTP in the midbrain alar plate. di, Diencephalon; mes, mesencephalon; met, metencephalon. D, Immunoprecipitation using an HA-specific antibody on RPTP-HA transfected HH16 midbrain tissue demonstrating coprecipitation of β-catenin with RPTP-HA. E, As in D, except tissue transfected with RPTP-IC-HA was used. No coprecipitation with β-catenin was observed. Top, Western Blot analysis with the HA-specific antibody detecting RPTP-HA (D) or RPTP-IC-HA (E), respectively. Bottom, Detection of β-catenin on the blots shown above after stripping and reprobing with a β-catenin-specific antibody. The blots were cut to remove the marker lane. Preclear, Supernatant after precipitation with an unspecific antibody or with agarose-coupled protein-G; sup, supernatant after precipitation with the HA-specific antibody; IP, immunoprecipitate. F–M, GFP fluorescence 24 h after transfection of a TOP:dgfp reporter into the MHB region. F–H, Different examples of GFP expression in embryos 24 h after electroporation with TOP:dgfp. I–K, Different examples of GFP expression after electroporation of TOP:dgfp together with RPTP-pMIW. L, M, Examples of GFP expression after electroporation of TOP:dgfp together with RPTP-IC-pMIW. F, G, I, J, L, and M are monochrome images. N, Quantification of the results: (1), percentage of embryos exhibiting GFP fluorescence 24 h after transfection with TOP:dgfp alone; (2), percentage of embryos exhibiting GFP fluorescence 24 h after transfection with TOP:dgfp together with RPTP-HA; (3), percentage of embryos exhibiting GFP fluorescence 24 h after transfection with TOP:dgfp together with RPTP-IC-HA. Scale bars: A, B, 200 µm; F, 500 µm; H, 250 µm.

 
The juxtamembrane domain of RPTP exhibits a high degree of homology to the cytoplasmic domain of E-cadherin (Wang et al., 1996). Moreover, the human homolog of RPTP, PCP2, colocalizes with β-catenin at sites of cell-to-cell contact in human adenocarcinoma cells and coprecipitates with β-catenin in PC-12 cells overexpressing both proteins (Yan et al., 2002). The interaction of both proteins occurs independently of the phosphorylation state of β-catenin, but β-catenin can be dephosphorylated on tyrosine residues by PCP2 (Yan et al., 2002). We therefore investigated whether β-catenin and RPTP may physically interact in the developing mesencephalon. We overexpressed either RPTP-HA or RPTP-IC-HA in the mesencephalic vesicle for 24 h and subsequently immunoprecipitated RPTP bound proteins using an antibody directed against the HA-epitope (Fig. 6D,E). Overexpressed RPTP-HA was detected as a discrete band of 185 kDa recognized by the HA-specific antibody (Fig. 6D, top panel, lysate). After stripping and reprobing of the blot, endogenous β-catenin expression could also be readily detected (Fig. 6D, bottom panel, lysate). Precipitation with an unspecific antibody or with agarose-coupled protein-G did not deplete RPTP-HA or β-catenin from the extracts (Fig. 6, preclear). After immunoprecipitation with the HA-specific antibody, the 185 kDa band was cleared from the supernatant (Fig. 6D, top panel, sup) and enriched in the immunoprecipitate (Fig. 6D, top panel, IP) (n = 5 of 5). β-Catenin was also present in the immunoprecipitates (Fig. 6D, bottom panel, IP) (n = 5 of 5). Neither β-catenin nor RPTP could be precipitated with an antibody not directed against HA or β-catenin (data not shown). When the truncated allele RPTP-IC-HA, which lacks the cytoplasmic part of the protein and thus the putative interaction domain with β-catenin, was electroporated into the mesencephalic vesicle, a specific band of 105 kDa was detected (Fig. 6E, top panel, lysate). After immunoprecipitation with the HA-specific antibody, RPTP-IC-HA could be successfully immunoprecipitated but failed to enrich β-catenin (Fig. 6E, IP) (n = 5 of 5). These data demonstrate that RPTP can coprecipitate β-catenin from mesencephalic extracts via its intracellular domain.

