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Keywords:

  • Prox1;
  • homeobox;
  • mouse;
  • expression;
  • subventricular zone;
  • hippocampus;
  • thalamus;
  • cerebellum

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Prox1, a homeobox gene related to the Drosophila gene prospero, is necessary for retina, lens, liver, pancreas, and lymphatics development. However, not much is yet known about Prox1 expression during central nervous system development. Here we provide a detailed analysis of Prox1 mRNA and protein expression during prenatal and postnatal murine brain development. Prenatally, Prox1 is expressed in the subventricular zone or in early differentiating regions of the brain. At these stages, Prox1 mRNA, but not Prox1 protein, was also detected in several regions of the prethalamus and hypothalamus. At an early postnatal stage, Prox1 expression is mainly detected in several nuclei of the thalamus, the cerebellum, and the hippocampus. In adulthood, Prox1 expression remains only in the hippocampus and cerebellum. These complex patterns of expression suggest that Prox1 activity is differentially required during brain development and adulthood. Developmental Dynamics 236:518–524, 2007. © 2006 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The homeobox gene Prox1 was originally cloned in mouse by homology to the Drosophila melanogaster gene prospero and consists of two main domains, the prospero domain and the homeodomain (Oliver et al.,1993; Burglin,1994). Prox1 homologues have been identified in other vertebrates, including Xenopus, zebrafish, chicken, and human (Tomarev et al.,1996; Glasgow and Tomarev,1998; Ny et al.,2005). In vertebrates, Prox1 is expressed in the developing neural retina, lens, cochlea, spinal cord, brain, skeletal muscle, heart, liver, pancreas, and in the endothelial cells that will give rise to the lymphatic vasculature (Oliver et al.,1993; Tomarev et al.,1996; Glasgow and Tomarev,1998; Wigle and Oliver,1999; Burke and Oliver,2002; Wigle et al.,2002; Wang et al.,2005; Bermingham-McDonogh et al.,2006).

In mice, functional inactivation of Prox1 demonstrated that this gene's activity is critical for the formation of a variety of organs and cell types, including the lens, retina, liver, pancreas, and lymphatic endothelial cells (Wigle and Oliver,1999; Wigle et al.,1999; Sosa-Pineda et al.,2000; Hong et al.,2002; Dyer et al.,2003; Harvey et al.,2005; Wang et al.,2005). In Drosophila, prospero is a cell-fate determinant in the asymmetric divisions of neuroblasts (Doe et al.,1991; Vaessin et al.,1991) and is required to maintain the mitotic potential of glial cells (Griffiths and Hidalgo,2004). Prospero is also necessary in the eye for distinguishing the cell fates among color photoreceptors (Cook,2003).

In the case of Prox1, its activity is required during neural retina development for the specification of horizontal cells (Dyer et al.,2003); however, no other functional role during vertebrate central nervous system development has been reported yet. Although intense expression of Prox1 is present in the developing spinal cord and brain (Oliver et al.,1993), the expression pattern and function of this gene during brain development in vertebrates remains unclear. It has been previously reported that early during neurogenesis, Prox1 is coexpressed with Mash1, a basic helix-loop-helix transcription factor, in the subventricular zone (SVZ) of the murine brain and spinal cord during the initial steps of neurogenesis (Torii et al.,1999). Prox1 levels are reduced in Mash1-mutant brains, and the overexpression of Mash1 in neural stem cells induces the expression of Prox1, suggesting that Prox1 expression during neurogenesis is Mash1 dependent (Torii et al.,1999). However, Prox1 expression is not restricted only to precursor cells; its expression is also in the mature granule cells of the dentate gyrus during development and in the adult brain (Bick-Sander et al.,2006; Navarro-Quiroga et al.,2006).

An initial description of the expression pattern of Prox1 during early stages of CNS development has been previously described (Oliver et al.,1993). In the spinal cord, Prox1 expression was initially detected at around E10.5, and at E12.5 its expression is seen in the SVZ of several brain regions (Oliver et al.,1993). In the forebrain, Prox1 is expressed in the ventral telencephalon, including the lateral ganglionic eminence (LGE), the medial ganglionic eminence (MGE), the thalamus, and the prethalamus. In the midbrain, Prox1 is expressed in the tegmentum, and in the hindbrain it is present in the SVZ of both the pons and medulla (Oliver et al.,1993). However, the expression pattern of Prox1 during later stages of vertebrate brain developmental and adulthood remains largely unknown.

