Fzd3 and Fzd6 deficiency results in a severe midbrain morphogenesis defect

Authors

  • Sebastian Stuebner,

    1. Institute of Developmental Genetics, Helmholtz Centre Munich, German Research Centre for Environmental Health, and Technical University Munich, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich/Neuherberg, Germany
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    • Sebastian Stuebner and Nilima Prakash contributed equally to this work.

  • Theresa Faus-Kessler,

    1. Institute of Developmental Genetics, Helmholtz Centre Munich, German Research Centre for Environmental Health, and Technical University Munich, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich/Neuherberg, Germany
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  • Thomas Fischer,

    1. Institute of Developmental Genetics, Helmholtz Centre Munich, German Research Centre for Environmental Health, and Technical University Munich, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich/Neuherberg, Germany
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  • Wolfgang Wurst,

    Corresponding author
    1. Institute of Developmental Genetics, Helmholtz Centre Munich, German Research Centre for Environmental Health, and Technical University Munich, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich/Neuherberg, Germany
    2. Max Planck Institute of Psychiatry, Munich, Germany
    • Helmholtz Centre Munich, German Research Centre for Environmental Health, Institute of Developmental Genetics, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany
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  • Nilima Prakash

    1. Institute of Developmental Genetics, Helmholtz Centre Munich, German Research Centre for Environmental Health, and Technical University Munich, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich/Neuherberg, Germany
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    • Sebastian Stuebner and Nilima Prakash contributed equally to this work.


Abstract

Wnt/β-catenin signaling controls the proper development of the mid-/hindbrain region (MHR) and of midbrain dopaminergic (mDA) neurons, but the Frizzled (Fzd) receptors transducing these signals are still unknown. Fzd3 is expressed throughout the mouse anterior neural tube, whereas Fzd6 is restricted to the MHR. We show that the MHR is properly established and mDA neurons develop normally in Fzd6−/− mutants, but the number of mDA neurons is initially reduced and recovers at later stages in Fzd3−/− embryos. Fzd3−/−; Fzd6−/− double mutants exhibit a severe midbrain morphogenesis defect consisting of collapsed brain ventricles, apparent thickening of the neuroepithelium, focal disruption of the ventricular basal lamina and protrusion of individual cells, and increased proliferation at later stages, despite a normal closure of the anterior neural tube and the rescue of the mDA defect in these embryos. Fzd3 and Fzd6 thus control proper midbrain morphogenesis by a yet unknown mechanism in the mouse. Developmental Dynamics 239:246–260, 2010. © 2009 Wiley-Liss, Inc.

INTRODUCTION

The Wnt (Wingless-related MMTV integration site) family of secreted, lipid-modified glycoproteins plays a fundamental role in many aspects of vertebrate and invertebrate development, including neural induction and patterning, cell proliferation, cell fate specification, cell polarization and migration, axon guidance, and synaptogenesis (Logan and Nusse,2004; Ille and Sommer,2005). The extracellular Wnt molecules signal into the cell via three different pathways: the “canonical” Wnt/β-catenin, and the “non-canonical” Wnt/planar cell polarity (PCP) and Wnt/Ca2+ pathways (Seifert and Mlodzik,2007; Huang and He,2008). Common to all three pathways is the binding of the Wnt ligand to the seven-pass transmembrane receptors of the Frizzled (Fzd) family, although Wnts can also bind to other single transmembrane receptors (Huang and Klein,2004; van Amerongen et al.,2008). The subsequent activation of one of the three signaling pathways appears to depend on the specific complement of Fzd receptors and co-receptors of the Low density lipoprotein (LDL) receptor-related protein (Lrp) family encountered on the surface of the receiving cell rather than on the Wnt ligand activating these receptors (van Amerongen et al.,2008). However, the identification of the individual Wnt/Fzd ligand/receptor pairs acting during the development of higher vertebrates has been complicated enormously by the fact that 19 Wnt ligands face 10 Fzd receptors encoded in the mammalian genome, thus allowing for potentially 190 different ligand/receptor pair combinations (van Amerongen et al.,2008).

The hallmark of the Wnt/β-catenin pathway is the stabilization of cytosolic β-catenin, which subsequently enters the cell nucleus and activates the transcription of Wnt target genes (Huang and He,2008). Apart from many other developmental processes in the mouse, the Wnt/β-catenin pathway is required for the maintenance of the isthmic organizer (IsO) at the mid-/hindbrain boundary (MHB), a key signaling center that controls the proper development of the midbrain and rostral hindbrain in the mouse embryo (Brault et al.,2001; Wurst and Bally-Cuif,2001). More recent data also indicate that the generation of dopamine-synthesizing (dopaminergic, DA) neurons in the ventral midbrain (VM), a very important neuronal population controlling motor behaviors and cognitive and affective brain functions, crucially relies on the activation of the Wnt/β-catenin pathway in their progenitors (Joksimovic et al.,2009; Tang et al.,2009). Due to the phenotypic resemblances of the corresponding mouse mutants, Wnt1 appears to be the Wnt ligand activating this pathway in both cases, although the participation of other Wnts cannot be completely ruled out (McMahon and Bradley,1990; Thomas and Capecchi,1990; Prakash et al.,2006). The identity of the Fzd receptors transducing the Wnt1 signal at the MHB and in midbrain dopaminergic (mDA) progenitors, however, still remains to be established. To this end, we have performed a detailed analysis of the mouse Fzd expression patterns at the MHB and in the cephalic flexure (the site of origin of mDA neurons) during midgestational stages of embryonic development (Fischer et al.,2007). This analysis revealed a striking pattern of Fzd6 expression in the caudal cephalic flexure and at the MHB in the developing mouse embryo (Fischer et al.,2007). We, therefore, hypothesized that the Fzd6 receptor might be a strong candidate to transduce the Wnt1 signal at these sites, although it might function in a redundant manner with the ubiquitously expressed Fzd3 receptor (Fischer et al.,2007).

To establish a function of Fzd6 and Fzd3 in mid-/hindbrain and mDA neuron development, we analyzed the development of these regions and neuronal populations in Fzd6−/− and Fzd3−/− single and double mutant mouse embryos. Fzd6−/− single mutants display hair-patterning defects, but a central nervous system (CNS) phenotype has not been reported in these mutant mice (Guo et al.,2004). Fzd3−/− single mutants, by contrast, have strong defects in commissural axon projection in the spinal cord and lack all major fiber tracts in the forebrain including the DA nigrostriatal projections, although mDA neurons still arise in these mutants (Wang et al.,2002; Lyuksyutova et al.,2003). The cephalic neural tube closure defect observed with a low penetrance in the Fzd3−/− single mutants (Wang et al.,2002), was reported to become a fully open neural tube with almost 100% penetrance in the Fzd3−/−; Fzd6−/− double mutants (Wang et al.,2006a). We show here that Fzd6 is not required for transduction of the Wnt1 signal at the MHB and in mDA progenitors, as the IsO was established correctly at the MHB and mDA neurons developed normally in the VM of Fzd6−/− single mutant mouse embryos. Notably, and although the MHB appeared morphologically normal in the Fzd3−/− single mutants, the number of mDA neurons was transiently reduced in these mutants at midgestation. Because cell proliferation and survival was not affected in the Fzd3−/− VM, this suggests that Fzd3 is required for the proper differentiation of mDA neurons. In addition, we describe a severe midbrain morphogenesis defect in the Fzd3−/−; Fzd6−/− double mutants consisting of a normal closure of the anterior neural tube but collapse of all brain ventricles together with an apparent thickening of the neuroepithelium. The focal disruption of the ventricular basal lamina and protrusion of individual cells, together with an increased proliferation in the double mutant midbrain at later stages, probably led to the massive protrusion of neural tissue and occlusion of the ventricular cavity of the midbrain observed in some double mutants. The increased proliferation in the Fzd3−/−; Fzd6−/− VM most likely also rescued the Fzd3−/− mDA phenotype, as the mDA neuron numbers were not significantly altered in the double mutants. This double mutant phenotype is different from the previously reported neural tube closure defects in the Fzd3−/− single and Fzd3−/−; Fzd6−/− double, and other Wnt/PCP pathway mutants (Copp et al.,2003; Wang et al.,2006a), and has to the best of our knowledge not been reported in the literature so far.