We next investigated whether the observed interaction of β-catenin with RPTP was biologically relevant for canonical Wnt signaling. β-Catenin plays an essential role in the structural organization of cells by linking the cytoplasmic domain of type I cadherins, such as E-cadherin, to the actin cytoskeleton and, in addition, functions as a key component of the canonical Wnt signaling pathway. Loss of E-cadherin-mediated cell adhesion increased canonical Wnt signaling, presumably because, in the absence of E-cadherin, more β-catenin is available for Wnt signal transduction (Orsulic et al., 1999; Stockinger et al., 2001). We therefore wondered whether overexpression of RPTP in the mesencephalon may sequester a portion of β-catenin at the plasma membrane, making it unavailable for Wnt signaling. To test this idea, we examined the activity of the β-catenin responsive reporter TOP:dgfp in the midbrain, alone or in the presence of ectopically expressed RPTP. TOP:dgfp contains four consensus Lef-binding sites and a minimal c-Fos promoter-driving expression of a destabilized form of GFP (dGFP) (Dorsky et al., 2002). Transgenic zebrafish that stably express dGFP under control of this promoter exhibit intense GFP expression throughout the entire midbrain alar plate, demonstrating that the canonical Wnt/β-catenin pathway is active in this region of the neural tube (Dorsky et al., 2002). When the TOP:dgfp reporter was introduced into the MHB region of HH11 chick embryos by in ovo electroporation, strong GFP expression in the midbrain alar plate could be observed 24 h later (at HH15–16), demonstrating that the midbrain alar plate is a domain of active Wnt/β-catenin signaling in the chick at this developmental age (Fig. 6F–H,N) (n = 8 of 8). Therefore, at HH15–HH16, neither RPTP nor Wnt1 are expressed in the mesencephalic alar plate, but the Wnt/β-catenin pathway is active. We therefore reasoned that misexpression of RPTP in the midbrain alar plate at HH15–H16 should allow us to analyze a possible influence of RPTP on canonical Wnt signal transduction that is independent of its ability to influence Wnt1 expression at the MHB. When RPTP was coelectroporated with TOP:dgfp, GFP fluorescence intensity was reduced severely (Fig. 6I–K,N) (n = 10 of 11). Transfection of RPTP-IC, which lacks the intracellular domain critical for coprecipitation of β-catenin, did not suppress GFP fluorescence (Fig. 6L–N) (n = 6 of 7). These results demonstrate that RPTP can suppress the activation of this β-catenin responsive reporter in vivo.

The normal growth and development of the mid-hindbrain region depends on activation of the canonical Wnt signaling pathway (McMahon and Bradley, 1990; Thomas and Capecchi, 1990; Brault et al., 2001; Panhuysen et al., 2004). To investigate whether ectopic RPTP expression or RNAi-mediated knock-down of expression altered midbrain growth, RPTP expression was targeted to the right side of the mesencephalic vesicle by in ovo electroporation into HH9 embryos, and the consequence of this manipulation on midbrain growth was assessed 24 h later (Fig. 7). We chose HH9 embryos for these experiments, because this developmental time is before the downregulation of endogenous RPTP expression in the mesencephalon, which occurs after HH12 (Fig. 6A–C) (Badde et al., 2005). Introduction of RPTP-pMES into the HH9 mesencephalic vesicle therefore maintains RPTP expression in the midbrain beyond HH12, whereas introduction of an RNAi targeting construct leads to precocious loss of RPTP expression. The homeodomain Meis2 was used as a marker for the tectal anlage, because its transcripts are excluded from the roof plate, allowing us to unambiguously determine the location of the dorsal midline and estimate the size of the left and right halves of the mesencephalic vesicle. After forced expression of GFP or of the truncated allele RPTP-IC-HA together with GFP, the right and left halves of the tectal anlage appeared to have normal size (Fig. 7A,B) (n = 5 of 5 for GFP; n = 6 of 6 for RPTP-IC-HA). However, in embryos that had been electroporated with an expression plasmid containing RPTP, the size of the right electroporated tectal anlage was reduced severely when compared with the left, nonelectroporated side of the same embryo (Fig. 7C,C') (n = 15 of 16). Conversely, the right tectal anlage was slightly enlarged in embryos transfected with the specific RNAi targeting construct RPTP_si-a (Fig. 7D) (n = 6 of 7).