Here we describe the results of a detailed analysis of the expression pattern of Prox1 mRNA and protein in the mouse brain during mid- and late-embryonic development, as well as during postnatal and adult stages.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

To determine the precise localization of Prox1 during murine embryonic brain development, we performed in situ hybridization and immunohistochemistry analyses on coronal and sagital sections of embryonic day (E) 14.5, E16.5, and E18.5 mouse brains.

Prox1 Expression in the E14.5 Mouse Brain

In the E14.5 telencephalon, Prox1 mRNA and protein were detected in the neuroepithelium of the dentate gyrus (Fig. 1A–E), the ganglionic eminences (Fig. 1A–C), and the amygdala (Fig. 1A,B). In the diencephalon, high levels of Prox1 expression were localized in the epithalamus and the dorsal thalamus (Fig. 1A–E). In the prethalamus (Fig. 1C,D), Prox1 transcripts were observed surrounding the reticular nucleus and the zona incerta. In the hypothalamus, Prox1 is expressed in the paraventricular nucleus (Fig. 1B,B'), in the preoptic area (Figs. 1B,B',E,E', 2E,F), and around the arcuate nucleus (Fig. 1D,D',E,E'). In the midbrain, Prox1 expression was confined to the pretectum, colliculus, and tegmentum (Fig. 1E,E'). In the hindbrain, Prox1 expression is observed in the pons and the medulla (Fig. 1E,E'). Interestingly, Prox1 mRNA but not Prox1 protein was detected in the prethalamus in the analyzed stages (compare Figs. 1C,D and 3C–H with Fig. 4C,D). These results were consistently reproduced when using a 3′ Prox1 mRNA probe (Oliver et al.,1993) in combination with any of the three available anti-Prox1 antibodies that recognize either the C-terminal (Fig. 1B–D) or the N-terminal (Figs. 3C–H, 4C,D) domains. These three antibodies yield the same expression pattern on mouse brain sections obtained from several developmental stages (not shown). This differential expression profile identified for Prox1 protein and Prox1 mRNA most likely is an indication of a postranscriptional regulatory mechanism.

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Figure 1. Comparative analysis of Prox1 mRNA and Prox1 protein in E14.5 mouse brain. Prox1 mRNA (left) and Prox1 protein (right) were observed in the subventricular zone (SVZ) and mantle zone (MZ) In this case, for Prox1 protein detection, a commercially available Prox1 antibody raised against its C-terminal region was used. A–D, A'–D': Anterior-to-posterior coronal sections. E-E': Sagital sections. A–E: In the SVZ, Prox1 mRNA was detected in the ganglionic eminence (GE), prethalamus (PT), paraventricular nucleus (PA), preoptic area (PO), arcuate nucleus (AN), colliculus (CL), tegmentum (TG), and pontine neuroepithelium (PNE). In the MZ, Prox1 was localized in the GE, amygdala (A), epithalamus (ET), dorsal thalamus (DT), prethalamus (PT), and pretectum (PTC). A'–E': The same expression pattern was observed for Prox1 protein, except it was not detected in the MZ of the prethalamus. A–E': Prox1 mRNA and protein were both expressed in the dentate gyrus neuroepithelium (DNE).

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Figure 2. Prox1 is expressed in postmitotic cells of the subventricular zone during mid-gestation. A: In the ganglionic eminence (GE) at E12.5, most Prox1-expressing cells are present in the subventricular zone (SVZ); only a few are detected in the differentiating &bgr'-tubulin+ cells of the mantle zone (MZ). B: Only a few Prox1-expressing cells are dividing, as indicated by coexpression of PH3. C,E: At E14.5, the number of postmitotic Prox1-expressing cells has increased in the whole brain. This is illustrated in the GE (C) and preoptic area (PO) (E). With the exception of Prox1-expressing cells in the DNE (G,H), none of the Prox1-expressing cells are dividing at this stage (D,F). Most sections are coronal with the ventricle to the left; however, in E and F, the ventricle is in the middle. The dotted line indicates the boundary between the ventricular zone (VZ) and the ventricle. White arrows, Prox1+;PH3+ cells; yellow arrows, Prox1+;β-tubulin+ cells; arrowheads, PH3-positive cells. For Prox1 protein detection, a commercially available Prox1 antibody raised against its C-terminal region and another one raised against the N-terminal portion were used.

