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Keywords:

  • Mpped1;
  • 239AB;
  • C22ORF1;
  • β-catenin;
  • neocortex;
  • hippocampus

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We report the expression of the mouse Mpped1 in the telencephalon through embryonic stages to adulthood. Using Northern blotting analysis and RNA in situ hybridization (ISH), our data show that Mpped1 is specifically expressed in the brain and is enriched in the cortical plate of the developing telencephalon. Postnatally, the expression of Mpped1 is reduced in the cerebral cortex relative to its levels in the embryonic dorsal telencephalon. Also, Mpped1 expression is sustained in the hippocampal CA1 region. Examination of the expression of Mpped1 and other cortical layer markers by ISH in a malformed β-catenin null dorsal telencephalon show that the Mpped1-, Cux2-, and Rorβ-expressing superficial cortical layers are reduced and form patchy patterns, and the Tbr-1-expressing deep-layer neurons are incorrectly located on superficial layers, indicative of a migration defect of cortical neurons in the absence of β-catenin. Developmental Dynamics 239:1797–1806, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The mammalian cerebral cortex is responsible for a range of integrative physiological responses, behaviors, and cognitions. It comprises a variety of projection neurons and interneurons and is organized into a six-layered cellular architecture (Peters and Jones,1984).

In mouse, the earliest neurons in the cortex are generated at embryonic day (E) 10.5. Specified neurons migrate outward from the ventricular zone (VZ) to form the preplate, which later splits and gives rise to the superficial marginal zone (MZ; the future layer I) and the subplate (Rice and Curran,2001; Campbell,2005; Molyneaux et al.,2007). Cells in the MZ expressing Reelin are required for subsequent proper lamination of the migrating neurons forming the cortical plate (CP) between the MZ and subplate (Rice and Curran,2001). From E11.5 onward, the cortical projection neurons arising from the progenitors in the proliferative zones, namely the VZ and the subventricular zone (SVZ), migrate radially in an inside-out manner (for reviews, see Marin and Rubenstein,2003; Dehay and Kennedy,2007; Molyneaux et al.,2007). In brief, the early-born neurons form the deeper layers (layers V and VI), whereas the late-born neurons pass over the earlier formed deep-layered neurons and form the upper layers (layers II to IV; Marin and Rubenstein,2003; Dehay and Kennedy,2007). Neurons in each layer share common features including birth-date, layer-specific gene expression, cellular morphologies, and connectivity (Hevner et al.,2003).

MPPED1 is an evolutionarily conserved gene present in various invertebrates and mammals including worms, flies, mice, and human (Schwartz and Ota,1997). Human MPPED1, also known as 239AB, has been reported to be expressed in the adult brain. Furthermore, the closely related gene MPPED2, also known as 239FB, has been shown to be expressed in the fetal human brain and is associated with WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, mental retardation; Schwartz and Ota,1997). The protein sequences of MPPED1 and MPPED2 share approximately 80% similarity and both contain a metallophosphoesterase domain (NCBI conserved domain search, pfam00149), which is also present in a diverse range of phosphoesterases. A recent study has reported that rat MPPED1 and MPPED2 both exhibit metal-dependent phosphodiesterase activity when characterized as in vitro purified recombinant proteins (Tyagi et al.,2009). Interestingly, rat MPPED1 acts as a unique cyclic nucleotide phosphodiesterase that can hydrolyze cAMP to form either 2′-AMP or 3′-AMP, whereas MPPED2 lacks this activity.

Differential expression of Mpped1 in mouse hippocampal CA1 neurons compared with CA3 neurons under normoxia and hypoxia conditions has been noted using a genome-wide approach (Newrzella et al.,2007). However, the precise expression patterns and molecular identity of Mpped1 in the brain are poorly understood at present. In this study, we specifically analyzed the ontogeny and distribution of Mpped1 in the normal mouse brain. Furthermore, we used Mpped1 expression as a readout for impaired neocortical neurogenesis in the β-catenin–deficient telencephalon, because previous studies have revealed that β-catenin has multiple roles and is required within the telencephalon (Machon et al.,2003; Backman et al.,2005; Junghans et al.,2005; Woodhead et al.,2006; Machon et al.,2007).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Ontogeny and Distribution of Mpped1 in the Mouse Brain

To characterize the expression profile of mouse Mpped1, Northern blot analysis was used on different tissues from varies developmental stages. Results showed that Mpped1 was expressed exclusively in the brain. Expression of Mpped1 should start between E10.5 and E14.5 (Fig. 1). Of interest, the intensity of the Mpped1 transcript seemed to be slightly reduced in the adult brain compared with the fetal brain (Fig. 1).