View larger version (89K): [in this window] [in a new window]   Figure 7. RPTP modulates mesencephalic growth. A–D, Whole-mount in situ hybridization with a Meis2-specific probe on HH15–HH16 embryos after electroporation with pMES (A, A'), RPTP-IC-pMES (B), RPTP-pMES (C, C'), or RPTP_si-a (D). In A', B, C', and D, the embryos are shown from the top. The dashed lines define the location of the dorsal midline as observed by the lack of Meis2 expression, and the dotted lines mark the perimeter of the right side of the mesencephalic vesicle. E, F, Cross sections through HH16 midbrains showing BrdU incorporation after ectopic expression of RPTP together with GFP (E–E'') or GFP alone (F–F''). E, F, Overlaid channels of E' and E'' or F' and F''. E', F', GFP expression. E'', F'', BrdU incorporation. G–I, TUNEL labeling of HH16 midbrains electroporated with nRFP together with RPTP (G) or nRFP alone (H). I, DNase treatment before TUNEL labeling as control for the validity of the experimental procedure. J, K, Cross sections through HH16 midbrains labeled for pH3 (red) and GFP (green) and counterstained with DAPI (blue). J, Misexpression of GFP. K, Misexpression of RPTP_si-a together with GFP. L, Cross section through an HH16 midbrain electroporated with RPTP together with GFP and stained for GFP (green), pH3 (red), and DAPI (blue). Fewer transfected (GFP+) cells are labeled for pH3 than nontransfected, neighboring (GFP–) cells. For more examples, see supplemental Figure 2 (available at www.jneurosci.org as supplemental material). M, Quantification of the percentage of pH3-positive cells transfected with combinations of the gene products indicated (gray bars) and the percentage of pH3-positive cells among the nontransfected, neighboring cell population under each condition (black bars). Error bars indicate SD. Scale bar (A), 500 µm.

 
To determine whether this growth reduction was the result of decreased cell proliferation and/or enhanced cell death, BrdU pulse-labeling experiments were performed. We delivered RPTP-HA using the electroporation vector pMES, which contains an IRES-GFP cassette allowing us to visualize individual RPTP-HA transfected cells by their GFP fluorescence (Swartz et al., 2001). Two hours after BrdU injection, 51% (±1.85 SEM) of the cells forced to express GFP, but only 27.2% (±1.41 SEM; p < 0.002, Student's t test) of RPTP/GFP expressing cells had incorporated the BrdU label (Fig. 7E–F''). Similar to mock transfected or nontransfected embryos, apoptotic cell nuclei were rarely observed 24 h after misexpression of RPTP, indicating that RPTP electroporation does not compromise cell survival within this time frame (Fig. 7G–I) (n = 3). Knock-down of RPTP expression at HH9 slightly increased the percentage of mitotic cells, visualized by detecting pH3 from 11.7% (±1.29 SEM) after introduction of GFP alone to 14.2% (±1.45 SEM) after transfection of the RNAi targeting construct RPTP_si-a together with GFP (Fig. 7J,K) (supplemental Fig. 2E, available at www.jneurosci.org as supplemental material) (n = 4). In conclusion, these results suggest that RPTP functions as negative modulator of mesencephalic growth primarily through modulation of progenitor cell proliferation.