RESULTS

Fzd6 Is Expressed in Wnt1+ Cells at the MHB and in Aldh1a1+ mDA Precursors

The starting point of our analyses was the finding of a highly restricted expression of the Fzd6 receptor in the caudal midbrain just abutting the MHB (Fig. 1) (Fischer et al.,2007). This Fzd6 expression domain appeared to overlap with Wnt1, one of the key IsO genes required for the proper development of the midbrain and rostral hindbrain (McMahon and Bradley,1990; Thomas and Capecchi,1990; Fischer et al.,2007). Moreover, mDA neurons develop from Wnt1+ and Aldh1a1+ (Aldehyde dehydrogenase family 1, subfamily a1; also known as Raldh1 or Ahd2) progenitors located in the ventral midline of the midbrain (Wallen et al.,1999; Zervas et al.,2004; Kittappa et al.,2007; Ono et al.,2007), the so-called floor plate (FP), and Wnt1 and the Wnt/β-catenin pathway are both necessary and sufficient for the generation of mDA neurons from the midbrain FP (Prakash et al.,2006; Joksimovic et al.,2009; Tang et al.,2009). Therefore, we first mapped in more detail the expression of Fzd6 in relation to these two marker genes, Wnt1 and Aldh1a1, at three crucial stages during the development of the mid-/hindbrain region (MHR) and of mDA neurons. The activity and gene expression patterns of the IsO are fully established at E10.5 (Wurst and Bally-Cuif,2001). Moreover, most mDA neurons are born at an equivalent stage in the rat embryo (Gates et al.,2006), but only very few cells express Tyrosine hydroxylase (Th), the rate-limiting enzyme in DA synthesis, at E10.5 in the mouse embryo. As reported before, Fzd6 expression is localized to the caudal cephalic flexure abutting the MHB ventrally at E10.5 (Fig. 1B), where it overlaps to a large extent with Wnt1 and Aldh1a1 expression (Fig. 1C,D). Fzd6 and Aldh1a1 expression also overlap in a dorsoventral (D/V) and medio-lateral position at this stage, i.e., both genes are expressed strongly in the lateral FP and basal plate (BP) of the midbrain but weakly in the medial FP (insets in Fig. 1B,D). Wnt1, by contrast, is expressed in two stripes within the BP and lateral FP abutting the medial FP and thus overlapping only partially with Fzd6 expression (insets in Fig. 1B,C). Fzd6 is not expressed in the dorsal midbrain (DM) at this stage (inset in Fig. 1B) (Fischer et al.,2007). A similar confinement of Fzd6 transcription to the caudal cephalic flexure and overlap with Wnt1 or Aldh1a1 expression in this region is seen at E11.5 and E12.5 (Fig. 1F,G,J,K and inset in Fig. 1H), when mDA precursors start to differentiate into mature Th+ mDA neurons, although the main patterning activity of the IsO might have already ceased at these stages (Wurst and Bally-Cuif,2001; Ang,2006). Notably, Fzd6 expression only overlaps with a caudal subset of postmitotic Th+ mDA neurons at E11.5 and not at all with these neurons at E12.5 (colored insets in Fig. 1H,L).

Figure 1.

Expression of Fzd6 overlaps with Wnt1 and Aldh1a1 in the cephalic flexure of midgestational mouse embryos. A–L: Representative close-up views of the cephalic flexure in midsagittal sections from E10.5 (A–D), E11.5 (E–H), and E12.5 (I–L) CD-1 mouse embryos hybridized with riboprobes for Fzd6 (B,F,J), Wnt1 (C,G,K), Aldh1a1 (D and inset in H), and Th (H,L). Dorsal is at the top, anterior to the left. A,E,I are brightfield images of D,H,L. Red broken lines in A,E,I indicate the plane of coronal sections at caudal midbrain levels shown in the bottom left corner insets in A–K. Black (A,E,I) and white (B–D,F–H,J–L) arrowheads point at the ventral position of the MHB. Top right corner insets in C,D,G,H,K,L are pseudo-colored overlays of consecutive sagittal sections hybridized with Fzd6 (red) and Wnt1 (green in C,G,K), Aldh1a1 (green in D), or Th (green in H,L); overlapping expression domains appear in yellow.

We concluded from these analyses that Fzd6 is expressed at crucial stages in Aldh1a1+ mDA progenitors and within at least a subset of, and in close vicinity to, Wnt1+ neuroepithelial cells at the MHB. The Fzd6 receptor might, thus, transduce the Wnt1 signal required for the development of the midbrain and of associated neuronal populations such as the mDA neurons.

Establishment of the IsO at the MHB and the Development of mDA Neurons Are Not Affected in Fzd6−/− Mutant Embryos

Based on the previous results, we first investigated whether the loss of the Fzd6 receptor in Fzd6−/− null mutants leads to a defective establishment of the MHB and/or patterning of the MHR at midgestational stages. Apart from Wnt1, most of the patterning activity of the IsO is conferred by only one molecule, the secreted fibroblast growth factor 8 (Fgf8) (Martinez,2001). The expression of Wnt1 and Fgf8 and the positioning of the corresponding domains at the rostral and caudal border of the MHB, respectively, was not changed in the Fzd6−/− embryos at E10.5, when this boundary is readily established in the wild-type (Fig. 2A–L). The expression of Wnt1 and Fgf8 on consecutive coronal sections of the midbrain and rostral hindbrain was also not changed in the Fzd6−/− embryos at E12.5 (Fig. 2M–T and data not shown), indicating that removal of the Fzd6 receptor does not affect the expression of these two IsO genes over time. As the Fzd6−/− mutant embryos did not exhibit any gross morphological and histological deficits in the MHR at E10.5, E12.5, and E17.5 (data not shown), we concluded that the positioning of the MHB, the activity of the IsO at this boundary, and consequently the patterning of the MHR are not affected by the loss of Fzd6 receptor function in the mouse embryo. The Wnt1 signal required for these processes is, therefore, likely to be transduced by an alternative or redundant Fzd receptor in the Fzd6−/− mutants.

Figure 2.

Wnt1 and Fgf8 expression at the MHB and the development of mDA neurons are not affected in Fzd6−/− embryos. Representative coronal (A–H, M–X) and midsagittal (I–L) sections from E10.5 (A–L) and E12.5 (M–X) Fzd6+/+ (A,B,E,F,I,J,M,N,Q,R,U,V) and Fzd6−/− (C,D,G,H,K,L,O,P,S,T,W,X) mouse embryos hybridized with riboprobes for Wnt1 (A–D,I,K,M–P) and Fgf8 (E–H,J,L,Q–T), or immunostained with antibodies for Th (green, U,W) and Pitx3 (red, V,X). Dorsal is at the top, anterior to the left. Arrowheads in I–L point at the caudal (I,K) or rostral (J,L) expression boundaries of the corresponding genes demarcating the position of the MHB. The section shown in J was cut at a more lateral level than the section shown in L; the ventral Fgf8 domain in the BP (sparing the FP) is thus visible in the section shown in J.

Although the establishment of the MHB was not influenced by the loss of Fzd6, the prominent expression of this receptor in Aldh1a1+ mDA precursors within the caudal cephalic flexure might have affected the development of these precursors into mDA neurons. Therefore, we analyzed the different stages of mDA neuron generation in Fzd6−/− mutant embryos. In addition to Aldh1a1, mDA postmitotic precursors express Nr4a2 (Nurr1), an orphan nuclear receptor required for the activation of the Th, vesicular monoamine transporter 2 (Vmat2/Slc18a2) and DA transporter (Dat/Slc6a3) genes (Perlmann and Wallen-Mackenzie,2004). At E10.5, Aldh1a1+ mDA progenitors and Nr4a2+ postmitotic precursors arose normally in the Fzd6−/− mutants (see Supp. Fig. S1, which is available online). Differentiation of these precursors into Th+, Pitx3+ (a homeodomain transcription factor [TF] required for the proper development and survival of a subset of mDA neurons; Smidt et al.,2004) and Dat+ mDA neurons at E12.5, and the subsequent survival of these neurons until E17.5, were not affected in the Fzd6−/− mutants (Fig. 2U–X, Fig. S1). The Pou4f1+ (Brn3a) domain in the E12.5 midbrain, giving rise to red nucleus (RN) neurons in the VM (Fedtsova and Turner,1995), and to glutamatergic neurons in the DM (Nakatani et al.,2007), was also not affected in the Fzd6−/− mutants (Fig. S1). This suggested that the lack of a neural and in particular mDA phenotype in the Fzd6−/− mutants might be due to the redundant function of another Fzd receptor.