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Figure 3. Expression of Prox1 mRNA and protein in the E16.5 mouse brain. In situ hybridization (A, C, F, J), immunohistochemistry (B, D, G, I, K), and double in situ immunohistochemistry staining for Prox1 (E, H). A–K: At this state, Prox1 mRNA and protein were both expressed in the pretectum (PTC), colliculus (CL), tegmentum (TG), pons (P), thalamus (T), paraventricular nucleus (PA), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), amygdala (A), pontine nucleus (PN), and pituitary (Pi). Prox1 was also observed in the subventricular zone (SVZ) of the cortex (small box in g) and in cells of the dentate migratory stream (DMS) (arrow in small box in D). The area surrounding the prethalamus (PT) (C–E), the subgeniculate nucleus (SGN), a ventral portion of the zona incerta (ZI) (F–H), and the external granular layer (EGL) of the cerebellum expressed Prox1 mRNA (arrows in C, F, J) but not Prox1 protein (arrows in D, G, I). H, hippocampus; CX, cortex; DNE, cortex neuroepithelium; ST, striatum; Cb, cerebellum; VZ, ventricular zone. For Prox1 protein detection, a antibody raised against the N-terminal portion was used.

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Figure 4. Prox1-positive cells are distributed along the cortex at late prenatal developmental stages (E18.5). Expression of Prox1 mRNA (A, C, E, I) and protein (B, D, F, G, H, small box in I) are shown at E18.5. Higher levels of Prox1 expression were observed in several nuclei of the thalamus (A–E). Similar to what was shown for E16.5 (Fig. 3), Prox1 protein was not detected in the area surrounding the prethalamus, in the subgeniculate nucleus (SGN), or in the ventral zona incerta (ZI) (arrows in A–D). At this stage, Prox1-expressing cells are also observed along the mantle zone (MZ) of the cortex and the colliculus (G, H), as well as in the external granule layer (EGL) of the cerebellum (small box in I; Prox1 in green). LDT, lateral dorsal thalamus; VMT, ventromedial thalamus; VLT ventrolateral thalamus; DMH, dorsomedial hypothalamus; PNT, posterior nucleus of thalamus; VMH, ventromedial hypothalamus; ZI, zona incerta; DG, dentate gyrus; A, amygdale; TG, tegmentum; CL, colliculus SVZ, subventricular zone; VZ, ventricular zone. For Prox1 protein detection, an antibody raised against the N-terminal portion was used.

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Similar to earlier stages, Prox1 expression at E14.5 was absent from the ventricular zone (VZ); instead, it was localized in the SVZ of the aforementioned structures, e.g., the preoptic area (Fig. 2E). It has been proposed that similar to the GMCs in Drosophila,Prox1-expressing precursor cells in the SVZ could retain some mitotic capacity at early stages of neurogenesis (Torii et al.,1999; Karcavich,2005). We identified some PH3+ cells in the Prox1-expressing population at E12.5 (Fig. 2B). At E14.5, cells coexpressing Prox1 and PH3 were found only in the neuroepithelium of the dentate gyrus (Fig. 2H) and in the SVZ of the pretectum (not shown). Prox1+ cells were also observed inside the mantle zone (MZ), and those cells coexpressed early postmitotic neuronal markers such as βTuj III (Fig. 2C–F) and MAP2 (Torii et al.,1999, not shown). However, some Prox1-positive cells present in the SVZ were negative for PH3 and did not express early postmitotic neural or radial glia markers (Torii et al.,1999, Fig. 2B,C, not shown), a result indicating that these cells are in an intermediate stage between a precursor and a postmitotic cell (Torii et al.,1999).