Because RPTP could precipitate (and possibly sequester) the Wnt-signaling intermediate β-catenin from mesencephalic extracts (Fig. 6D,E), we wondered whether cotransfection of β-catenin or Wnt1 together with RPTP would rescue the proliferation defect observed after introduction of RPTP alone. To test this idea, we introduced either pMES (expressing only GFP), RPTP-pMES (expressing RPTP together with GFP), RPTP-Wnt1-pMEC together with GFP-pMIW (which results in coexpression of RPTP, Wnt1, and GFP), or RPTP-pMES together with caβ-catenin-pMIW (which results in coexpression of RPTP, GFP, and a stabilized form of β-catenin) into the mesencephalic vesicle at HH9 and determined the mitotic index of midbrain progenitor cells 24 h later (Fig. 7L,M) (supplemental Fig. 2A–D, available at www.jneurosci.org as supplemental material) (n = 9 for pMES, RPTP-pMES; n = 3 for RPTP-Wnt1-pMEC, RPTP-pMES plus β-catenin-pMIW). Transfection of RPTP together with GFP significantly reduced the percentage of mitotic cells compared with GFP transfected controls. Cotransfection of Wnt1 or stabilized β-catenin together with RPTP and GFP partially restored the mitotic index (Fig. 7M, gray bars). Therefore, the negative influence of RPTP on midbrain progenitor cell proliferation could, at least in part, be alleviated by cotransfection of Wnt1 or of the Wnt1-signaling intermediate β-catenin. To determine whether RPTP modulates midbrain progenitor cell proliferation in a cell-autonomous or noncell-autonomous manner, we compared the percentage of pH3-positive cell nuclei in the transfected (GFP expressing) cell population under each of these experimental conditions with the percentage of pH3+ cells in nontransfected (GFP-negative) neighboring cells (Fig. 7M, black bars). The mitotic index of cells that were located in close proximity to cells transfected with RPTP plus GFP did not differ from that of the neighbors of GFP transfected cells, suggesting that RPTP acts cell autonomously on midbrain progenitor cell proliferation. We also did not observe a significant difference in the percentage of pH3+ cell nuclei in neighbors of RPTP/GFP/β-catenin transfected cells compared with neighbors of GFP-expressing cells. In contrast, the percentage of pH3+ nuclei in cells located near RPTP/Wnt1/GFP transfected cells was clearly increased. This observation is consistent with the notion that the secreted molecule Wnt1 may influence cell proliferation in a cell-nonautonomous manner.

Our finding that overexpression of RPTP interfered with Fgf8-mediated induction of Wnt1 expression at the MHB as well as with the activation of the Wnt/β-catenin-dependent TOP:dgfp reporter in the mesencephalon raises the question of whether the reduced TOP:dgfp reporter activity in RPTP transfected embryos may be a secondary consequence of their diminished Wnt1 expression or, as suggested above, may reflect a more direct inhibition of Wnt/β-catenin signaling, presumably through interaction of RPTP with β-catenin. To distinguish between these possibilities, we first decreased Wnt1 expression at the MHB through RNAi-mediated knock-down and monitored TOP:dgfp activity in the mesencephalic vesicle 24 h later (supplemental Fig. 3A–H', available at www.jneurosci.org as supplemental material) (n = 15). Although electroporation of Wnt1-specific RNAi targeting constructs reduced Wnt1 expression in vivo, GFP expression of the TOP:dgfp reporter appeared normal. Second, we blocked Wnt signaling in the midbrain through forced expression of the known signaling antagonist Axin (Zeng et al., 1997). Wnt1 expression at the MHB was normal 24 h after transfection (supplemental Fig. 3I,J, available at www.jneurosci.org as supplemental material) (n = 10). Based on these observations, we suggest that RPTP may modulate Wnt1 expression and signal transduction in the midbrain through two separate mechanisms.