The Number of Th+ mDA Neurons Is Reduced in the Fzd3−/− Embryos at E12.5

Fzd3 is expressed throughout the anterior neural tube (Fischer et al.,2007), and this made it a strong candidate to act redundantly with Fzd6 in the development of the MHR and of mDA neurons. Fzd3−/− mutants do not have gross morphological abnormalities in the MHR except for an open neural tube phenotype at low penetrance (Wang et al.,2002; and data not shown). Even though the generation of Aldh1a1+ and Nr4a2+ mDA precursors did not appear to be affected in the Fzd3−/− embryos at E10.5 (Fig. S2), the number of Th+ mDA neurons was significantly reduced by an average of 42% in the Fzd3−/− mutants at E12.5 (Fig. 3A–G). The number of Pitx3+ cells was also reduced in the Fzd3−/− embryos at this stage, but this reduction did not reach statistical significance (data not shown). The Th+ cell numbers appeared to recover at E13.5, and although a slight reduction of Th+ mDA neurons was still apparent at E17.5 in the Fzd3−/− mutants, this difference was statistically not significant (Fig. 3G). Apart from the reduced mDA neuron numbers, a medio-lateral broadening of the Th+, Pitx3+, Nr4a2+, and Dat+ mDA domain, particularly in the caudal midbrain, was clearly evident in the Fzd3−/− mutants at E12.5 and E13.5 (Fig. 3A–F, Fig. S2), but was not detected at later embryonic stages (Fig. S2 and data not shown). The transient reduction in mDA neuron numbers and broadening of the mDA domain in the Fzd3−/− mutants did not appear to affect other neuronal populations such as the Pou4f1+ RN neurons arising from an adjacent domain in the VM (Fig. 3H,I).

Figure 3.

The number of Th+ mDA neurons is significantly reduced in Fzd3−/− embryos at E12.5. A–F, H–M: Representative coronal midbrain sections from E12.5 (A–F,H,I,L,M) and E10.5 (J,K) wild-type (wt [Fzd3+/+ or Fzd3+/−]; A,C,E,H,J,L) and Fzd3−/− (B,D,F,I,K,M) mouse embryos immunostained with antibodies for Th (red, A–F), Pitx3 (green, A–F), Pou4f1 (Brn3a; H,I), and phospho-Histone H3 (pH3; J–M). Dorsal is at the top. Sections in A–F were taken at successive anterior (A,B) to posterior (E,F) levels of the midbrain. Double arrows in A–F depict the broadening of the mDA domain in Fzd3−/− embryos, in particular in the caudal midbrain. Pou4f1+ red nucleus (RN) neurons are not affected in the Fzd3−/− mutants (H,I). White broken lines in J–M indicate the approximate dorsal limit of the VM area used for quantification of pH3+ cells. G: Quantification of the number of Th+ mDA neurons in wt and Fzd3−/− embryos at E12.5, E13.5, and E17.5 (significant interaction effect in the two-way ANOVA, P = 0.025). (E12.5: wt, 2,040 ± 494; Fzd3−/−, 1,169 ± 340 mean ± s.e.m.; * P = 0.031 in the paired t-test; n = 3 pairs of mice; E13.5: wt, 3,524 ± 282; Fzd3−/−, 3,816 ± 473 mean ± s.e.m.; ns, not significant, P = 0.425; n = 3 pairs of mice; E17.5: wt, 1,1730 ± 255; Fzd3−/−, 10,252 ± 277 mean ± s.e.m.; ns, not significant, P = 0.159; n = 2 pairs of mice). N: Quantification of mitotic (pH3+) cells/μm2 in the wt and Fzd3−/− VM at E10.5 and E12.5 (E10.5: wt, 0.000456 ± 0.0000330x; Fzd3−/−, 0.000486 ± 0.0000418 mean ± s.e.m.; ns, not significant, P = 0.774 in the one-way ANOVA for repeated measurements; n = 3 mice in each group with 7–8 sections each; E12.5: wt, 0.000243 ± 0.0000195; Fzd3−/−, 0.000215 ± 0.0000253 mean ± s.e.m.; ns, not significant, P = 0.425; n = 2 pairs of mice with 7–8 sections each).

The reduced numbers of Th+ mDA neurons in the E12.5 Fzd3−/− embryos could have been the consequence of an impaired proliferation of their progenitors or survival of the postmitotic neurons. We, therefore, assessed the number of cells expressing phosphorylated Histone H3 (pH3), a mitotic marker, or cleaved (activated) Caspase3 (cCasp3), an apoptotic marker, in the Fzd3−/− VM at E10.5, E12.5, E13.5, or E17.5 (Fig. 3J–M and data not shown). The number of mitotic (pH3+) cells was not significantly changed in the Fzd3−/− VM at E10.5 or E12.5 (Fig. 3N), the time period during which most mDA neurons are presumably born in the mouse embryo. Moreover, only very few if any apoptotic (cCasp3+) cells were detected in the Fzd3−/− VM at E12.5, E13.5, and E17.5, similar to the wild-type (data not shown). We thus concluded that the loss of the Fzd3 receptor in the VM in general and in mDA precursors in particular has no influence on the proliferation and survival of these cells, but appears to affect the differentiation of mDA precursors into Th+ mDA neurons.

Fzd3−/−; Fzd6−/− Double Mutant Mouse Embryos Have a Severe Midbrain Morphogenesis Defect

The lack of an overall MHB phenotype in the Fzd6−/− and Fzd3−/− single mutants, and the transient mDA phenotype in the Fzd3−/− embryos, suggested a redundant function of the two receptors in these processes. We, therefore, proceeded with the analysis of Fzd3−/−; Fzd6−/− double mutants. Due to the low frequency of double mutant embryos derived from our breeding scheme, we focused our analyses on embryonic stages E10.5 (when the IsO is already established at the MHB) and E12.5 (when different neuronal populations are emerging in the midbrain). Five out of seven (71%) double mutant Fzd3−/−; Fzd6−/− and two Fzd3−/−; Fzd6+/− embryos dissected at E12.5, as well as two out of three (67%) double mutant Fzd3−/−; Fzd6−/− embryos dissected at E10.5, had the outer appearance of an open neural tube (Fig. 4A,B). Furthermore, all Fzd3−/−; Fzd6−/− and Fzd3−/−; Fzd6+/− mutants dissected at E10.5 and E12.5 had a curly tail (Fig. 4A,B), similar to what was reported by Wang et al. (2006a). Upon closer inspection of these embryos, however, we noted that their anterior neural tube was closed and in some cases it exhibited several bulges along the antero-posterior (A/P) axis (Fig. 4A,B). Nissl-stained coronal sections at the level of the forebrain, midbrain, and hindbrain, indeed, confirmed that the anterior neural tube of these mutants was completely closed and covered by mesenchyme (Fig. 4D–I). These sections also revealed a massive growth of dorsal neural tissue into the mesencephalic (third) ventricle in three out of seven (43%) double mutant Fzd3−/−; Fzd6−/− embryos dissected at E12.5 (Fig. 4D,E). In fact, the third ventricle was almost entirely occluded by the protruding neural tissue, which had also fused in part with VM neuroepithelium (Fig. 4D,E). All other Fzd3−/−; Fzd6−/− double mutants analyzed at E10.5 or E12.5 did not display this massive protrusion of dorsal neural tissue, but showed a collapse of the brain ventricles and an apparently thickening of the neuroepithelium and of the mesenchymal layer surrounding it, particularly in the dorsal MHR (Fig. 4F–I). Moreover, the Fzd3−/−; Fzd6−/− double mutant midbrain was broadened laterally and tended to be shortened along the D/V axis at E12.5 (Fig. 4C). Altogether, the simultaneous loss of Fzd3 and Fzd6 leads to a severe midbrain morphogenesis defect that is not an open (exencephalic) anterior neural tube. Since such a neurulation phenotype has to the best of our knowledge not been described before, we tried to characterize in more detail the midbrain defect in the Fzd3−/−; Fzd6−/− double mutants.

Figure 4.

A severe midbrain morphogenesis defect is already evident at E10.5 in Fzd3−/−; Fzd6−/− double mutant embryos. A,B: Lateral views of an Fzd3+/+; Fzd6−/− “wild-type” (A) and an Fzd3−/−; Fzd6−/− double mutant (B) embryo at E12.5. Black arrow in B points at the mid-/hindbrain “bulge” in the double mutant embryo. Note a similar bulge from the caudal forebrain/midbrain in this embryo. Open arrow in B points at the curly tail of the double mutant. C: Quantification of the dorsoventral (D/V) and left-right (lateral, L) extent of the midbrain in E12.5 wild-type (wt) and Fzd3−/−; Fzd6−/− double mutant embryos, as depicted by red double arrows in D. Significant interaction effect in the two-way ANOVA, P = 0.002. (D/V: wt, 1741.4 ± 46.6 μm; Fzd3−/−Fzd6−/−, 1,559.6 ± 75.0 μm mean ± s.e.m.; ns, not significant, P = 0.053 in the one-way ANOVA for repeated measurements; L: wt, 2305.6 ± 88.9 μm; Fzd3−/−Fzd6−/−, 2,596.5 ± 68.9 μm mean ± s.e.m.; *P = 0.018; n = 3 pairs of embryos with 4 sections each). D–I: Representative Nissl-stained coronal sections at midbrain (D,E), forebrain (F,G), and anterior hindbrain (H,I) levels from Fzd3+/+; Fzd6−/− or Fzd3+/−; Fzd6+/− “wild-type” (D,F,H) and Fzd3−/−; Fzd6−/− double mutant (E,G,I) embryos at E12.5 (D,E) and E10.5 (F–I). Dorsal is at the top. Black staining in F–I are radioactive in situ hybridization signals for Wnt1. L, II, III, IV: lateral (telencephalic), second (diencephalic), third (mesencephalic), and fourth (metencephalic) brain ventricles, respectively.

Cell Proliferation Is Not Increased in the Fzd3−/−; Fzd6−/− Double Mutant Midbrain at E10.5

The collapse of the brain ventricles and apparently thickening of the neuroepithelium in the MHR of the Fzd3−/−; Fzd6−/− embryos already at E10.5, which in its most extreme case might lead to the massive protrusion of dorsal neural tissue and occlusion of the third ventricle at E12.5, suggested an increased cell proliferation in the double mutant midbrain. Wnts are known to regulate cell proliferation and in particular the transition between G1 to S phase of the cell cycle (Megason and McMahon,2002). We therefore analyzed the distribution and number of BrdU-labeled cells in the midbrain neuroepithelium of wild-type and Fzd3−/−; Fzd6−/− embryos at E10.5. The distribution of BrdU+ cells was similar in wild-type and double mutant embryos at this stage (Fig. 5A–F,H). For the quantification of BrdU+ cells, we had to account for the fact that even in the wild-type embryo, cell proliferation is higher in the DM compared to the VM (Fig. 5G). We thus defined the border between the DM and VM areas in wild-type and double mutant embryos roughly at the position of the dorsolateral hinge-points, as depicted in Figure 5A,B. Although the total number of BrdU+ cells in the DM and VM tended to be increased in the Fzd3−/−; Fzd6−/− double mutants, this increase did not reach statistical significance (data not shown). We obtained a similar result when we calculated the density of the BrdU-labeled cells in the DM and VM of wild-type and Fzd3−/−; Fzd6−/− embryos (Fig. 5G), indicating that cell proliferation, and in particular cells in the S phase of the cell cycle, are not significantly increased in the double mutant midbrain at E10.5.

Figure 5.

Cell proliferation is not significantly increased but cellular protrusions are evident at E10.5 in the neuroepithelium of Fzd3−/−; Fzd6−/− double mutant embryos. A–F, H: Representative coronal midbrain sections from Fzd3+/−; Fzd6+/− “wild-type” (wt; A,C,E) and Fzd3−/−; Fzd6−/− double mutant (B,D,F,H) embryos pulse-labeled with BrdU for 2 hr at E10.5. Dorsal is at the top. C,E and D,F are higher magnifications of the DM (C,D) and VM (E,F) shown in A and B, respectively. Black arrows in D,H point at the sites where the basal lamina at the ventricular surface of the neuroepithelium appeared to be disrupted by filopodia-like cellular protrusions and cell nuclei extending into the ventricular cavity of the double mutant midbrain. G: Quantification of BrdU+ cells/μm2 in the VM (area delineated with a red line in A,B) and DM (area delineated with a black line in A,B) of wt and Fzd3−/−; Fzd6−/− double mutant embryos at E10.5 (VM: wt, 0.00267 ± 0.00028; Fzd3−/−Fzd6−/−, 0.00292 ± 0.00019 mean ± s.e.m.; DM: wt, 0.00476 ± 0.00025; Fzd3−/−Fzd6−/−, 0.00534 ± 0.00024 mean ± s.e.m; Genotype effect in two-way ANOVA without interaction not significant (ns), P = 0.096, post-hoc tests not appropriate; n = 2 pairs of embryos with 4 sections each).

The nuclear staining of proliferating BrdU+ cells located at the ventricular surface of the neuroepithelium, however, revealed another defect in the double mutants. The basal membrane at the ventricular surface of the neuroepithelium appeared to be focally disrupted by filopodia-like cellular protrusions and BrdU+ cell nuclei extending into the remaining ventricular cavity of the double mutant midbrain (Fig. 5D,H). These focal disruptions occurred more often in the DM than in the VM, and were never observed in the wild-type midbrain (Fig. 5C–F,H). We thus propose that these focal disruptions of the basal membrane and cellular protrusions might be the sites at which a later “fusion” of dorsal and ventral neural tissues occurs, especially if they face each other on both sides of the remaining ventricular cavity, as depicted in Figure 5D.

D/V Patterning Is Not Affected in the Fzd3−/−; Fzd6−/− Double Mutant Midbrain

We next assessed whether the midbrain morphogenesis defect in the Fzd3−/−; Fzd6−/− embryos might be due to a defective D/V patterning. The dorsal (roof plate, RP) and ventral (FP) midline of the midbrain is characterized by the expression of Wnt1 (Wilkinson et al.,1987), Lmx1a (Millonig et al.,2000; Chizhikov and Millen,2004b), and Lmx1b (Chizhikov and Millen,2004a). At E10.5, Wnt1 and Lmx1a were expressed in a perpendicular stripe of cells across the thickened dorsal neuroepithelium, at a position that might correspond to the RP of the double mutant midbrain (Fig. S3). A similar pattern of Wnt1 and Lmx1a expression as in the FP of the wild-type embryo was observed in the Fzd3−/−; Fzd6−/− double mutant VM at E10.5, except for a slightly aberrant morphology and broadening of their expression domains due to the thickened and protruding ventral neural tissue in the double mutants (Fig. S3). Moreover, the A/P extent of the Wnt1+ and Lmx1a+ domains did not appear to be affected in the double mutants at E10.5, as assessed on consecutive coronal sections of the wild-type and double mutant midbrain (Fig. S3). The alar plate (AP) is the DM domain adjacent to the RP and characterized by the expression of Dbx1 and Pax7, the BP is the VM domain adjacent to the FP and characterized by the expression of Nkx6-1 and Nkx6-2, and an intermediate zone located at the ventrolateral sulcus of the midbrain is characterized by the expression of Nkx2-2 (Prakash et al.,2009). The spatial relationship of the Dbx1+ and Pax7+ domains dorsally, and of the Nkx2-2+ and Nkx6-2+ domains ventrally, was preserved in the Fzd3−/−; Fzd6−/− embryos at E10.5, despite the altered morphology of the double mutant midbrain (Fig. S3).

The D/V positioning and A/P extent of the Wnt1+, Lmx1a+, Lmx1b+, Dbx1+, Nkx2-2+, Pax7+ and Nkx6-2+ domains were still preserved in the Fzd3−/−; Fzd6−/− double mutant midbrain at E12.5 (Fig. 6A–N and data not shown). At this stage, the ventral boundary of the dorsal Dbx1+ domain abuts the Nkx2-2+ domain in the mantle zone (MZ) of the lateral midbrain, and the ventral boundary of the dorsal Pax7+ domain abuts the dorsal Nkx6-2+ domain in the ventricular (VZ)/subventricular (SVZ) zone of the lateral midbrain (Fig. 6G,H,K,L). These D/V boundaries of expression were not altered in the double mutants, indicating that D/V positional information is conserved in the Fzd3−/−; Fzd6−/− midbrain despite the severe morphogenesis defect.

Figure 6.

D/V patterning is not affected in the Fzd3−/−; Fzd6−/− double mutant midbrain at E12.5. A–N: Representative coronal midbrain sections from E12.5 “wild-type” (wt) (Fzd3+/+; Fzd6−/− or Fzd3+/−; Fzd6+/−; A,C,E,G,H,K,L) and Fzd3−/−; Fzd6−/− double mutant (B,D,F,I,J,M,N) embryos hybridized with riboprobes for Wnt1 (A,B), Lmx1a (C,D), and Lmx1b (E,F), or immunostained with antibodies for Dbx1 (red) and Nkx2-2 (green) (G–J), or Nkx6-2 (red) and Pax7 (green) (K–N). Dorsal is at the top. The section in B was taken at a more anterior level than the section in A. Red broken lines in A and C delimit the roof plate (RP) and the floor plate (FP), respectively. Black and white arrows in B,D,F point at the stripe of Wnt1+, Lmx1a+, and Lmx1b+ cells extending from the dorsal surface ventrally across the protruding dorsal neural tissue. H,J,L,N are higher magnifications of the right half of the midbrain shown in G,I,K,M, respectively. O–R: Representative coronal midbrain sections from Fzd3+/−; Fzd6−/− “wild-type” (wt; O,P) and Fzd3−/−; Fzd6+/− (Q,R) embryos pulse-labeled with BrdU for 2 hr at E12.5. Dorsal is at the top. P and R are higher magnifications of O and Q, respectively. Black broken lines in O,Q indicate the approximate position of the border between the DM and VM areas used for the quantification of pH3+ and BrdU+ cells in E12.5 embryos. Quantification of BrdU+ cells/μm2 in these embryos is shown in Figure 7N.

A Reduction of Fzd6 Gene Dosage on an Fzd3−/− Background Is Sufficient to Cause the Severe Midbrain Morphogenesis Defect

In the absence of an obvious D/V patterning defect in the Fzd3−/−; Fzd6−/− embryos, we then asked whether the number of proliferating cells, and in particular S phase cells, was increased in the double mutant midbrain at E12.5, thereby leading to the massive growth of dorsal neural tissue and occlusion of the third ventricle observed in three out of seven (43%) double mutant embryos at this stage. Due to the low frequency of double mutants expected from our breeding scheme, we did not obtain BrdU pulse-labeled Fzd3−/−; Fzd6−/− embryos at E12.5. However, we noted that embryos genotyped as Fzd3−/−; Fzd6+/− also had the outer appearance of an open neural tube at this stage, and we therefore proceeded with the analysis of these embryos. We used their Fzd3+/−; Fzd6−/− littermates for comparison and for the assessment of a gene dosage effect. Coronal sections through the anterior neural tube revealed a brain morphogenesis defect in the Fzd3−/−; Fzd6+/− but not in the Fzd3+/−; Fzd6−/− embryos that was indistinguishable from the defect in two out of seven (29%) and two out of three (67%) Fzd3−/−; Fzd6−/− double mutants analyzed at E12.5 and E10.5, respectively (data not shown). At E12.5, the Fzd3−/−; Fzd6+/− embryos showed a similar collapse of the third ventricle and thickening of the midbrain neuroepithelium as the Fzd3−/−; Fzd6−/− double mutants (Fig. 6O–R), and we therefore assessed the number and distribution of BrdU-labeled cells in their midbrain. Even though the total number of BrdU+ cells was significantly (P < 0.001) increased in the VM but not in the DM of the Fzd3−/−; Fzd6+/− embryos (data not shown), their density was not significantly increased in these embryos (Fig. 7N). Two conclusions were drawn from these experiments: firstly, the reduction of Fzd6 gene dosage on an Fzd3 null background is sufficient to cause the severe midbrain morphogenesis defect, but not vice versa. Secondly, the tendency towards an increased number of proliferating S phase cells in the VM of the Fzd3−/−; Fzd6+/− embryos at E12.5 might be the first sign of an increased proliferation in these mutants, and might contribute to the protrusion of dorsal and ventral neural tissues and to the rescue of mDA neuron numbers in the double mutants (see below).

Figure 7.

Cell proliferation is significantly increased at E12.5 in the VM and DM of Fzd3−/−; Fzd6−/− embryos with protrusion of dorsal neural tissue. A–L: Representative coronal midbrain sections from E12.5 Fzd3+/+; Fzd6−/− “wild-type” (A,C,E,G,I,K) and Fzd3−/−; Fzd6−/− double mutant (B,D,F,H,J,L) embryos immunostained with antibodies for phospho-Histone H3 (pH3; A,B), cyclin D1 (Ccnd1; C,D), p27Kip1 (Cdkn1b; E,F), Sox2 (G,H), Mash1 (Ascl1; I,J), and NeuN (K,L). Dorsal is at the top. Double mutant embryos in B,F,H,J,L show already a massive protrusion of dorsal neural tissue into the third ventricle, whereas dorsal neural tissue is beginning to protrude in the embryo shown in D. White arrow in F points at the stripe of Cdkn1b+ cells extending from the dorsal surface ventrally across the protruding dorsal neural tissue. M: Quantification of mitotic (pH3+) cells/μm2 in the VM and DM (delimitation of areas as in Fig. 6) of wt and Fzd3−/−; Fzd6−/− embryos with protruding dorsal neural tissue at E12.5. Significant genotype and region effect in the one-way ANOVA without interaction, both P<0.0001. (Post-hoc genotype comparisons with P value adjustment: VM: wt, 0.000211 ± 0.0000134; Fzd3−/−Fzd6−/−, 0.000323 ± 0.0000191 mean ± s.e.m.; ***P = 0.0006 in the one-way ANOVA for repeated measurements; DM: wt, 0.000573 ± 0.0000201; Fzd3−/−Fzd6−/−, 0.000687 ± 0.0000337 mean ± s.e.m.; ***P<0.0001; n = 3 pairs of embryos with 11–16 sections each). N: Quantification of BrdU+ cells/μm2 in the VM and DM of wt and Fzd3−/−; Fzd6+/− embryos without protruding dorsal neural tissue at E12.5. Significant interaction effect in the two-way ANOVA, P = 0.009. (Region-specific genotype comparisons: VM: wt, 0.00135 ± 0.0000965; Fzd3−/−Fzd6+/−, 0.00344 ± 0.000439 mean ± s.e.m.; ns, not significant, P = 0.053 in the one-way ANOVA for repeated measurements; DM: wt, 0.00506 ± 0.000182; Fzd3−/−Fzd6+/−, 0.00516 ± 0.000526 mean ± s.e.m.; ns, not significant, P = 0.928; n = 2 pairs of embryos with 5 sections each).