Prox1 Expression in the SVZ of the E16.5 Cortex

At E16.5, the distribution of Prox1 transcripts and protein was maintained in most of the regions described at the E14.5 stage, though there were several significant changes. In the telencephalon, Prox1 expression remained in the amygdala region (Fig. 3E,H); however, it was greatly reduced in the ganglionic eminences (Fig. 3C–E). In the region of the dentate gyrus, the Prox1+ cells of the neuroepithelium joined the dentate migratory stream (DMS) and had migrated dorsally towards the anlage of the dentate gyrus, near the pial surface (small box in Fig. 3D) (reviewed in Forster et al.,2006). Prox1-positive cells were detected also at E16.5 in the SVZ of the neocortex (Fig. 3G). At this stage, precursor cells in the SVZ of the neocortex give rise to late-born neurons (mostly of layers IV to II) and glial cells (Tarabykin et al.,2001). The onset of Prox1 expression in this area suggests that Prox1 is involved in this later neurogenic process. In the thalamus, Prox1 expression was detected in the epithalamus and the dorsal thalamus (Fig. 3A–H). In the prethalamus, Prox1 mRNA was observed surrounding the zona incerta and the reticular nucleus (Fig. 3C,E), in the subgeniculate nucleus, and in a ventral region of the zona incerta (Fig. 3F,H). Prox1 expression was also observed in the paraventricular, dorsomedial, and ventromedial hypothalamic nuclei (Fig. 3C–H). In the midbrain, Prox1 expression localized in the pretectum, colliculus, and tegmentum (Fig. 3A,B). In the hindbrain, Prox1-positive cells were found in the EGL in the cerebellum (Fig. 3I,J), pontine gray nucleus (Fig. 3K), and the pons and medulla (Fig. 3A,B). As in the prethalamus, EGL cells expressed Prox1 mRNA but not protein at this stage (Fig. 3I,J), suggesting the existence of a similar postranscriptional regulation mechanism. Prox1 was observed also in the posterior lobe of the pituitary at this stage (Fig. 3K). Some Prox1-expressing cells remained in the SVZ (e.g., the neocortex, small box in Fig. 3G) and Prox1-positive cells located inside the MZ still coexpressed early postmitotic neuronal markers such as βTuj III and MAP2 (not shown).

Prox1-Positive Cells in the MZ of the E18.5 Cortex

At the end of prenatal brain development (E18.5), Prox1 expression in the telencephalon was detected in the amygdala (Fig. 4C,D), dentate gyrus (Fig. 4A–F), and neocortex (Fig. 4G). In the neocortex, Prox1-positive cells are no longer restricted to the SVZ but are also detected in the MZ (Fig. 4G). These cells most likely are migrating dorsally from the SVZ region into the cortical plate and are giving rise to neurons or glia. In the thalamus, high levels of Prox1 mRNA and protein were detected in the laterodorsal, ventrolateral, ventromedial, and posterior thalamic nuclei (Fig. 4A–F). Prox1 expression was already observed in all of these thalamic locations at E16.5, except in the ventrolateral nuclei (compare Fig. 3C–H with Fig. 4A–D). In the hypothalamus, Prox1 was also detected in the dorsomedial and ventromedial nuclei. In the midbrain, Prox1 expression remained in the colliculus (Fig. 4H) and tegmentum (Fig. 4I). At this stage, in the colliculus, as in the neocortex, Prox1-positive cells are also located in the cortical plate (Fig. 4G). In the hindbrain, both Prox1 mRNA (Fig. 4I) and protein (small box in Fig. 4I) were detected in the EGL of the cerebellum. At this stage, Prox1-positive cells in the MZ coexpressed late neuronal postmitotic markers as NeuroN and NeuroF200 (not shown).

Characterization of Prox1 Expression During Postnatal Brain Development and Adulthood

To analyze the distribution of Prox1 mRNA and protein during postnatal mouse brain development, we compared results from in situ hybridization, immunohistochemistry, and X-Gal staining of coronal and sagital sections of brains from mice at postnatal day (P) 0, P5, P10, P15, P30, and adult (12 months) wild-type and Prox1 +/lacZ mouse brains. The Prox1 +/lacZ mice have an insertion of the lacZ gene into the Prox1 locus and β-galactosidase expression recapitulates the expression pattern of the endogenous Prox1 gene (Wigle et al.,1999, see Fig. 6E–H, not shown).

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Figure 6. Characterization of Prox1 expression in postnatal (P30) and adult brains. X-Gal staining (A–E, G) and immunohistochemistry (F, H, I) analyses of P30 (A–C) and adult (D, H, I) mouse brains. Prox1 expression remained only in the dentate gyrus (DG) of the hippocampus (A, B, D–F), the thalamus (T), and in cells of the internal granule layer (IGL), molecular layer (ML), and white matter (WM) of the cerebellum. The Prox1-positive cells of the white matter also expressed GFAP (I).