The midbrain alar plate undergoes massive expansion between HH12 and HH19 when RPTP expression is absent from this tissue. Cells around the MHB in embryonic day 10.5 (E10.5) to E11.5 mouse embryos are less mitotically active than cells in the midbrain and rhombomere 1 (Trokovic et al., 2005). A similar domain of reduced proliferation also exists at the chick MHB at HH15 and HH20 (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). Although the ring of RPTP expression seen during this developmental period does not cover this domain entirely, RPTP expression nevertheless appears to localize within this domain.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here, we present evidence based on gain-of-function and knock-down experiments that RPTP can counteract transcriptional activation of Wnt1 in the embryonic midbrain. In addition, we show that RPTP can physically interact with β-catenin in the midbrain, inhibit the activity of a β-catenin responsive promoter, and suppress mesencephalic cell proliferation. RPTP therefore emerges as a potential modulator of Wnt1 expression and signaling at the MHB organizer.

RPTP is part of the complex molecular interactions that maintain and shape the MHB region
A complex spatiotemporal pattern of gene expression has been described at the developing mid-hindbrain boundary. The MHB is bordered by a narrow, transverse ring of Wnt1 expression at its anterior side and by a ring of Fgf8 expression at its posterior side. Several proteins, including Pax2/5 and En1/2, are expressed in nested, symmetric gradients across the MHB region. Fgf8 signaling induces activation of the Ras-MAP kinase pathway, which is required for isthmic organizer activity (Sato and Nakamura, 2004). Fgf feedback inhibitors, such as Spry2, Sef, and Mkp3, interfere with Ras-MAP kinase signaling downstream of receptor tyrosine kinases at different steps within the signal transduction pathway and are thought to limit the lateral expansion of the Fgf signal at the MHB (Gross et al., 2001; Nutt et al., 2001; Fürthauer et al., 2002; Lin et al., 2002; Tsang et al., 2002; Kawakami et al., 2003; Echevarria et al., 2005). Maximal Ras-MAP kinase activation occurs within the domain of Fgf8 expression but tapers off into the mesencephalic and metencephalic vesicles (Corson et al., 2003; Sato and Nakamura, 2004). The shapes of the Pax2/5 and En1/2 expression domains reflect the gradient of Ras-MAP kinase activity at the MHB. In contrast, Wnt1 expression is confined to a tight ring of cells in the mesencephalon and is absent from the metencephalon. Metencephalic tissue is not permissive for Wnt1 expression, explaining the asymmetric nature of the Wnt1 expression domain (Kikuta et al., 2003). It is yet unclear, however, as to why Wnt1 expression is restricted to such a discrete band of cells and does not, like that of Pax2/5 or En1/2, taper off into the mesencephalic vesicle. As we show here, the receptor tyrosine phosphatase is expressed in a ring of cells rostral to the Wnt1 domain and inhibits Wnt1 expression when overexpressed. RPTP thus has the potential to restrict Wnt1 expression to its characteristic tight ring at the MHB.

RPTP expression itself is induced by Fgf8b in a concentration-dependent manner. When Fgf8-induced expression of Wnt1 and RPTP was monitored successively in the same specimen, ectopic expression of RPTP could always be observed anterior to the ring of Wnt1 expression. Induction of Wnt1 and RPTP expression by Fgf8 thus mimics their normal spatial relationship at the MHB, where the Wnt1 expression also separates the domain of RPTP expression from that of Fgf8. In addition, after implantation of Fgf8b releasing beads, ectopic RPTP expression could be observed at a Fgf8b concentration that was not sufficient to induce Wnt1 expression. We conclude from these results that different dosages of the Fgf8 signal from the MHB are required for transcriptional induction of RPTP and Wnt1, respectively. Examples for dosage dependency of Fgf function during embryogenesis have been described previously and include telencephalic and cerebellar development, patterning of the foregut, and skeletal development (Sato et al., 2001; Storm et al., 2003, 2006; Hajihosseini et al., 2004).