Increased Cell Proliferation and Delayed Cell Cycle Exit Might Be the Cause for the Massive Growth of Dorsal Neural Tissue in Some Fzd3−/−; Fzd6−/− Embryos at E12.5

Because of the previous findings, we next investigated whether the massive protrusion of dorsal neural tissue in three out of seven (43%) Fzd3−/−; Fzd6−/− double mutant embryos dissected at E12.5 might be due to the excessive proliferation of dorsal neuroepithelial cells in their midbrain. Although mitotic (pH3+) progenitors were mostly confined to the VZ/SVZ lining the occluded third ventricle and pH3+ cells were not found inside the protruding tissue mass (Fig. 7A,B), their cell density was significantly increased in the VM and DM of these double mutants (Fig. 7M). Moreover, the protruding dorsal neural tissue contained mostly cells expressing cyclin D1 (Ccnd1, a marker for cells in the cell cycle) and only very few cells expressing the cyclin-dependent kinase inhibitor p27Kip1 (Cdkn1b, a marker for cells exiting the cell cycle) (Fig. 7C–F), suggesting that cell cycle exit was also delayed in the protruding dorsal neural tissue of the double mutants. In line with this finding, the TFs Sox2 and Mash1 (Ascl1), markers for proliferating neural progenitors in the midbrain (Kele et al.,2006), were widely expressed across the protruding dorsal neural tissue (Fig. 7G–J), whereas expression of NeuN (Neuna60), a marker for postmitotic neurons, was mostly confined to the outer margin (MZ) of the DM and hardly detected within the protruding tissue (Fig. 7K,L). The number of apoptotic (cCasp3+) cells, by contrast, was not increased in the double mutant midbrain (data not shown). We therefore concluded that the massive growth of neural tissue in some double mutant embryos at E12.5, leading to the occlusion of the third ventricle, was most likely a consequence of the increased proliferation and delayed cell cycle exit of neural progenitors at this later stage.

Th+ and Pitx3+ mDA Neurons Are Correctly Specified in the Fzd3−/−; Fzd6−/− Double Mutants

Finally, we wanted to know if the further reduction of Fzd gene dosage and the midbrain morphogenesis defect in the Fzd3−/−; Fzd6−/− double mutants might have affected the normal development of the mDA and other midbrain neuronal populations. Th+ and Pitx3+ mDA neurons were detected in the Fzd3−/−; Fzd6−/− VM at E11.5 and E12.5, and although their numbers tended to be reduced in the double mutants, this reduction did not reach statistical significance (Fig. 8A–G and data not shown). However, the Th+ cell density was significantly reduced and a medio-lateral broadening of the mDA domain, similar to the Fzd3−/− single mutants, was detected in the caudal midbrain of the double mutants at E12.5 (Fig. 8E,F,H). Wnt1, Lmx1a, and Lmx1b are expressed in proliferating mDA progenitors/postmitotic precursors in the midbrain FP and required for the generation of mDA neurons (Smidt et al.,2000; Zervas et al.,2004; Andersson et al.,2006; Prakash et al.,2006; Ono et al.,2007). The expression of these factors in the Fzd3−/−; Fzd6−/− embryos (Fig. 6A–F, Fig. S3) also indicated the correct specification of mDA precursors in the double mutant VM. Pou4f1+ RN neurons emerged from the double mutant VM, but the appearance of this domain was more affected in the Fzd3−/−; Fzd6−/− embryos (Fig. 8I,J). The Pou4f1+ RN domain was elongated along the D/V axis and exhibited a cellular defect in its ventral part (Fig. 8I,J) that might be due to the massive up-folding of ventral neural tissue in the double mutants.

Figure 8.

Th+ and Pitx3+ mDA and Pou4f1+ RN neurons are specified correctly in the Fzd3−/−; Fzd6−/− double mutant embryos at E12.5. A–F,I,J: Representative coronal midbrain sections from E12.5 Fzd3+/−; Fzd6+/− and Fzd3+/+; Fzd6−/− “wild-type” (wt; A,C,E,I) and Fzd3−/−; Fzd6−/− double mutant (B,D,F,J) embryos immunostained with antibodies for Th (red, A-F), Pitx3 (green, A-F), and Pou4f1 (Brn3a; I,J). Dorsal is at the top. Sections in A–F were taken at successive anterior (A,B) to posterior (E,F) levels of the midbrain. Double arrows in E,F depict the broadening of the mDA domain in the Fzd3−/−; Fzd6−/− caudal midbrain. White arrows in J point at the cellular defects within the Pou4f1+ RN domain. G: Quantification of the number of Th+ mDA neurons in wt and Fzd3−/−; Fzd6−/− double mutant embryos at E11.5 and E12.5 (E11.5: wt, 438 ± 177; Fzd3−/−; Fzd6−/−, 349 ± 18 mean ± s.e.m.; n = 2 pairs of mice; E12.5: wt, 2,080 ± 847; Fzd3−/−; Fzd6−/−, 1,537 ± 536 mean ± s.e.m.; n = 4 pairs of mice; genotype effect in two-way ANOVA without interaction not significant (ns), P = 0.181, post-hoc tests not appropriate). H:Quantification of the density of Th+cells/μm2 in wt and Fzd3−/−; Fzd6−/− double mutant embryos at E11.5 and E12.5. Significant genotype effect in the one-way ANOVA without interaction, P<0.004. (Post-hoc genotype comparisons with P value adjustment: E11.5: wt, 0.00183 ± 0.000292; Fzd3−/−; Fzd6−/−, 0.00124 ± 0.0000606 mean ± s.e.m.; ns, not significant, P = 0.593 in the paired t-test; n = 2 pairs of mice; E12.5: wt, 0.00286 ± 0.000303; Fzd3−/−; Fzd6−/−, 0.00184 ± 0.000186 mean ± s.e.m.; *P = 0.0021; n = 4 pairs of mice).

Based on these results, we concluded that the loss of Fzd6 expression in an Fzd3−/− background does not affect the specification of mDA and RN neurons. However, their density and distribution was altered in the double mutant VM, most likely because of the midbrain morphogenesis defect. We also concluded that the transient reduction of Th+ mDA neuron numbers, observed in the Fzd3−/− single mutants at E12.5, might be compensated in the Fzd3−/−; Fzd6−/− embryos by the increased proliferation detected in the double mutant VM at this stage.

DISCUSSION

Here we investigated the potential function of the two Fzd receptors, Fzd3 and Fzd6, in midbrain development, based on their prominent expression in this brain region of the midgestational mouse embryo and on the known requirement of Wnt/β-catenin signaling for this process. Our results indicate that the establishment and maintenance of the IsO at the MHB, and the patterning of the midbrain along the D/V axis are not affected in the Fzd6−/− and Fzd3−/− single, and Fzd3−/−; Fzd6−/− double mutant embryos. The mDA neurons developed normally in the Fzd6−/− embryos, but their number was transiently reduced in the Fzd3−/− single mutants at E12.5 and recovered at later stages. This mDA phenotype was rescued in the Fzd3−/−; Fzd6−/− embryos, probably due to the increased proliferation detected in the double mutant VM at this stage. We, therefore, concluded that Fzd3 and Fzd6 are not necessary for the A/P and D/V patterning of the midbrain, and that Fzd3 but not Fzd6 plays a role in mDA neuron differentiation. The Fzd3−/−; Fzd6−/− double mutants, however, displayed a severe midbrain morphogenesis defect from the earliest stage (E10.5) analyzed, suggesting that these two receptors are involved in early morphogenetic processes required for the proper shaping of the anterior neural tube in the mouse embryo.

Fzd6 Does Not Transmit the Wnt1 Signal Required for MHB and mDA Neuron Development

We initially hypothesized that Fzd6 might be a strong candidate to transduce the Wnt1 signal required for the establishment and maintenance of the IsO at the MHB, and for the proper development of mDA neurons, because of its striking expression at the MHB and in the caudal cephalic flexure. However, the analyses of the Fzd6−/− single and Fzd3−/−; Fzd6−/− double mutants showed that this receptor is not involved in these processes. The expression of Wnt1 or Fgf8 was neither altered along the D/V and A/P axes of the neural tube nor developmentally regulated in the Fzd6−/− and Fzd3−/−; Fzd6−/− embryos. Moreover, Th+ and Pitx3+ mDA neurons arose normally in these mutants. Fzd6 is, therefore, unlikely to transmit the Wnt1 signal required for the proper development of the MHR and of mDA neurons. The analyses of the Fzd3−/−; Fzd6+/− and Fzd3−/−; Fzd6−/− embryos, however, revealed a previously unsuspected function of Fzd6 in early midbrain morphogenesis. Heterozygocity for Fzd6 on an Fzd3−/− background was sufficient to cause what appears to be an early neurulation defect. The contribution of Fzd6 to this phenotype is indeed enigmatic, given that Fzd6 expression is initially (at E9.5) confined to small and defined regions in the anterior neural tube, and becomes even more restricted at later embryonic stages (Fischer et al.,2007). Fzd6, however, is strongly expressed in the mesenchyme surrounding the neural tube at these midgestational stages (Fischer et al.,2007). The identity of the cells or tissues within or outside the neural plate/tube expressing the Fzd6 receptor at earlier developmental stages and contributing to the double mutant phenotype, therefore, still needs to be established.

Fzd3−/− Mutants Show a Transient Delay in mDA Neuron Differentiation: A Link to Schizophrenia?

The ubiquitous expression of Fzd3 in the mouse anterior neural tube and the severe axon outgrowth defects in the CNS of the Fzd3−/− single mutants (Wang et al.,2002; Lyuksyutova et al.,2003; Wang et al.,2006b; Fischer et al.,2007), made it another candidate to act either alone or redundantly with Fzd6 in the development of the MHR and of mDA neurons. The morphogenesis (including cell proliferation and survival) of the MHR and the generation of Pou4f1+ RN and tectal neurons, however, were not affected in the Fzd3−/− embryos, suggesting a minor role of Fzd3 in the development of this region. Nevertheless, and despite an apparently normal expression of Aldh1a1 and Nr4a2 in their precursors at E10.5, the numbers of Th+ mDA neurons were conspicuously reduced in the Fzd3−/− single mutants at E12.5. Although this reduction of Th+ mDA neurons was only transient and appeared to recover already one day later in development, it suggested that the differentiation of Aldh1a1+ and Nr4a2+ mDA precursors into Th+ mDA neurons was delayed in the Fzd3−/− mutants. In addition, we noted a transient medio-lateral broadening of the Th+/Pitx3+ mDA domain in the Fzd3−/− embryos, that was most pronounced in the caudal midbrain and disappeared at approximately the same time when the Th+ mDA neuron numbers recovered in these mutants. At first sight, the mDA defect in the Fzd3−/− embryos appears to resemble the phenotype of the Wnt5a−/− mutants (Andersson et al.,2008). A closer look, however, reveals several differences between the Fzd3−/− and Wnt5a−/− mutant phenotype: first, the number of Th+ mDA neurons is transiently increased and not decreased in the Wnt5a−/− mutants; second, proliferation in the FP region was also increased in the Wnt5a−/− embryos; and third, the medio-lateral broadening of the mDA domain was most pronounced in the rostral and not in the caudal midbrain of the Wnt5a−/− mutants and persisted throughout development (Andersson et al.,2008). As midbrain morphogenesis was also not altered in the Fzd3−/− single mutants, in contrast to the Wnt5a−/− mice (Andersson et al.,2008), it is unlikely that Fzd3 transduces the “non-canonical” Wnt5a/PCP signal. Remarkably, Lrp6−/− mutants also display a transient delay in mDA neuron differentiation without any alterations in cell proliferation, survival, or midbrain patterning (Castelo-Branco et al.,2009, pages 211–221, this issue), thus resembling closely the Fzd3−/− mutant phenotype. Moreover, the persistent reduction of Th+ mDA neuron numbers is a characteristic phenotype of other mouse mutants for the “canonical” Wnt1/β-catenin pathway (Prakash et al.,2006; Joksimovic et al.,2009; Tang et al.,2009), and Xenopus Fzd3 and Wnt1 strongly interact during neural crest development in the frog (Deardorff et al.,2001). The less severe and transient reduction of Th+ mDA neurons in the Fzd3−/− and Lrp6−/−X mutants, therefore, suggests that this receptor and co-receptor are involved in the transduction of the Wnt1/β-catenin signal required for mDA neuron differentiation, but they might be functionally compensated by other Fzd/Lrp receptors/co-receptors during mouse development (see also the report by Castelo-Branco et al.,2009, pages 211–221, this issue).

The requirement of Fzd3 for the proper differentiation of mDA neurons in the mouse embryo, as described here, might also have some relevance for the pathogenesis of human neuropsychiatric disorders, in particular of schizophrenia. Polymorphisms in the human FZD3 gene have been associated with schizophrenia, although these findings could not be replicated in all studies (reviewed by De Ferrari and Moon,2006). The functional implications of these FZD3 polymorphisms are still unclear, but it might now be hypothesized that apart from an aberrant neurotransmission due to the disruption of major fiber tracts (including the corticothalamic, thalamocortical and nigrostriatal pathways) (Wang et al.,2002,2006b), a delayed differentiation of mDA neurons could also contribute to the pathogenesis of this disease in carriers of FZD3 mutations.

Fzd3 and Fzd6 Control Midbrain Morphogenesis in the Mouse by an Unknown Mechanism

Our main finding is an improper morphogenesis of the early mouse brain in the Fzd3−/−; Fzd6+/− and Fzd3−/−; Fzd6−/− double mutants. This phenotype differs from the neural tube closure defects (exencephaly and craniorachischisis) reported before in the Fzd3−/− single and Fzd3−/−; Fzd6−/− double mutants (Wang et al.,2002,2006a). In fact, the anterior neural tube comprising fore-, mid-, and rostral hindbrain was properly closed in all (100%) double mutants at midgestation. The main defect consisted of a collapse of all brain ventricles leaving only a small gap between the neural tissues, and was observed at a penetrance of around 70% in the Fzd3−/−; Fzd6−/− embryos. This defect was already evident at E10.5 and was accompanied by a thickening of the neuroepithelium and of the overlying mesenchyme in some regions, particularly in the dorsal MHR, that did not appear to be due to an increased proliferation in this region. Dorsal midbrain tissue had protruded and fused with ventrolateral tissues in some (43%) double mutants at E12.5, probably as a consequence of the focal disruption of the ventricular basal lamina and protrusion of individual cells into the remaining ventricular cavity of the midbrain observed at E10.5. These mutants also showed an increased proliferation in the DM and VM that might contribute to the occlusion of the mesencephalic (third) ventricle by the protruding neural tissue. The primary defect in the Fzd3−/−; Fzd6+/− and Fzd3−/−; Fzd6−/− double mutant brain is, therefore, not a neural tube closure defect, which is mainly characterized by the failure of neural fold elevation (Wallingford,2005). It should be noted that apart from the proper elevation and fusion of the dorsal neural folds, the elongation (by convergent extension) of the neural tube, the formation of the median and dorsolateral hinge-points, and the orientation of neuroepithelial cells were not affected in the double mutant midbrain. These observations argue against a “classical” PCP or convergent extension phenotype in the Fzd3−/−; Fzd6+/− and Fzd3−/−; Fzd6−/− midbrain, characteristic for several mutants of the non-canonical Wnt/PCP pathway (Copp et al.,2003; Andersson et al.,2008). Moreover, expression of Wnt1 and of one of its putative target genes, Lmx1a, along the A/P axis, and D/V patterning were not altered in the double mutant midbrain, indicating that patterning of the MHR proceeded independently of the morphogenetic defect. However, the analysis of earlier embryonic stages (at the beginning of neurulation) will be necessary to unravel the mechanism underlying the brain morphogenesis defect in the double mutants. At this point, we can only speculate about possible mechanisms: although neural fold elevation and fusion proceeds normally to form the neural tube, accompanying or subsequent cell migration and adhesion within the elevating neuroepithelium and/or within the surrounding mesenchyme might be aberrant in the double mutants. Alternatively or in addition, the ventricular cavities in the double mutant brain might collapse as a consequence of a reduced or missing internal pressure due to the closure defect in the caudal neural tube of the Fzd3−/−; Fzd6+/− and Fzd3−/−; Fzd6−/− embryos.

The molecular details of Fzd3 and Fzd6 function in mouse brain morphogenesis are completely unknown and remain to be investigated, but their involvement in midbrain morphogenesis is somewhat enigmatic for the following reason. Neither single mutant displays a midbrain morphogenesis defect, but a reduction of Fzd6 gene dosage in the complete absence of Fzd3 was sufficient to cause this defect, albeit at a reduced penetrance. This suggests a redundant function of Fzd3 and Fzd6, together with other (unknown) genetic modifiers. Expression of these two receptors, however, is mostly confined to two different compartments in the midgestational mouse head: Fzd3 is strongly expressed in the neuroepithelium of the anterior neural tube and only very weakly in the surrounding mesenchyme, whereas Fzd6 expression is strongest in the surrounding mesenchyme and highly restricted and weaker in the anterior neural tube (Fischer et al.,2007). Moreover, our data suggest a participation of Fzd3 in the transduction of the Wnt1/β-catenin signal required for mDA neuron development in the VM, whereas the human FZD6 receptor was reported to transmit a signal antagonizing the canonical Wnt/β-catenin pathway (Golan et al.,2004). It, therefore, remains to be established whether Fzd3 and Fzd6 cooperate within the same pathway, as implied by previous reports (Wang et al.,2002,2006a; Guo et al.,2004), or act in antagonistic pathways during midbrain morphogenesis. Alternatively, these two Fzd molecules might operate independently of any Wnt signals in the morphogenesis of the early mouse brain (Seifert and Mlodzik,2007; van Amerongen et al.,2008).

EXPERIMENTAL PROCEDURES

Mutant Mice

Outbred CD-1 mice were purchased from Charles River (Kisslegg/Germany). Generation and genotyping of Fzd3−/− and Fzd6−/− mice was described by Wang et al. (2002) and Guo et al. (2004). Mutant mouse lines were maintained on a mixed C57BL/6 × 129/SVJ background (Wang et al.,2006a). Male Fzd3+/−; Fzd6−/− mice were reported to have a strongly reduced fertility, but in our hands, male Fzd3+/−; Fzd6−/− mice did not produce any sperm at all (data not shown), although we kept the same genetic background as Wang et al. (2006a). The infertility of our male Fzd3+/−; Fzd6−/− mice forced us to change our breeding scheme and to use double heterozygote Fzd3+/−; Fzd6+/− males intercrossed with Fzd3+/−; Fzd6+/− or Fzd3+/−; Fzd6−/− females. Fzd3−/−; Fzd6−/− double mutant embryos were obtained by natural matings or by in vitro fertilization according to standard procedures. Fzd3+/+; Fzd6−/− and Fzd3+/−; Fzd6+/− embryos were used as “wild-type” controls as these embryos do not display a neural tube phenotype (this report; Wang et al.,2006a). Collection of embryonic stages was done from timed-pregnant females as indicated in the text. Noon of the day of vaginal plug detection was designated embryonic day (E) 0.5. Animal treatment was conducted under federal guidelines for the use and care of laboratory animals and was approved by the HMGU Institutional Animal Care and Use Committee.

Radioactive In Situ Hybridization (ISH) and Immunohistochemistry

Paraffin sections (8 μm) of mouse embryos were processed for radioactive ISH or for immunohistochemistry as described in Prakash et al. (2006) and Fischer et al. (2007). Riboprobes for Fzd6, Wnt1, Aldh1a1, Th, Fgf8, Nr4a2, Dat, Lmx1a, and Lmx1b were reported by a number of authors (Niederreither et al.,2002; Brodski et al.,2003; Puelles et al.,2003; Fischer et al.,2007; Prakash et al.,2009). Monoclonal antibodies used were mouse Th (1:600; Chemicon), Pou4f1 (Brn3a) (1:100; Santa Cruz Biotechnology), Nkx2-2 (1:100), Pax7 (1:50) (both obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242), p27Kip1 (Cdkn1b) (1:2,000; BD Transduction Laboratories), Ascl1 (Mash1) (1:40; BD Pharmingen) and NeuN (1:300; Chemicon), rat BrdU (1:100; AbD Serotec), and rabbit-cleaved Caspase 3 (cCasp3) (1:100; Cell Signaling). Polyclonal antisera used were rabbit Th (1:150; Chemicon), Pitx3 (1:300; Zymed/Invitrogen), phosphorylated Histone H3 (pH3) (1:500; Upstate), Dbx1 (1:5,000; gift from A. Pierani), cyclin D1 (Ccnd1) (1:150; Thermo Scientific) and Sox2 (1:200; Santa Cruz Biotechnology), and guinea pig Nkx6-2 (1:5,000; gift from J. Ericson). Secondary antibodies (donkey anti-mouse, -rat, -rabbit, and -guinea pig) were either fluorescently labeled (Cy3, Jackson ImmunoResearch; Ale- xaFluor 488 and 594, Molecular Probes), or coupled to biotin/streptavidin-horseradish-peroxidase (Jackson ImmunoResearch) and detected using the Vectastain ABC System (Vector Laboratories). Brightfield, darkfield, and fluorescent images were taken on an Axioplan2 or Axiovert 200M microscope and StemiSV6 stereomicroscope equipped with AxioCam MRc or HRc camera (Zeiss), or on a confocal laser scanning microscope (LSM 510 META, Zeiss). Images were processed with AxioVision 4.6 (Zeiss) and Adobe Photoshop CS3 (Adobe Systems Inc.) software.

5-Bromo-2′-Deoxyuridine (BrdU) Treatments

Pregnant dams were injected intraperitoneally with 10 μg BrdU (Sigma)/g body weight on E10.5 or E12.5. Embryos were dissected 2 hr later and processed for immunodetection of BrdU.

Unbiased Stereology, Cell Countings, and Length Measurements

Th+ and Pitx3+ cells were evaluated by the optical fractionator method, and BrdU+ and pH3+ cells were counted using the StereoInvestigator 5.05.4 software (MBF Bioscience, Williston, VT) on every sixth section from serial coronal paraffin sections (8 μm) through the midbrain of E10.5, E11.5, E12.5, E13.5, and E17.5 embryos. The D/V and left-right (lateral) extension of the midbrain was measured on 4 consecutive Nissl-stained paraffin sections from E12.5 embryos using AxioVisionLE (Zeiss) software.

Statistical Analyses

For comparisons between wild-type and mutant animals from the same litters, methods for paired or grouped data were applied, namely the paired t-test and repeated-measurements ANOVA. Repeated-measurements methods were also applied for comparisons between wild-type and mutant embryos with several sections per embryo. For analysis of the effect of two factors simultaneously, first a two-way ANOVA with interaction was fitted. If the interaction effect was significant, one-way ANOVAs were performed for subgroups. Otherwise a two-way ANOVA without interaction effect was fitted. In case of a significant genotype effect, post-hoc one-way ANOVAs with P value correction according to HOLM were calculated to assess the genotype effect in subgroups (Holm,1979). For all calculations, the R software with the nlme package was used (Pinheiro et al.,2008; R Development CoreTeam,2008).

Acknowledgements

The authors thank J. Nathans for providing the Fzd3 and Fzd6 mutant mice, A. Pierani and J. Ericson for antibodies, A. Simeone for discussions, and S. Laass for excellent technical assistance. This work was funded by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN+ Functional Genomics of Parkinson Disease FKZ 01GS08174), by Virtual Institute on Neurodegeneration and Ageing (VH-VI-252), by the Initiative and Networking Fund in the framework of the Helmholtz Alliance of Systems Biology and of Mental Health in an Ageing Society (HA-215), Bayerischer Forschungsverbund “ForNeuroCell” (F2-F2410-10c/20697), European Union (mdDANEURODEV FP7-Health-2007-B-222999, EuTRACC LSHG-CT-2006-037445), and Deutsche Forschungsgemeinschaft (DFG) SFB 596, WU 164/3-2, and WU 164/4-1.

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