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During postnatal development (P0-P30), Prox1 was detected in nearly all of the same brain regions described for E18.5 mice, but most likely as a reflection of the end of cell differentiation, a progressive overall reduction in the number of Prox1-positive cells and in the intensity of its staining was observed. In the telencephalon, Prox1 was detected in the amygdala (Fig. 5E), cortex (Fig. 5D), corpus callosum (Fig. 5A,B), fimbria (not shown), and dentate gyrus (Fig. 5G,H). Prox1 protein was observed in neurons of the outer layers of the cortex but not all the Prox1-positive cells coexpressed neuronal markers. This finding suggests that some of these cells may give rise to glial cells (Fig. 5D). It has been described that in vitro Prox1-positive cells are able to differentiate into both neuronal and glial cells (Torii et al.,1999). However, we were not able to detect the expression of the glial markers Olig1/2 and GFAP in any Prox1-positive cells in the cortex (not shown). In the corpus callosum and fimbria, Prox1-positive cells were first detected at around P10 (Fig. 5B), and some coexpressed the neuronal marker calretinin (Fig. 5A). The function of this calcium-binding protein has not yet been fully established, although mice deficient in calretinin exhibited alterations in motor coordination due to changes in granule cell excitability in the cerebellum (Bearzatto et al., 2006).

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Figure 5. Expression of Prox1 during postnatal brain development. Prox1 is expressed in neurons of the corpus callosum (CC) (A:Prox1 mRNA blue; calretinin brown; B: X-Gal staining of the Prox1-expressing cells), thalamus (C: Prox1, green; NeuroN, red), and cortex (D: Prox1, brown; NeuroN, light blue). In the habenula (Hb) (F), anteroventral nucleus (AVN) (E, F), thalamic paraventricular nucleus (PVN) (G, lower inbox in G), and in the reuniens nucleus (RN) (E, F, small inbox in E) of the thalamus, Prox1 is coexpressed with calretinin (Prox1 mRNA, blue; calretinin, brown). In the hippocampus, Prox1 is localized in the dentate gyrus (DG) and the subgranular zone (SGN) (top small box in G: Prox1 mRNA, blue; calretinin, brown; H: Prox1, green; NeuroN, red). Some Prox1-positive cells in the SGN coexpressed GFAP (I: Prox1, green: GFAP, red). Prox1 is also detected in the external granular layer (EGL) at P0 (J, Prox1 in red) and P5 (K, Prox1 in green). Prox1 is also observed in the granular layer (GL) (J, K), internal granular layer (IGL) (L), and white matter (WM, K) of the cerebellum. A, amygdala; ZI, zona incerta. Arrows: Prox1 cells co-expressing a neuronal or glial marker. Arrowheads: no coexpressing Prox1-positive cells. For Prox1 protein detection, the three available Prox1 antibodies were used indistinctly.

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In the dentate gyrus, Prox1 is expressed in the mature granule cells and in the subgranular zone (Fig. 5H) with the calretinin+ fibers from the mossy cells projecting into the surrounding inner molecular layer (small box in Fig. 5G). These granule cells were also surrounded by astrocytes, and a few GFAP+;Prox1+ cells were observed in the subgranular zone (Fig. 5H,I). These cells may arise from radial glial precursors that differentiated to granule cells but did not express all neuronal markers (Fig. 5H,I). In the diencephalon, Prox1 was still observed in the thalamus. This was a location in which the continuous strong and persistent postnatal Prox1 expression suggested a possible association with adult neurons. In this region, Prox1 expression pattern closely resembled that of calretinin. When double in situ immunohistochemistry analysis was performed on P15 mouse brain sections, double staining was observed in the habenula (Fig. 5F), the anterioventricular nucleus (Fig. 5E,F), the paraventricular nucleus (Fig. 5G, small box in G), and the reuniens nucleus in the thalamus (Fig. 5E,F, small box in E). In the prethalamus, Prox1 mRNA was expressed in the zona incerta (Fig. 5E–G), and in the dorsomedial and ventromedial nuclei of the hypothalamus (not shown). We also detected Prox1 coexpression with neuronal markers NeuroN and NeuroF200 near the dorsomedial and ventromedial nuclei of the hypothalamus (not shown). In the cerebellum, the pattern of Prox1 expression changed as differentiation advanced. At P0, Prox1 was detected in the premigratory granule cells of the cerebellum that are massively generated just after birth (Fig. 5J). These cells will differentiate after migrating to deeper areas inside the cerebellum. Later, at P5, Prox1-positive cells were not only observed in the EGL and the immediate internal granule layer (IGL) in formation, but also in the white matter (Fig. 5K,L). In the EGL cell layer, Prox1 expression was detected as late as P15 (not shown).