RPTP specifically inhibited Wnt1 expression but not that of other MHB marker genes when overexpressed in the MHB region. In addition, when cells in the midbrain were forced to express RPTP, they failed to upregulate Wnt1 expression after subsequent stimulation by Fgf8, yet they readily expressed En1, Pax2, or Pax5 under identical experimental conditions. RPTP therefore appears to specifically interfere with Fgf8-induced transcriptional activation of Wnt1 at the MHB. How can this high degree of functional specificity be achieved mechanistically? Expression of many MHB marker genes, including Otx2, Gbx2, Pax2/5, En1/2, and Wnt1, is negatively affected by overexpression of a dominant-negative form of Ras (Sato and Nakamura, 2004). Yet, we observed recently after transfection of different concentrations of a constitutive active form of Ras (caRas) that transcriptional activation of Wnt1 at the MHB could only be induced at a narrow concentration range of caRas, whereas transcriptional activation of En1 was observed over a concentration range spanning more than an order of magnitude (Vennemann et al., 2008). Induction of Wnt1 at the organizer thus appears to be more sensitive to changes in the strength of Ras-MAP kinase pathway activation than other MHB marker genes. This raises the possibility that tight, local regulation of Ras-MAP kinase activity may be a prerequisite for Wnt1 expression at the MHB and that RPTP may contribute to this regulation through modulation of Ras-MAP kinase signaling. Notably, in mouse embryos in which Fgf8 expression was specifically lost in the MHB territory from the five somite stage onwards, the transverse ring of Wnt1 expression in the caudal mesencephalon was also absent, whereas the expression domains of Fgf8, Otx2, Pax2, or En1 appeared normal at early stages of embryogenesis (Chi et al., 2003). Genetic loss of Fgf8 at the MHB, however, caused massive cell death, an effect we did not observe within 24 h after RPTP transfection. We cannot rule out that RPTP inhibits Wnt1 expression by interfering with still unknown factor(s) that are specifically required for transcriptional Wnt1 activation in Fgf8 responding cells. Regardless of the exact molecular nature of this inhibition, the experiments described here suggest a model in which Fgf8 induces Wnt1 and RPTP expression in the midbrain at different signaling thresholds and consequently at different distances to the MHB, so that the Wnt1 domain is always bordered by RPTP-expressing cells at its rostral side. One function of RPTP at the MHB may thus be to act as a Ras-MAP kinase feedback inhibitor to restrict the anterior expansion of the transverse Wnt1 domain (Fig. 8).

View larger version (30K): [in this window] [in a new window]   Figure 8. Proposed model for the function of RPTP at the MHB. The MHB (dashed vertical line) is located at the border between the expression domains of Otx2 (rostral) and Gbx2 (caudal). Once the MHB is established during early somitogenesis, Fgf8, Wnt1, En2, Pax2, and Pax5 function in an interdependent, positive feedback loop, which is necessary for organizer maintenance. RPTP expression is induced by Fgf8 at a signaling threshold that differs from that required for Wnt1 induction, resulting in a band of RPTP expression anterior to the Wnt1 domain. RPTP counteracts Fgf8-mediated transcriptional activation of Wnt1, which constrains the Wnt1 domain to its typical tight ring anterior to the Otx2-Gbx2 boundary. Presumably, as a consequence of the high degree of homology between the juxtamembrane domain of RPTP with cadherins, RPTP can bind to and sequester β-catenin and thereby negatively influence canonical Wnt signaling.

 
RPTP binds to β-catenin and modulates canonical Wnt1 signaling at the MHB
In addition to restricting Wnt1 expression at the MHB, RPTP also seems to play a role in modulating Wnt1 signal transduction. In the canonical or Wnt/β-catenin pathway, Wnt binding to frizzled receptors and LRP coreceptors inhibits degradation of the cytoplasmic pool of β-catenin, allowing it to translocate to the nucleus, where it acts as coactivator for LEF/TCF transcription factors to promote target gene expression (Logan and Nusse, 2004). In addition to its role in Wnt signaling, β-catenin is a structural link between cadherins/-catenin and the actin cytoskeleton. Cadherin bound β-catenin can be freed to participate in Wnt signaling and vice versa, a switch that appears to be regulated by phosphorylation of β-catenin on tyrosine residues (Brembeck et al., 2004; Nelson and Nusse, 2004; Schambony et al., 2004; Lilien and Balsamo, 2005). It was shown previously that β-catenin interacts with the juxtamembrane domain of the human homolog of RPTP and can be dephosphorylated by this protein in vitro (Yan et al., 2002). As shown here, a similar interaction of RPTP and β-catenin also occurs in the embryonic chick midbrain. In addition, when full-length RPTP was overexpressed, we found that two functional readouts of canonical Wnt signaling in the midbrain, activation of a β-catenin responsive promoter and neuroepithelial cell proliferation, were reduced. This proliferation defect could be partially reversed by cotransfection of Wnt1 or of a stabilized form of β-catenin. It is intriguing to speculate that RPTP may antagonize canonical Wnt signaling through binding and potentially dephosphorylation of β-catenin and ultimately serve as a negative regulator of cell proliferation in the developing midbrain (Fig. 8). Decreasing Wnt1 expression at the MHB through transfection of Wnt1-specific RNAi targeting constructs did not notably reduce β-catenin responsive reporter activity in the midbrain, nor did misexpression of Axin, a known inhibitor of the Wnt signaling pathway, in the mesencephalon disturb the transverse ring of Wnt1 expression at the organizer. These observations suggest that RPTP acts on Wnt1 expression and signal transduction through two independent mechanisms.

How can this dual effect be explained? Protein tyrosine phosphatases antagonize the activity of protein tyrosine kinases and influence a broad spectrum of physiological processes. For instance, RPTP (also termed RPTP) is required for Delta/Notch-dependent oscillatory gene expression in the presomitic mesoderm as well as for convergent extension during gastrulation through an as yet unknown mechanism (Aerne and Ish-Horowicz, 2004). It is therefore possible that RPTP impinges on multiple signaling pathways through dephosphorylation of different signaling components. In addition, the ring of RPTP expression at the MHB may coincide with that of another receptor tyrosine phosphatase, RPTP/β, a keratan sulfate modified protein, which has been suggested to modify signal transduction at the MHB through influencing the diffusion of morphogenetic signals of the Wnt and Fgf families (Canoll et al., 1993; Hamanaka et al., 1997). Although the precise role of RPTP in any of these processes will only be fully understood once its substrates are identified, our results suggest that RPTP is part of the complex molecular network that governs development of the midbrain region.

	Footnotes

 
Received July 4, 2007; revised March 21, 2008; accepted April 17, 2008.

A. Badde's present address: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Munich, Germany.

This work was supported by the International Max-Planck Research School Program in Structure and Function of Biological Membranes (Frankfurt, Germany) (A.B.). We thank C. Tabin (Harvard Medical School, Boston, MA), C. Logan (University of Calgary, Calgary, Alberta, Canada), C. Krull (University of Michigan, Ann Arbor, MI), H. Rohrer (Max-Planck Institute for Brain Research, Frankfurt, Germany), F. Costantini (Columbia University Medical Center, New York, NY), and R. Dorsky (University of Utah, Salt Lake City, UT) for reagents; C. Ziegler for excellent technical assistance; G. O'Sullivan for critically reading this manuscript; and A. Vennemann, E. Dohle, and A. Poplawski for experimental help.

Correspondence should be addressed to Dorothea Schulte, Department of Neuroanatomy, Max Planck Institute for Brain Research, Deutschordenstrasse 46, 60528 Frankfurt, Germany. Email: schulte{at}mpih-frankfurt.mpg.de

Copyright © 2008 Society for Neuroscience 0270-6474/08/286152-13$15.00/0

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