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Figure 1. Northern blot analysis of Mpped1 expression in embryonic and adult organs. Top: A 3.2-kb Mpped1 transcript was specifically detected in embryonic (embryonic day [E] 14.5, E16.5, and E18.5) and adult brains, but not in other organs. Bottom: The 28S and 18S ribosomal RNAs were visualized by ethidium bromide and used as loading controls for the total RNA used.

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To precisely define the earliest time point and location of Mpped1 expression in the developing brain, we performed in situ hybridization (ISH) analysis on brain sections at E12.5–E14.5 (Supp. Fig. S1, which is available online). The sense probe of Mpped1 served as a negative control for ISH staining with the Mpped1 antisense probe (Supp. Figs. S1, S2). Mpped1 expression was not detected in the telencephalon at E12.5 (Supp. Fig. S1). It first appeared in the lateral wall of neocortex (NCX) at E13.5 (Supp. Fig. S1). At E14.5, the expression of Mpped1 was detected in the CP (Fig. 2AA′; Supp. Fig. S1EE′) located immediately beneath Reelin-positive MZ (Fig. 2BB′). The expression of Mpped1 was undetectable in the lateral ganglionic eminences (LGE) (E14.5; Fig. 2A), the striatum, and thalamus in all stages (E12.5–E18.5) we tested (data not shown).

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Figure 2. Expression of Mpped1 is enriched within the cortical plate (CP) of the developing neocortex. A,E,G:Mpped1 in situ hybridization (ISH) revealed the presence of intense Mpped1 staining mainly located within the CP of the embryonic day (E) 14.5, E16.5, and E18.5 developing neocortex (NCX). Mpped1 could also be detected in the CA1 of the developing hippocampus at E18.5 (G). LGE, lateral ganglionic eminences. B:Reelin ISH represent Cajal-Retzius cells of the MZ at E14.5. C:Tbr-1 ISH revealed early-born neurons of the CP at E14.5. D,F,H: IF staining for TBR-1 (green) revealed post-mitotic neurons of the CP, whereas Ki67 (red) labeled cells were the proliferative neuronal progenitors of the intermediate zone (IZ), subventricular zone (SVZ) and ventricular zone (VZ). Higher magnifications of the cropped boxes marked in A–H are showed in A′–H′. Arrows indicated positive-stained areas. Scale bars = 200 μm in A–H, 100 μm in A′–H′.

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To characterize Mpped1-expressing cells, we compared the expression patterns of Mpped1 with T-Brain-1 (Tbr-1), a marker for postmitotic neurons (Bulfone et al.,1995) and Ki67, a marker for proliferative progenitors (Fujimoto et al.,2009). We showed that the Mpped1-stained layers and the Tbr-1-stained layers both reside in the CP at E14.5 (Fig. 2CC′ vs. AA′), and these Tbr-1-positive cells do not overlap with Ki67-expressing cells (Fig. 2DD′). At E16.5, Mpped1 was detected in the entire CP uniformly (Fig. 2EE′), whereas TBR-1 was restricted in the deeper layers of the CP and was separated from Ki67-labeled VZ and SVZ (Fig. 2FF′, HH′). At E18.5, Mpped1 was strongly expressed in the upper layers and its expression was reduced in the deep layers (Fig. 2GG′), where the TBR-1 was strongly expressed (Fig. 2HH′). In addition, Mpped1 was detected in the CA1 region of the developing hippocampus at E18.5 (Fig. 2G). Our results suggest that Mpped1 is expressed in the postmitotic neurons that form the CP during corticogenesis.

Through ISH, we observed that the Mpped1 staining in the CP was reduced postnatally as compared to the perinatal stages (Fig. 3A–C; Supp. Fig. S2C) relative to its levels in the perinatal (E18.5–postnatal day [P] 0) CP (Fig. 2G; Supp. Fig. S2A). Notably, Mpped1 was strongly expressed in the hippocampal CA1 and to a lesser extent in the CA3 (Fig. 3 AA′–CC′). These findings are in agreement with the expression pattern described in the Allen Brain Atlas (http://mouse.brain-map.org/brain/Mpped1.html) and with an earlier report (Newrzella et al.,2007) showing differential expression of Mpped1 in CA1 compared with CA3 under both normoxia and hypoxia conditions.

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Figure 3. Mpped1 expression is maintained in the CA1 of the hippocampus but is reduced with dynamic expression patterns in the neocortex of the postnatal mouse brains. A–C:Mpped1 in situ hybridization (ISH) revealed two major stained areas, the neocortex and the hippocampal CA1, within postnatal day (P) 7, P14, and adult brains. A′–C′: Higher power views of the hippocampus in A–C (dash lined boxes) revealed a strong Mpped1 ISH stained pattern (purple) with a sharp boundary (red arrows) in CA1 field but weak or faint staining in CA3 or the dentate gyrus (DG). A″–C″: Higher power views of the cropped areas (solid lined boxes) of the neocortex identified as A–C reveal the dynamic appearance of the Mpped1 ISH stained patterns (purple in A′ to C′). D–I: The cortical laminations of layers II/III, IV, V, and VI were defined by comparing with the Rorβ (D–F) and TBR-1 (G–I) enriched layers IV and VI, respectively. D–F:Rorβ ISH revealed layer IV neurons within the neocortex, which are indicated by arrowheads. G–I: TBR-1 IHC (green) revealed deeper-layered neurons (layers VI and part of layer V) within the neocortex, which are indicated by arrowheads. Scale bars = 500 μm in A–C, 200 μm in A′–C′,D–I.

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To further delineate Mpped1 expression in cerebral cortex layers, we examined its expression at P7 and P14 after cortical lamination is thought to be completed. Expressions of Mpped1 were compared with that of Rorβ (Schaeren-Wiemers et al.,1997) and TBR-1 (Bulfone et al.,1995), which are specific to layer IV and layer VI plus partial layer V, respectively. When compared with the Rorβ distribution within layer IV (Fig. 3D–F) and the TBR-1–expressing deeper layers of the postnatal cerebral cortex (Fig. 3 G–I), we found that Mpped1 was predominately expressed within layers II/III at P7 and P14 (Fig. 3A″,B″), and was later weakly distributed in the layers II/III and V of the adult cerebral cortex (Fig. 3C″).

Mpped1 Expression in the β-Catenin–Deficient Dorsal Telencephalon

In an earlier report, Campos et al. had demonstrated cortical malformation and increased seizure susceptibility in a mouse model, in which β-catenin was specifically ablated in the dorsal telencephalon by Emx1-cre (Campos et al.,2004). Although the role of β-catenin in seizure susceptibility has been extensively analyzed, it remains unclear how the cortex becomes malformed in the absence of β-catenin. If corticoneogenesis is aberrantly regulated, the expression or distribution of Mpped1 and other layer-specific genes may be able to provide an entry point to understand the potential defects of β-catenin–deficient dorsal telencephalon. Thus, we generated mutant mice with a malformed neocortex and hippocampus, referred to Emx1-Cre;Ctnnb1fx/fx mice (see the Experimental Procedures section). We found that the Emx1-Cre;Ctnnb1fx/fx mice at E17.5 (Fig. 4A) and at birth (data not shown) had a cleft cheek with an exposed tongue, a phenotype that was not previously reported. The cleft cheek appearance might be due to Cre activity, in addition to its expected forebrain expression, detected around the maxillary process at E12.5 (Fig. 4B). To confirm the neocortex-specific loss of β-catenin expression in the Emx1-Cre;Ctnnb1fx/fx brain, we performed immunofluorescence (IF) staining for β-catenin and found that the loss of β-catenin is neocortex-specific, with expression of β-catenin remaining in the thalamus and hypothalamus (Fig. 4G).

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Figure 4. Use of Mpped1 as a marker for analyzing defects within the β-catenin–deficient dorsal telencephalon. A: A representative Emx1-Cre;Ctnnb1fx/fx (Cre;fx/fx, right panel) embryo at embryonic day (E) 17.5 displayed a cleft cheek and an exposed tongue (white arrow) in comparison with a gross view of a Emx1-Cre;Ctnnb1fx/+ (Cre;fx/+, left panel) control embryo. B: An E12.5 embryo Emx1-Cre/+;R26R/+ (Cre;R26R) stained for β-galactosidase activity, which was present in the forebrain (white arrow) and maxillary process (black arrow) of the embryo. C: Brain anatomy of a Emx1-Cre;Ctnnb1fx/fxfetus (right panel) at postnatal day (P) 0 revealed a reduced cerebral hemisphere size and an uncovered superior colliculus (arrow) in comparison with the Emx1-Cre;Ctnnb1fx/+ (left panel) control fetus. D: Whole-mount in situ hybridization (ISH) staining for Mpped1 revealed a patched and reduced intensity within the cerebral hemispheres of the P0 Emx1-Cre;Ctnnb1fx/fx fetus (right panel) compared with the strong and homogenous staining of the Emx1-Cre;Ctnnb1fx/+ fetus (left panel). OB, olfactory bulb; CH, cerebral hemispheres; MB, mid-brain. E:Mpped1 ISH (arrowheads) revealed a patched and reduced intensity in a rostral coronal section of the P0 mutant neocortex (NCX, right panel) compared with the control (left panel). F: ISH using Mpped1 sense probe was served as the negative control. G: β-catenin IF staining (green) revealed NCX-specific loss of β-catenin expression in the mutant NCX (right panel) compared with the control (left panel). Cc, corpus callosum; Scale bars = 1 mm in C,D, 500 μm in E–G.

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Due to the perinatal lethality of these mutant mice, we investigated the defects in these mutant brains at P0. We analyzed the gross anatomy of the Emx1-Cre;Ctnnb1fx/+ and Emx1-Cre;Ctnnb1fx/fx brains (Fig. 4C), followed by whole-mount ISH for Mpped1 expression (Fig. 4D). We found that the mutant brain was smaller than the control brain (Fig. 4C) and the overall intensity of Mpped1 was weaker within the cerebral hemispheres of mutant brains at P0, whereas intense staining of Mpped1 signal was uniformly distributed in the control brains (Fig. 4D). We further performed Mpped1 ISH on coronal sections of mutant versus control brains to examine if there are lamination defects in the mutant brains. In the control (Emx1-Cre;Ctnnb1fx/+) forebrain, Mpped1 expression appeared within the superficial layers of the neocortex, whereas in the malformed Emx1-Cre;Ctnnb1fx/fx neocortex, the expression of Mpped1 was drastically reduced and was patched within the dorsal neocortex (Fig. 4E). The Mpped1 sense probe was used for ISH on Emx1-Cre;Ctnnb1fx/fx neocortex as a negative control (Fig. 4F).

Next, we closely examined the lamination defects of the β-catenin–deficient neocortex at P0 by comparing the expression of Mpped1 with that of layer-specific markers Reelin (layer I), Cux2 (layers II/III), Rorβ (layer IV), and Tbr-1 (layers V/VI) on Emx1-Cre; Ctnnb1ffx/+ and Emx1-Cre;Ctnnb1fx/fx brain sections. In addition, IHC using antibodies against TBR-1 and Ki67 was performed to define the deeper layers and proliferative cells in the VZ. We found, in Emx1-Cre;Ctnnb1fx/fx brains, that Reelin-positive were aberrantly scattered throughout the cortex (Fig. 5AA′,BB′). Furthermore, we observed that Mpped1 was strongly expressed in the superficial layers of the Emx1-Cre;Ctnnb1fx/+ neocortex (Fig. 5CC′) and was expressed at a much reduced level and in an uneven and patched staining pattern in the Emx1-Cre;Ctnnb1fx/+ neocortex (Fig. 5DD′). Likewise, upper layer markers such as Cux2 (Zimmer et al.,2004) in the layers II/III and Rorβ in the layer IV were also reduced (Fig. 5FF′,HH′) compared with the control (Fig. 5EE′,GG′). We further observed that some Cux2-expressing cells were aberrantly located in the deep layers (arrowhead in Fig. 5F′).

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Figure 5. Evaluation of aberrant cortical laminations defined by the expression of Reelin, Mpped1, Cux2, Rorβ, and Tbr-1 together with Ki67 proliferative marker in the β-catenin–deficient cerebral cortex at postnatal day (P) 0. A,B:Reelin in situ hybridization (ISH) revealed intensely stained layer I (arrows) of both Emx1-Cre;Ctnnb1fx/+ (Cre;fx/+) and Emx1-Cre;Ctnnb1fx/fx (Cre;fx/fx) cortical coronal sections. More scattered Reelin positively stained cells were also observed in other layers (B′; arrowheads), in addition to in the layer I, of Emx1-Cre;Ctnnb1fx/fx neocortex. C:Mpped1 ISH revealed positively stained superficial layers (arrows) of the Emx1-Cre;Ctnnb1fx/+ neocortex. D:Mpped1 ISH revealed focal patched areas (arrows) within the Emx1-Cre;Ctnnb1fx/fx neocortex. E:Cux2 ISH was used to label layers II/III neurons of the Emx1-Cre;Ctnnb1fx/+ neocortex. F:Cux2 ISH revealed discontinuous patched areas in the superficial layers (arrows) and in the deeper layers (arrowheads) of the Emx1-Cre;Ctnnb1fx/fx neocortex. G:Rorβ ISH served as a molecular marker to label cortical layer IV of the Emx1-Cre;Ctnnb1fx/+ neocortex. H:Rorβ-positive staining by ISH was reduced and showed focal patched pattern in the Emx1-Cre;Ctnnb1fx/fx neocortex. I:Tbr-1 ISH was predominately stained in the deeper layers (arrows) and weakly stained in the superficial layers of the Emx1-Cre;Ctnnb1fx/+ neocortex. J:Tbr-1 ISH revealed focal expanded or aberrant laminations of the Emx1-Cre;Ctnnb1fx/fx neocortex. K: TBR1 and Ki67 were expressed in the deep-layered post-mitotic neurons and in the proliferative ventricular progenitors of Emx1-Cre;Ctnnb1fx/+ neocortex, respectively. L: TBR-1 IF staining revealed expansion toward the superficial layers of the Emx1-Cre;Ctnnb1fx/fx neocortex compared with the control. The Ki67-labeled cells were also expanded and partially overlapped with the TBR-1-positive deep layers of the Emx1-Cre;Ctnnb1fx/fx neocortex. A′–L′: Higher-power view of the cropped areas of A–F. Scale bars = 200 μm.

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We next examined the deep-layer neurons using ISH for Tbr-1 RNA expression (Fig. 5 II′,JJ′) or IF staining for TBR-1 protein distribution in conjunction with Ki67 expression (Fig. 5KK′,LL′). When compared with the control sections (Fig. 5 II′,KK′), we found that the Tbr-1-expressing deep-layer neurons were aberrantly distributed throughout the cortex and accounted for the majority of the neurons in the Emx1-Cre;Ctnnb1fx/+ neocortex (Fig. 5JJ′,LL′). We observed many cell-dense ectopically formed Tbr-1-expressing neurons in the superficial layers in the mutant brains (arrowheads in Fig. 5JJ′). Interestingly, the Ki67-labeled cells could be detected in the VZ/SVZ of the Emx1-Cre;Ctnnb1fx/+ neocortex (Fig. 5L′), indicating that proliferation of cortical precursors in the mutant cortex continues to proceed. Taken together, our data demonstrate that β-catenin is required for the development of proper lamination of neocortex. In β-catenin–deficient cortices, the expression of superficial layer marker genes is reduced and patchy and the distribution of deep-layer neurons is disrupted.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In the present study, we first characterized the expression patterns of Mpped1 in the mouse brain from embryonic stages to adulthood. This finding is controversial to an earlier report showed that human MPPED1 (239AB) is solely expressed in the adult brain (Schwartz and Ota,1997). Although this contradiction remains unresolved, species-specific differences in the expression of MPPED1 between humans and mice cannot be excluded.

The expression of Mpped1 is detected in postmitotic neurons during corticoneurogenesis. We found that Mpped1 is expressed in the CP of the neocortex before birth; its expression levels are decreased in the adult brain. In contrast to the cortex, Mpped1 exhibits sustained expression in the CA1 of the hippocampus throughout adulthood. Although the in vivo role of Mpped1 remains unclear, our expression analyses and previous biochemical studies (Tyagi et al.,2009) suggest that MPPED1 may regulate the development or the function of the cortical and hippocampal neuronal cells through MPPED1's potential enzymatic activity, namely metallophosphodiesterase activity.

β-Catenin has been shown to be required within the telencephalon for cell–cell adhesion (Junghans et al.,2005), cortical precursor proliferation (Machon et al.,2003; Woodhead et al.,2006), neuronal cell-fate specification (Machon et al.,2003,2007), and dorsal–ventral patterning (Backman et al.,2005). In this report, we investigated a mouse model where the loxP-flanked β-catenin exon2-6 was excised under the control of Emx1-Cre PAC transgene, which caused a telencephalon-specific ablation of β-catenin. Our results indicate that there was mislocalization of early-born neurons labeled by TBR-1 together with a drastic reduction of superficial layers, as detected by Mpped1, Cux2, and Rorβ, in β-catenin–deficient neocortex at birth. These changes are possibly caused by impaired migration of cortical neurons in the absence of β-catenin. Interestingly, Emx1-Cre;Ctnnb1fx/fx in this report also has manifest craniofacial defects causing neonatal lethality, mainly because the Cre activity of the Emx1-Cre PAC Tg1 mice (Iwasato et al.,2004) ectopically appeared at the maxillary process. This early postnatal lethality restricted our analysis to perinatal stages. In contrast, the Emx1-Cre;Ctnnb1fx/fx mice generated by Campos et al. were able to survive until adulthood and were available for functional testing in terms of seizure susceptibility and epileptogenesis. The increased seizure susceptibility of Emx1-Cre;Ctnnb1fx/fx mice may be indirectly caused by their massive neuronal connectivity defects that affect the neocortex, corpus callosum, hippocampus, and other circuits (Campos et al.,2004).

Recently, Mao et al. showed that β-catenin stability can be modulated by the Disrupted in Schizophrenia 1 (DISC1) protein through direct inhibition of GSK3β activity (Mao et al.,2009), which raises the importance of the DISC1 and the GSK3β/β-catenin signaling pathway in psychiatric disorders. Although the role of β-catenin in psychiatric disorders remains unclear, future studies are needed to address whether loss of β-catenin in the mouse brain is able to recapitulate the clinical manifestations of schizophrenia or other psychiatric disorders. Interestingly, human MPPED1 resides on chromosome 22q13.3, where various abnormalities have been linked to schizophrenia (Liang et al.,2002; Takahashi et al.,2005; Zheng et al.,2006; Bulayeva et al.,2007; Condra et al.,2007), bipolar disorders (Tiziano et al.,2008; Shimojima et al.,2009), and brain malignancies (Rey et al.,1993; Ino et al.,1999; Oskam et al.,2000; Prowald et al.,2005; Modena et al.,2006). This suggests that MPPED1 is possibly involved in the pathogenesis of these corresponding diseases. Future studies will involve the ablation of Mpped1 in the mouse to determine its biological function or pathological role in the neurogenesis of the neocortex and hippocampus.

In summary, we have demonstrated the dynamic expression of Mpped1 in the mouse forebrain through embryogenesis and into the adult stage and have used Mpped1, in conjunction with other genes (Reelin, Cux2, Rorβ, TBR-1, and Ki67), as a marker for superficial cortical lamination with the aim of elucidating corticogenesis defects in the absence of β-catenin.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Northern Blotting Analysis

A total of 30 μg of RNA was isolated from each of the various homogenized tissues, mixed with equal volume of glyoxal, and incubated at 50°C for 30 min. The denatured RNAs were then separated by gel electrophoresis and transferred to Hybond-N+ membrane (GE healthcare, Taiwan). This membrane was prehybridized with hybridization buffer (Ambion Inc, TX) at 65°C for at least 30 min and then hybridized for overnight with 10 ng Mpped1 probe, spanning nucleotides 727-1197 (NCBI, UniGene, NM_172610.3), which was labeled with psoralen-biotin compound (Ambion Inc) using irradiation with long wave ultraviolet light (365 nm) for 45 min. Next, the hybridized membrane was washed with low stringency buffer (2× standard saline citrate [SSC], 0.1% sodium dodecyl sulfate [SDS]) and then high stringency buffer (0.1× SSC, 0.1 SDS). This was followed by visualization using the BioDetect system (Ambion Inc).

Mice

The ICR mouse strain was purchased from BioLASCO (BioLASCO Inc., Taipei, Taiwan). The floxed allele of β-catenin (Ctnnb1fx/fx) on the C57BL/6 congenic background (B6.129-Ctnnb1tm2Kem) was initially generated by Kemler's laboratory (Brault et al., 2001) and was purchased from the Jackson Laboratory (the Jackson Laboratory, Bar Harbor, ME). Emx1-Cre Tg1 mice (Iwasato et al.,2004) on the B6 background were imported from the RIKEN biological resource center (Experimental Animal Division, RIKEN BRC, Ibaraki, Japan) and were originally generated by Itohara's laboratory. The Emx1-Cre transgenic mice were bred with Ctnnb1fx/fx to obtain Emx1-Cre/+; Ctnnb1fx/+ mice, which were then backcrossed with Ctnnb1fx/fx to obtain the mice bearing the Emx1-Cre/+; Ctnnb1fx/fx genotype. For the embryonic analysis, of the presence of a vaginal plug between morning and noon was considered to be 0.5 days post coitum (dpc). The date of birth was considered to be P0. All animals were housed in microisolator cages (up to five mice per cage) using specific pathogen-free husbandry. All experiments with mice were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at National Yang-Ming University.

Brain Sampling

The E12.5–E14.5 whole embryos, E16.5 brains, and E18.5 brains were dissected and fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C overnight. P0, P7, P14, and adult mice were anesthetized and perfused with 4% PFA/PBS by means of the intracardial route, and then the brains were dissected. For whole-mount in situ hybridization, the brains were fixed with 4% PFA/PBS overnight, dehydrated with a graded series of methanol in PTW (PBS containing 0.1% Tween-20), and then stored in 100% methanol at −20°C until use. For paraffin section in situ hybridization or immunofluorescence staining, brains were fixed with 4% PFA/PBS overnight, dehydrated with 70% ethanol at 4°C overnight, and then the brains were dehydrated in a graded series of ethanol, cleared in xylene, and infiltrated in paraffin. Subsequently, the brains were embedded in paraffin and stored at room temperature. Serial sections of 10-μm thickness were then cut using a HM315 microtome (Microm international GmbH).

Digoxigenin-Labeled Riboprobe Preparation

The plasmid, pGEM-T-Mpped1(nucleotide 727-1197, the NCBI accession number NM_172610), was obtained by TA-cloning of pGEM-T (Promega, USA) and the Mpped1 DNA fragment by reverse transcriptase-polymerase chain reaction (RT-PCR) amplifying from the brain cDNA using primers (5′-gcatctacctgcaggactcagaagg-3′ and 5′-gccagacatacagagctgacatgg-3′). For antisense RNA probe preparation, pGEM-T-Mpped1 was digested with SalI. Also, other layer-specific probes (kindly provided by Dr. O'Leary) included Reelin (nucleotide 9742-11698, the NCBI accession no. NM_011261), Cux2 (nucleotide 270-1157, the NCBI accession no. NM_007804), Rorβ (nucleotide 365-2651, the NCBI accession no. NM_001043354), and Tbr-1 (nucleotide 163-2301, NCBI accession no. NM_009322) were digested with EcoRI, XbaI, XhoI, and EcoRI, respectively. These linearized plasmids served as DNA templates and were mixed with the digoxigenin (DIG) -RNA labeling kit (Roche, Germany) to synthesize DIG-labeled RNA probes. The antisense RNA probes of Mpped1 and Tbr-1 were generated by T7 RNA polymerase, whereas the RNA probes of Reelin, Cux2, and Rorβ were generated by T3 RNA polymerase. After incubation at 37°C for 2 hr, the solutions were treated with DNase I to remove the DNA template. Subsequently, the DIG-labeled RNA probes were precipitated by adding one-tenth volume of 3M NaOAC and 2.75-fold volume of 100% ethanol followed by storage in a −20°C refrigerator for at least 30 min. After centrifugation, the precipitated pellets were rinsed twice with 75% ethanol, air dried, and then finally resuspended in DEPC-treated ddH2O.

RNA ISH

PFA-fixed paraffin-embedded tissue sections were heated at 65°C for 2 hr, and then deparaffinized with xylene, rehydrated with a graded series ethanol, and washed with PBS for 5 min. Subsequently, proteinase K (20 μg/ml) digestion was performed at 37°C for 5 min. After proteinase K digestion, the tissue sections were post-fixed with 4% PFA and then were washed with PBS twice. The tissue sections were further equilibrated in 0.1 M TEA (triethanolamine, pH 8.5) and then incubated with acetic anhydride. The tissue sections were dehydrated through a graded series ethanol and then air dried for 1 hr. Later, the tissue sections were prehybridized with prehybridization buffer (50% deionized formamide, 2× SSC) at 65°C for 2 hr. During the prehybridization step, the DIG-labeled RNA probes were denatured in hybridization solution (50% deionized formamide, 2× SSC, 10% dextran sulfate sodium salt, 1× Denhardt's solution, 10 mM ethylenediaminetetraacetic acid [EDTA], 10 mM DTT, 1 mg/ml yeast-tRNA) at 80°C for 5 min. After prehybridization, the sections were incubated with hybridization solution containing the individual denatured RNA probes at 65°C overnight. On the second day, the slides were rinsed in 5× SSC and then washed in wash buffer (50% deionized formamide, 2× SSC) at 65°C for 30 min. Subsequently, the sections were equilibrated in STE buffer (4× SSC, 20 mM Tris-HCl, 1 mM EDTA, pH 8.0) at 37°C for 10 min. The tissue sections were then incubated in RNaseA solution (20 μg/ml in STE buffer) at 37°C for 30 min. After RNaseA treatment, the samples were washed with STE buffer for 10 min and then washed with FS buffer again at 65°C for 30 min. Next, the slides were washed with 2× SSC, 0.1× SSC and then ddH2O. Subsequently, the sections were equilibrated with 1× MABT (1× MAB: 0.1 M maleic acid, 0.15 M NaCl, pH 7.5, containing 0.1% Tween-20), incubated with blocking solution (10% sheep serum and 2% Roche blocking reagent in 1× MABT) at room temperature for 2 hr, and finally the alkaline phosphatase (AP) -conjugated anti-DIG antibody (1:1,000, Roche, Germany) was added for overnight incubation at 4°C. After labeling, the sections were washed three times (30 min each) with 1× MABT containing 2mM levamisole. Subsequently, the sections were washed and equilibrated in NTMT solution (20 mM NaCl, 100 mM Tris-HCl, 2 mM levamisole, 0.1% Tween-20, pH 9.5) and then color (dark purple) developed using NBT (nitroblue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3-indolyl-phosphate) chromogenic stain for 24 hr. Finally, the sections were washed with PBS, dehydrated with serial concentration of ethanol, cleared in xylene and mounted for viewing.

IF Staining

The detailed procedure has been described previously (Lu et al.,2007). The sections were incubated with primary antibodies, including Ki67 (1:200, BD-Pharmingen, USA), TBR-1 (1:400, Millipore, USA) at 4°C overnight. After washing the sections three times with PBS, secondary antibodies of Alexa-488 conjugated goat anti-rabbit IgG against the TBR-1 antibody and of Alexa-568 conjugated goat anti-mouse IgG against the Ki67 antibody (Molecular Probes, USA) were applied at room temperature for 1 hr. Finally, the slides were counterstained with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI; 0.5 ng/ml) for 3 min and then mounted in fluorescent mounting medium (DakoCytomation).

Whole-Mount X-gal Staining

The detailed experimental procedures have been described previously (Lu et al.,2007; Liang et al.,2009).

Whole-mount In Situ Hybridization

The brains at P0 were rehydrated with a graded series of methanol in PTW and finally washed in PTW. Next, the brains were digested in proteinase K (10 μg/ml) at 30°C for 20 min. After proteinase K digestion, brains were washed with glycine solution (5 mg/ml in PTW) followed by post-fixation with 4% PFA/PBS. After washing with PTW, the brains were prehybridized in hybridization buffer (50% deionized formamide, 5× SSC, 50 μg/ml yeast tRNA, 1% SDS, 50 μg/ml heparin) and gently rolled at 65°C for 2 hr. During the prehybridization step, each DIG-labeled RNA probe (1 μg/ml) was denatured in hybridization buffer at 65°C for 20 min. After prehybridization, the brains were transferred into the hybridization solution, which contained the denatured RNA probes, then incubated at 65°C overnight. After overnight incubation, the brains were washed three times with Solution I (50% deionized formamide, 4× SSC, 1% SDS) at 70°C and then washed three times with Solution II (50% deionized formamide, 2× SSC) at 65°C for 30 min each. Subsequently, the brains were washed twice with TBST, and then incubated in blocking solution (TBST plus 10% normal sheep serum) at room temperature for 2 hr. The brains then moved to the blocking solution containing AP conjugated anti-DIG antibody (1:1,000; Roche, Germany), which had been preabsorbed with E14.5 embryonic extracts, at 4°C overnight. On the next day, the AP-conjugated anti-DIG antibody stained brains was washed with TBST four times (1 hr for each) at 4°C and then washed with TBST at 4°C overnight. Then, the brains were washed with PTW at room temperature five times (10 min for each). Subsequently, the brains were then washed with NTT solution (20 mM NaCl, 100 mM Tris-HCl, 0.1% Tween-20, pH 9.5) for 15 min and then equilibrated in NTMT solution (20 mM NaCl, 100 mM Tris-HCl, 50 mM MgCl2, 0.1% Tween-20, pH 9.5) for 1 hr. The brains were further incubated in NTMT solution containing 5 mM levamisole (Sigma, USA) for 1 hr. Color developing was performed in NBT/BCIP solution (Roche, Germany) at room temperature, this was done for several min to several hr until brain samples were properly stained. Then, the brains were washed with PBS containing 10mM EDTA for 10 min and post-fixed with 4% PFA/PBS at 4°C overnight. Finally, the brain samples were stored in PBS containing 0.1% sodium azide at 4°C. Photographs were taken using a stereomicroscope (MZ6, Leica, Germany).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Drs Ming-Ji Fann, Yi-Ping Hsueh, and Yu-Ting Yan for their helpful comments on this study, and Drs Shen-Ju Chou, Carl Brown, Ralph Kirby, and anonymous reviewers for editing our manuscript. We also thank Drs Shen-Ju Chou and Dennis O'Leary for kindly providing ISH probes and protocols. We thank Drs. Rolf Kemler and Shigeyoshi Itohara for generously distributing mouse resources to the research community. Authors also thank Yi-Ru Yu and Hsiao-Lin Wu for their initial assistance. Also we thank RIKEN BRC and the Jackson Laboratory for maintenance and delivery the mouse strains. C.-M. C. was funded by the National Research Program of Genomic Medicine and the Taiwan Mouse Clinic was funded by the NSC.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22293_sm_suppfigS1.eps13825KSupp. Fig. S1. Expression of Mpped1 begins at the embryonic day (E) 13.5–E14.5 cortical plate (CP). A,C,E: In situ hybridization (ISH) of Mpped1 antisense probe (AS). B,D,F: ISH of Mpped1 sense probe (S); A,B: ISH of Mpped1 AS and S probes revealed background staining levels on sagittal section of E12.5 forebrain. C,D: ISH of Mpped1 (AS) revealed a thin positive-stained layer (arrows) within E13.5 neocortex, compared with background levels stained by Mpped1 (S) shown in (D). E, F: ISH of Mpped1 (AS) could evidently reveal a thin positive-stained layer (arrows) within E14.5 neocortex, compared with background levels stained by Mpped1 (S) shown in F. A′–F′: Higher power view of the cropped areas of A–F. Scale bars = 200 μm in A–F, 100 μm in A′–F′.
DVDY_22293_sm_suppfigS2.eps5477KSupp. Fig. S2. Mpped1 is reduced in cerebral cortex, but is enriched in the hippocampal CA1 at postnatal day (P) 7 compared with at P0. A: In situ hybridization (ISH) of Mpped1 antisense probe (AS) revealed two stained areas, the neocortex and the developing hippocampal CA1 (arrow), in the P0 coronal brain section. C: The expression of Mpped1 is easily detected at CA1 (arrow) compared with other regions, such as NCX or thalamus, of the P7 coronal brain section. B,D: Mpped1 sense (S) RNAs were used as the negative control for the background staining level on P0 and P7 coronal brain sections. Scale bars = 200 μm.

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