At the end of mouse postnatal brain development, Prox1 expression was greatly reduced and disappeared from almost all of the structures already mentioned. At P30, X-Gal staining was detected in the granule cells of the dentate gyrus, the thalamus, some scattered cells of the internal granular layer (IGL), and in the white matter in the cerebellum (Fig. 6A–C). Similarly, in the adult brain, Prox1-positive cells were detected only in the dentate gyrus, as previously described (Bick-Sander,2006; Navarro-Quiroga et al.,2006) and in the IGL and white matter in the cerebellum (Fig. 6D–H). As suggested by the coexpression with GFAP (Fig. 6I), the Prox1-positive cells detected in the white matter most likely correspond to glial cells.

Together, these data demonstrate that during murine brain development Prox1 is expressed in most of the locations in which neurogenesis and glial formation occur during middle and late prenatal and postnatal stages. The expression data suggest that there is more than one mechanism through which Prox1 controls cell fate. Although most of the expression data suggest that Prox1 is involved in the control of cell cycle (e.g., its expression in the EGL of the cerebellum), its activity could also be necessary to fulfill additional roles. For example, in the dentate gyrus Prox1 expression is constantly detected during embryonic and postnatal stages in both precursors and in mature postmitotic cells. This expression could reflect a role of Prox1 in the maintenance of the identity of these cells or for their proper function.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

In Situ Hybridization

Embryonic (E12.5, E14.5, E16.5, E18.5) and postnatal (P0, P5, P10, P12, P15, P30, and 12 months) mouse brains were perfused with 4% PFA, cryopreserved in 30% sucrose, embedded in TissueTek, and sectioned in a cryostat at 12 μm. In situ hybridization was performed as described previously in Schaeren-Wiemers and Gerfin-Moser (1993). The Prox1 probe used in this study has been previously described in Oliver et al. (1993). The RNA probe was prepared using the DIG RNA labeling kit (Roche) according to the manufacturer's protocol.

Immunohistochemistry

Embryonic and postnatal mouse brains were prepared as described above and sectioned in a cryostat at 12 μm. Immunostaining was performed as described in Lavado et al. (2006), with the difference that primary antibodies were incubated overnight at room temperature. Sections were incubated with the following primary antibodies: rabbit anti-Prox1 (1:500, AngioBio) and guinea pig anti-Prox1 (1:100) (our own) (both antibodies recognize the C-terminal end of Prox1), anti-NH-Prox1 (which recognizes the N-terminal end of Prox1) (1:1,000) (a gift from B. Sosa-Pineda), mouse anti-Nestin (1:250, Chemicon, Temecula, CA), mouse anti-MAP2 (1:200, Roche), mouse anti-βTuj III (BabCO), mouse anti-PH3 (Upstate), mouse anti-NeuroN (1:100, Chemicon), mouse anti-NeuroF200 (1:80, Sigma, St. Louis, MO), rabbit anti-GFAP (1:100, Sigma), mouse anti-Olig1/2 (1:100, R&D), and rabbit anti-Calretinin (1:4000, Chemicon). These antibodies were followed by the appropriate secondaries: goat anti-rabbit Alexa 488, goat anti-mouse Alexa 488, goat anti-guinea pig Alexa488 (Molecular Probes, Eugene, OR), donkey anti-mouse Cy3, donkey anti-rabbit Cy3 (Jackson Immunoresearch, West Grove, PA). DAB immunostaining was performed on sections hybridized with the desired primary antibodies, then incubated with the appropriate anti-rabbit or anti-mouse biotin-conjugated secondary antibody (Jackson Immunoresearch) and developed with DAB as a substrate (ABC kit, Vector, Burlingame, CA).

In Situ and Immunohistochemistry Double Staining

In situ hybridization on brain sections was followed by DAB immunohistochemistry as described above.

X-Gal Staining

X-Gal staining was performed on Prox1 +/lacZ brain sections (Wigle and Oliver,1999) as described in Montoliu et al. (1995). Briefly, postnatal mouse brains were perfused in 2% PFA, 0.2% glutaraldehyde, cryoprotected in 30% sucrose, and cryosectioned at 20 μm. Sections were stained with X-Gal at 30 or 37°C for 2 to 12 hr.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

A. L. holds a postdoctoral fellowship (MEC/Fulbright) from the Spanish Government. The authors are grateful to Beatriz Sosa-Pineda for the anti-N-Prox1 antibody, Miriam Dillard for the critical revision of the manuscript, and Dr. Anastassia Stoykova for advice.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES