The transcription factor Otx2 is required to determine mesencephalic versus metencephalic (cerebellum/pons) territory during embryogenesis. This function of Otx2 primarily involves positioning and maintaining the mid-hindbrain organizer at the border between midbrain and anterior hindbrain. Otx2 expression is maintained long after this organizer is established. We therefore generated conditional mutants of Otx2 using the Cre/loxP system to study later roles during rostral brain development. For inactivation of Otx2 in neuronal progenitor cells, we crossed Otx2flox/flox animals with Nestin-Cre transgenic animals. In Nestin-Cre/+; Otx2flox/flox embryos, Otx2 activity was lost from the ventral midbrain starting at embryonic day 10.5 (E10.5). In these mutant embryos, the mid-hindbrain organizer was properly positioned at E12.5, although Otx2 is absent from the midbrain. Hence, the Nestin-Cre/+; Otx2flox/flox animals represent a novel mouse model for studying the role of Otx2 in the midbrain, independently of abnormal development of the mid-hindbrain organizer.
Our data demonstrate that Otx2 controls the development of several neuronal populations in the midbrain by regulating progenitor identity and neurogenesis. Dorsal midbrain progenitors ectopically expressed Math1 and generate an ectopic cerebellar-like structure. Similarly, Nkx2.2 ectopic expression ventrally into tegmentum progenitors is responsible for the formation of serotonergic neurons and hypoplasia of the red nucleus in the midbrain. In addition, we discovered a novel role for Otx2 in regulating neurogenesis of dopaminergic neurons. Altogether, these results demonstrate that Otx2 is required from E10.5 onward to regulate neuronal subtype identity and neurogenesis in the midbrain.
- neuronal identity
- dopaminergic neurons
- serotonergic neurons
- conditional inactivation
Specification of neuronal fates in the mesencephalic-metencephalic (mes-met) region begins with the acquisition of anterior-posterior (A-P) and dorsal-ventral (D-V) identities instructed by signals from underlying tissues and subsequently from organizing centers within the CNS (for review, see Lumsden and Krumlauf, 1996; Jessell, 2000). The mid-hindbrain (MHB) organizer, marked by the expression of Fgf8 and Wnt1 at the isthmic constriction, is crucial for patterning and growth of the mes-met region (for review, see Simeone, 2000; Liu and Joyner, 2001; Nakamura, 2001; Rhinn and Brand, 2001; Wurst and Bally-Cuif, 2001; Echevarria et al., 2003; Raible and Brand, 2004). Mesencephalic and metencephalic progenitors are subsequently programmed by D-V signals from the roof plate and floor plate, including Bmps and Shh (Tanabe and Jessell, 1996). These cell-cell signaling events ultimately result in progenitors expressing distinct codes of transcription factors that endow them with the capacity to develop into distinct types of neurons.
Mes-met specification begins with the induction of gene expression that distinguishes the mesencephalon from the metencephalon. Loss of function of Otx2 and Gbx2 in mice indicates that these genes are required cell intrinsically for specification of the midbrain and cerebellum, respectively (Wassarman et al., 1997; Rhinn et al., 1998). In addition, Otx2 and Gbx2 act antagonistically in the mes-met region to position the MHB organizer (Broccoli et al., 1999; Millet et al., 1999; Katahira et al., 2000). In contrast to Otx2, other transcription factors required for development of the mes-met region, such as the homeobox-containing genes Pax2, Pax5, En1, and En2, are required for the development of both the midbrain and cerebellum (Wurst et al., 1994; Hanks et al., 1995; Schwarz et al., 1997; Bouchard et al., 2000). Hence, among the critical mes-met determination genes, only Otx2 is specifically required for the development of the mesencephalon.
To investigate later roles of Otx2 in the mes-met region, we generated a conditional mouse mutant of Otx2 using the Cre/loxP system and inactivated Otx2 activity starting at embryonic day 10.5 (E10.5). Our studies revealed later roles for Otx2 in regulating neuronal identity and neurogenesis that are distinct from its earlier role in A-P patterning and in positioning the MHB organizer. Strikingly, an ectopic cerebellar-like structure developed at the position of the colliculi that is preceded by changes in expression of genes involved in cerebellar development. Similar changes in cell fate occur in the ventral midbrain in which ectopic serotonergic neurons developed rostral to Fgf8 expression, likely at the expense of Pou4f1+ red nuclei (RN) neurons. In addition, hypoplasia of midbrain dopaminergic (DA) neurons is attributable to reduced Mash1 and Ngn2 and consequently decreased neurogenesis from the ventral midline. These results showed that some midbrain progenitors in Otx2 conditional knock-out (Otx2-CKO) embryos adopt a hindbrain differentiation program (cerebellar granule and serotonergic neurons). Altogether, these data demonstrate novel roles for Otx2 from E10.5 onward in regulating general neuronal and subtype differentiation program in the midbrain.
Materials and Methods
Generation and genotyping of mutants embryos and animals. Nestin-Cre transgenic and the Otx2flox/flox mouse strains were maintained in an out-bred MF1 background. The Nkx2.2 mutant strain was maintained in a mixed MF1-C57BL/6 background. To obtain conditional Otx2 mutants, we crossed Nestin-Cre transgenic mice (Isaka et al., 1999) to animals homozygous for the Otx2flox allele (Puelles et al., 2003). Nestin-Cre/+; Otx2+/flox male animals were then mated to Otx2flox/flox females. Nestin-Cre/+; Otx2+/flox males were also mated with Nkx2.2+/- females to generate Nestin-Cre/+; Otx2+/flox; Nkx2.2+/- male animals. Simultaneously, Otx2flox/flox males were mated with Nkx2.2+/- females to generate Otx2+/flox; Nkx2.2+/- female animals. Nestin-Cre/+; Otx2flox/flox; Nkx2.2-/- mutants were obtained by crossing Nestin-Cre/+; Otx2+/flox; Nkx2.2+/- males with Otx2+/flox; Nkx2.2+/- or Otx2flox/flox; Nkx2.2+/- females. The Otx2flox allele was detected by PCR (Puelles et al., 2003), whereas the Cre transgene was detected by using a pair of primers (5′ ATC CGA AAA GAA AAC GTT GA 3′ and 5′ ATC CAG GTT ACG GAT ATA GT 3′) and PCR as described by Indra et al. (1999). A null mutation in the Nkx2.2 gene was generated by eliminating the entire coding region (Sussel et al., 1998). For genotyping of Nkx2.2 alleles, two sets of PCR primers were used. To detect the wild-type allele, a pair of primers (forward, 5′-CCC CCA GTA CCC GAC AGC ACA-3′; reverse, 5′-GGG GCC GGT TGG TCC TTT CTC-3′) and the PCR program includes an initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min. To detect the mutant allele, a pair of primers (forward, 5′-AGA GGC TAT TCG GCT ATG ACT-3′; reverse, 5′-CCT GAT CGA CAA GAC CGG CTT-3′) and the PCR program includes an initial denaturation at 95°C for 5 min, followed by 30 cycles of 94°C for 1 min, 58°C for 2 min, and 72°C for 3 min. At all times, animals were handled according to the Society of Neuroscience Policy on the Use of Animals in Neuroscience Research, as well as the European Communities Council Directive.
Histological analysis. Postnatal brains were fixed in Bouin's fixative solution for 48 h at room temperature and then stored in 70% alcohol until embedding in paraffin. Paraffin sections (7 μm) were cut on a microtome and counterstained with hematoxylineosin or cresyl violet.
Whole-mount in situ hybridization, in situ hybridization, and immunohistochemistry of brain sections. Embryos or dissected brains were fixed for 2 h at 4°C in 4% paraformaldehyde in 0.1 m PBS and either stored in methanol at -20°C or cryoprotected with 30% sucrose in PBS, embedded in OCT compound (VWR International, Poole, UK), and cryosectioned on a cryostat (CM3050S; (Leica, Nussloch, Germany). Section and whole-mount in situ hybridization were performed as described previously (Schaeren-Wiemers and Gerfin-Moser, 1993 and Conlon and Herrmann, 1993, respectively). The following mouse antisense RNA probes have been used: Otx1 (Puelles et al., 2003), Otx2Δ (Puelles et al., 2003), Otx2 (Rhinn et al., 1998), Fgf8 (Crossley and Martin, 1995), Wnt1 (Bally-Cuif et al., 1995), Gbx2 (Bouillet et al., 1995), Shh (Echelard et al., 1993), Patched1 (Ptch1) (Puelles et al., 2003), Gli1 (Hui et al., 1994), Pax3 (Goulding et al., 1991), Gdf7 (Puelles et al., 2003), Msx1 (Hill et al., 1989), Math1 (Helms and Johnson, 1998), Lmx1b (Chen et al., 1998), Nr4a2 [previously named Nurr1 (Zetterstrom et al., 1997)], Ptx3 (Puelles et al., 2003), tyrosine hydroxylase (TH) (Grima et al., 1985), Pou4f1 [previously named Brn3a (Puelles et al., 2003)], Ephrin-A5 (Zarbalis and Wurst, 2000), Dll1 (Bettenhausen et al., 1995), Hes5 (Akazawa et al., 1992), Mash1 (Guillemot and Joyner, 1993), and Ngn2 (Fode et al., 1998). For each probe, a minimum of three control and three mutant embryos were analyzed.
For immunohistochemistry, sections were incubated overnight at 4°C with the appropriate primary antibody diluted in 0.1% Triton X-100 (TX-100) and 10% normal goat serum in PBS. Sections were then extensively washed in PBS plus 0.1% TX-100 and incubated 1 h at room temperature with a secondary antibody conjugated with a fluorochrome. Sections were then washed and mounted in Vectashield H-1000 (Vector Laboratories, Burlingame, CA). The following primary antibodies were used: rabbit anti-Otx (1:1000) (Baas et al., 2000), rabbit anti-serotonin (S5545, 1:5000; Sigma, St. Louis, MO), rabbit anti-Nkx2.2 (1:100) (Briscoe et al., 1999), rabbit anti-calbindin D-28k (CB38a, 1:1000; Swant, Bellinzona, Switzerland), rat anti-bromodeoxyuridine (BrdU) (OBT0030S, 1:10; Oxford Biotechnology, Kidlington, UK), rabbit anti-TH (AB152, 1:200; Chemicon, Temecula, CA), mouse anti-Pou4f1 (sc-8429, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-tubulin isotype III (SDL.3D10, 1:100; Sigma). All images were collected on a Zeiss (Oberkochen, Germany) LSM510 microscope or Leica TCS SP2 confocal microscope and processed with Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). TH+ and Pou4f1+ cell counting were done after immunohistochemistry using anti-TH or anti-Pou4f1 antibodies, respectively. Positive cells were numbered along the whole A-P axis of the midbrain every 50 μm. Altogether, 10 sections were analyzed at E12.5 and 14 sections at E15.5. Student's one-tailed t test was used to determine statistical significance.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling. Cryostat sections were washed once for 5 min in PBS-0.1% TX-100, permeabilized in ice-cold 0.01 m citrate buffer and 0.1% TX-100 for 2 min, and washed again in PBS-0.1% TX-100. The enzymatic reaction was then performed at 37°C according to the protocol of the manufacturer (1 684 795; Roche Diagnostics, Mannheim, Germany).
BrdU labeling. Pregnant females were injected intraperitoneally with a solution of BrdU (B-5002, at 10 mg/ml in physiological serum; Sigma) at 100 mg for 1 g of body weight and killed 1 h later. Proliferating cells were revealed by immunohistochemistry on frozen sections after in situ hybridization with an Shh antisense probe. BrdU+ cells in the Shh+ domain were counted on four adjacent sections per embryo at the level of DA and red nuclei neurons. Student's one-tailed t test was used to determine statistical significance.
Nestin-Cre mediated inactivation of the Otx2flox allele in the CNS
In Nestin-Cre transgenic animals, the Cre recombinase gene driven by the Nestin promoter and enhancer is specifically expressed in neural precursor cells (Isaka et al., 1999). To analyze Cre activity during embryogenesis, Nestin-Cre/+; Otx2flox/+ mice were crossed with the R26R reporter mouse strain (Soriano, 1999). In Nestin-Cre/+; R26R/+ embryos, the cells in which Cre is active constitutively expressed the β-galactosidase enzyme. Cre activity was first detected in the ventrolateral domain of the CNS at E10.5 and becomes progressively activated throughout the midbrain and most regions of the CNS by E12.5 (data not shown) in which Otx2 is expressed.
To study the role of Otx2 during neurogenesis, we generated Nestin-Cre/+; Otx2flox/flox embryos (hereafter referred to as Otx2-CKO mutants). We analyzed Otx2 inactivation using an anti-Otx antibody (Baas et al., 2000) or an RNA probe to detect exon 2, which is deleted on Cre recombination of the Otx2flox allele (see Materials and Methods). The anti-Otx antiserum that we used likely recognizes both Otx1 and Otx2 proteins. The expression of Otx proteins observed in Otx2-CKO embryos at E10.5 and E12.5 corresponds to the remaining expression of Otx1 (Fig. 1B′,B″,D′,D″,F′,F″,H′,H″).
In agreement with the analyses using R26R animals, expression of exon 2-containing transcripts and Otx2 protein were first lost in the ventrolateral midbrain at E10.5 but no activation occurred yet at the level of the floor plate and the isthmus (Fig. 1B,F). Subsequently, Otx2 was progressively inactivated in the whole midbrain between E11.5 (data not shown) and E12.5. At E12.5, expression of Otx2 protein was almost completely missing in the midbrain of Otx2-CKO mutant embryos (Fig. 1D,D′,H,H′). The residual strong signal in the roof plate and ventral part of the midbrain (Fig. 1H′) correlated with the remaining sites of Otx1 expression (Fig. 1H″). By comparison, the Nestin:Cre/+; Otx2flox/+ control (hereafter called control) embryos never showed any loss of Otx2 protein in the domain of Cre activity (Fig. 1G,G′) because one functional allele of Otx2 remains.
In summary, the Otx2-CKO animals allow us to study any requirements of Otx2 in the developing midbrain from E10.5 onward.
Otx2-CKO mice showed an ectopic cerebellar structure in the dorsal midbrain and abnormal development of tegmentum nuclei
Otx2-CKO mutants are found with the expected Mendelian ratio until birth, indicating that loss of Otx2 activity in the CNS from E10.5 onward does not result in embryonic lethality as in homozygous Otx2 null mutants. (Acampora et al., 1995; Matsuo et al., 1995; Ang et al., 1996). Later, 30% of Otx2-CKO pups die during the first 3 weeks of their postnatal life, and the remaining 70% survive until adulthood. It remains unclear what is the cause of the postnatal lethality in Otx2-CKO mutants, but some postnatal animals that died showed growth defects. Histological analysis of pups at birth [postnatal day 0 (P0)], 1 week (P7), and 1 month (P28) revealed two major macroscopic defects in Otx2-CKO brain. First, an ectopic cerebellar-like structure formed at the dorsal midline (Fig. 2A,A′,D,D′,E,E′) but not in the lateral domain of the mutant midbrain at all stages analyzed (Fig. 2B,B′). Two dense layers of cell nuclei were observed in the ectopic structure, resembling the external granular cell layer (EGL) and Purkinje cell layer in the normal cerebellum. Confirmation of the Purkinje cell layer was obtained by specific staining for calbindin protein at E17.5 (data not shown) and P28 (Fig. 2F′). However, in the ectopic cerebellar structure, the Purkinje cell layer was less organized than in the endogenous cerebellum (Fig. 2, compare F, F′). Second, compared with the control, the endogenous cerebellum in Otx2-CKO mutant embryos appeared truncated in its most posterior part in both vermis and hemispheres at all stages analyzed (Fig. 2 and data not shown). In addition, the remaining foliae showed an abnormal foliation pattern. The defects in the cerebellum will be the focus of another study. The formation of the ectopic cerebellum disrupted the dorsal midbrain structure, in particular the colliculi. Both superior and inferior colliculi could not be observed any more in adult Otx2-CKO animals (data not shown). Sagittal and coronal sections (Fig. 2A′,B′) of the brain of Otx2-CKO embryos also showed a severe reduction of these structures in the dorsal midbrain already by P0.
In the ventral midbrain, several groups of neurons are organized into nuclei that are visible by cresyl violet staining such as the RN and the oculomotor (OM) neurons. The OM neurons appeared normal, although there was a consistent reduction of the RN throughout the midbrain at birth (Fig. 2C,C′). In summary, the histological analyses revealed that Otx2 is required for proper formation of neuronal subtypes in the midbrain.
Environmental signals along A-P and D-V axes are not modified in Otx2-CKO embryos
Previous genetic analyses in mice have shown that mouse mutants with rostral shifts of the MHB organizer develop an enlarged cerebellum at the expense of the midbrain (Acampora et al., 1997; Suda et al., 1997). Hence, we examined whether the formation of an ectopic cerebellar-like structure in the midbrain may reflect an alteration of the position and/or function of this organizer. We therefore assessed the status of key regulatory molecules required for MHB organizer function in Otx2-CKO mutants. At E10.5 (data not shown) and E12.5, Otx2 (Fig. 3A,A′), Gbx2 (Fig. 3D,D′), Wnt1 (Fig. 3C,C′), and Fgf8 (Fig. 3B,B′) showed identical patterns of expression in mutant and control embryos, indicating that the MHB organizer was correctly positioned and maintained in the Otx2-CKO embryos.
We had also shown previously that the dosage of Otx proteins regulates the expression of Shh, a gene coding for a secreted molecule crucial for D-V patterning of the CNS (Puelles et al., 2003). Therefore, we examined D-V patterning in the midbrain by determining the expression of Shh and its ventral targets Gli1 and Ptch1. At E12.5, the domain of Shh expression in Otx2-CKO embryos was identical to control embryos; however, the expression pattern appeared uniform, whereas in control embryos, Shh expression was slightly more intense at its dorsal limit (Fig. 3E,E′). This uniform expression pattern of Shh in the midbrain of Otx2-CKO embryos was similar to the expression pattern observed in the metencephalon of control embryos (see Fig. 5B″). We also observed an alteration in the morphology of the ventral midbrain. Specifically, the Shh-expressing cells showed a narrower V-shaped ventricular zone in Otx2-CKO compared with control embryos (Fig. 3E,E′), but the number of cells expressing Shh was similar in control and Otx2-CKO embryos. In addition, the alar-basal boundary of the midbrain, normally delimited by a sulcus, in control embryos was less apparent in Otx2-CKO embryos (arrows in Fig. 3E-H and E′-H′ respectively). The expression domain of Ptch1 (Fig. 3F,F′) and Gli1 (Fig. 3G,G′) also appeared unchanged compared with control embryos, suggesting that the reception of Shh signaling is not modified in Otx2-CKO embryos. Consistent with the idea that Shh signaling is normal in these embryos, the expression pattern of Pax3 (Fig. 3H,H′), a paired homeodomain-con-taining gene that is normally repressed by Shh signaling in ventral regions of the neural tube, was also not altered. Hence, we did not detect any evidence of abnormal Shh expression and signaling in these mutants.
Dorsally, the roof plate also functions as a signaling center secreting TGFβ-related superfamily molecules, including Gdf7 and Bmp6 that influence the differentiation of dorsal neurons. Expression of these molecules was identical in control and Otx2-CKO embryos (Fig. 3I,I′ and data not shown). We also determined the response of dorsal midbrain cells to BMP signals in Otx2-CKO mutants. Expression of Msx1, a downstream target of BMP signaling, appeared slightly broader in the dorsal neural tube of Otx2-CKO mutants (Fig. 3J′) compared with its expression in control embryos (Fig. 3J).
Altogether, these results demonstrate that that the expression of A-P and D-V patterning signals is primarily unaffected in Otx2-CKO mutants. However, the shape of the ventral midbrain was modified, and, in particular, the sulcus marking the alar-basal boundary was affected.
Midbrain progenitors express transcriptional codes similar to hindbrain progenitors in Otx2-CKO embryos
Next, we investigated whether Otx2 might alter the fate of midbrain precursors by modifying progenitor code in the midbrain. The cerebellum is derived from the alar plate of the metencephalon (Wingate, 2001). The rhombic lip, located at the posterior dorsal edges, of the metencephalon gives rise to cerebellar granule cells, whereas the ventricular layer of the anterior metencephalon produces all other cerebellar cell types (for review, see Hatten and Heintz, 1995; Wang and Zoghbi, 2001). Math1, a basic helix-loop-helix (bHLH) transcription factor, is expressed in cerebellar granule progenitors in the rhombic lip and is required for the generation of cerebellar granule neurons (Ben-Arie et al., 1997). We found that Math1 was ectopically expressed in the dorsal midbrain of E11.5, E12.5, and E15.5 Otx2-CKO mutants (Fig. 4A′-D′ and data not shown) compared with control embryos (Fig. 4A-D).
Mouse embryos with mutations in the netrin receptor Unc5h3 and the transcription factor Pax6 (Przyborski et al., 1998; Engelkamp et al., 1999; Goldowitz et al., 2000) also showed an ectopic Math1+ EGL in the midbrain. However, their phenotype differed from Otx2-CKO embryos in three ways. First, the ectopic EGL in the former mutant embryos was observed when the endogenous EGL has covered the surface of the cerebellum after migrating out of the rostral rhombic lip between E13.0 and E16.0 in mouse. In Otx2-CKO mutants, the first ectopic Math1+ cells in dorsal midbrain were detected at E11.5 (Fig. 4A′, inset) before the time when granule neuron progenitors are starting to migrate out of the rhombic lip at approximately E12.5-E13.0 (Miale and Sidman, 1961; Hatten and Heintz, 1995; Wang and Zoghbi, 2001). Second, two separate sites of ectopic granule cells appeared in the dorsal midbrain of Otx2-CKO mutants at E12.5 (Fig. 4B,B′), namely the posterior third of the midbrain and at the border with the diencephalon far away from the endogenous site of Math1 expression. Third, we never observed any continuity between the endogenous EGL and the ectopic Math1+ cells in histological sections of the midbrain (Fig. 2A′ and data not shown). All of these data strongly suggest that ectopic Math1+ cells are not coming from the cerebellum via migration through the isthmus but are likely induced in midbrain neuronal progenitors after Otx2 inactivation.
We also observed that midbrain neuronal progenitors of Otx2-CKO embryos exhibited expanded ventral expression of the homeobox gene Nkx2.2 into a region expressing Shh when compared with control embryos (Fig. 5A′,B′). These progenitors lie adjacent to ectopic Lmx1b+ and 5-HT+ post-mitotic neurons (Fig. 5C′,D′), as observed in the hindbrain of control embryos (Fig. 5C″,D″). Normally, coexpression of Shh and Nkx2.2 is only observed in hindbrain progenitors that generate 5-HT neurons starting at E10.5 (Puelles et al., 2004) (Fig. 5A″,B″). These results suggest that ectopic Nkx2.2 may be responsible for the formation of 5-HT neurons abnormally in the midbrain. In agreement with this hypothesis, removal of Nkx2.2 activity resulted in the disappearance of midbrain serotonergic neurons in Otx2-CKO; Nkx2.2-/- embryos (Fig. 6E,H) at E12.5. Because the ectopic 5-HT neurons were found at the same D-V position as the endogenous Pou4f1-positive cells in the RN (Fig. 5D′,E′), hypoplasia of the RN (Table 1) may also be attributable to the abnormal expression of Nkx2.2 by RN progenitors. This hypothesis is supported by the fact that the RN neurons are found in normal numbers in Otx2-CKO; Nkx2.2-/- (Fig. 6G, Table 1) compared with control (Fig. 6A, Table 1) embryos. Importantly, we also found that Nkx2.2-/- single mutants do not display any defects in the development of OM and RN neurons at E12.5 (data not shown).
In summary, some midbrain progenitors exhibited a hindbrain-like progenitor code. This change in progenitor identity is responsible for the abnormal development of serotonergic neurons from the midbrain. It is noteworthy that, despite these fate transformations midbrain identity is not completely abolished because the undeleted portion of the Otx2 gene (Fig. 3A′) and EphrinA5 (data not shown) are still normally expressed in the midbrain of Otx2-CKO embryos.
Otx2 is required for the differentiation of ventral midline DA neurons
DA neurons that contribute to the substantia nigra and ventral tegmentum area are also generated from progenitors in the ventral midbrain (Zervas et al., 2004). To determine whether Otx2 has a role in regulating the development of DA neurons, we examined the expression of genes that mark the DA lineage and are required for its development, such as Lmx1b, which is normally expressed in DA progenitors and DA neurons in the ventral midbrain (Smidt et al., 2000) (Fig. 5C). Expression of Lmx1b does not appear to be affected in DA progenitors but was reduced in the DA field of the ventral midbrain (Fig. 5C′), suggesting a reduction in the number of DA neurons. To confirm whether reduced Lmx1b expression in ventral region is attributable to a reduction in DA neurons, we also determined the expression Nr4a2, Ptx3, and TH (Smidt et al., 1997; Zetterstrom et al., 1997) in the midbrain of Otx2-CKO mutants at E12.5 and E15.5. Expression of all three genes was significantly reduced in the DA field at E12.5 and more severely reduced at E15.5 (Figs. 7, 8A,B,C,C′,F,G,H,H′).
Nkx2.2 expression expands ventrally into the DA neuron progenitor domain presumably marked by Lmx1b expression in Otx2-CKO embryos (Fig. 5A′,C′). The resulting change in progenitor code may affect the differentiation of Lmx1b+ progenitors into DA neurons. To test this hypothesis and directly determine the contribution of ectopic Nkx2.2 expression to the DA neuron deficit in Otx2-CKO mutants, we examined the expression of TH in DA neurons in Otx2-CKO mutants in an Nkx2.2 null background. Reduction in the number of DA neurons was maintained in Otx2-CKO; Nkx2.2-/- embryos (Fig. 8K,L,M,M′) like in Otx2-CKO embryos (Fig. 8F,G,H,H′). We also determined the expression of TH in Nkx2.2-/- embryos and found normal numbers of TH+ DA neurons (data not shown). This observation indicates that Nkx2.2 ectopic ventral expression alone cannot explain the reduction in the number of DA neurons in Otx2-CKO embryos.
We next determined whether loss of DA neurons might be attributable to a defect in cell proliferation or cell death by BrdU labeling and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling analysis, respectively. No difference in the number of cells undergoing apoptosis was observed at E11.5 and E12.5 (data not shown). The percentage of BrdU+ cells did not change at E11.5 but showed a 25% decrease in the mutants at E12.5 compared with controls (Table 2). Birth-dating analysis has shown that the first DA neurons are born at E10.5, with a peak of production at E11.5 and E12.5 in mouse (Bayer et al., 1995). The reduction in proliferation observed at E12.5 is therefore unlikely to be sufficient to explain the 40% reduction of TH+ DA neurons observed at the same stage (Table 3). These results suggest that the loss of DA neurons at E12.5 is not primarily attributable to proliferation or apoptosis.
Because the expression of DA neuronal markers was almost completely missing beneath the ventral medial midbrain (Fig. 8A′-D′), this raised the possibility that neurons may be missing in this region. We therefore examined whether neurogenesis is affected in the ventral midline of the midbrain by analyzing the expression of a general neuronal marker, β-tubulin. β-Tubulin+ neurons surrounded the ventricular surface including the floor plate in the midbrain of control embryos (Fig. 8D,E). In contrast, β-tubulin-labeled neuronal cell nuclei (detected by Toto-3 labeling) were missing from the ventral midline in the midbrain of mutants (Fig. 8J), although β-tubulin+ fibers could still be detected (Fig. 8I). The observation of this acellular gap below the medial neural tube suggests a failure of neurogenesis in this region of Otx2-CKO mutants.
Proneural bHLH genes have been implicated in regulating neurogenesis in the CNS and PNS (Bertrand et al., 2002). Both Mash1 (Fig. 8N) and Ngn2 (Fig. 8O,P) proneural genes are expressed in the ventral midbrain of wild-type embryos at E11.5. In Otx2-CKO embryos, Mash1 expression was extinguished (Fig. 8S), whereas Ngn2 expression was severely reduced in proliferating ventricular zone progenitors (Fig. 8T,U) of Otx2-CKO embryos. We then examined two components of the Notch signaling pathway, Dll1 and Hes5, whose expression are dependent on proneural gene activity in the neuroepithelium. Dll1, the earliest known marker of cell cycle exit in the neuroepithelium (Henrique et al., 1995), is a ligand of Notch and is probably a direct transcriptional target of proneural proteins (Bertrand et al., 2002), whereas Hes5, an effector of notch signaling, is induced when newborn neurons activate Notch in neighboring cells (Kageyama and Ohtsuka, 1999). Dll1, normally expressed in a salt and pepper pattern throughout the ventricular zone (Henrique et al., 1995) (Fig. 8R), was no longer detectable at the ventral midline (Fig. 8W), and Hes5 expression was also abolished at the same level in Otx2-CKO embryos (Fig. 8V). Altogether, these results indicate that loss of Otx2 leads to a specific loss of proneural genes in the medial floor plate. This results in a failure of neurogenesis at the ventral midline that correlates well spatially and precedes the absence of the medial DA neurons, suggesting a causal relationship between these events. Additional loss of TH-positive neurons between E12.5 [40% reduction (Table 3)] and E15.5 [50% reduction (Table 3)] determined by immunohistochemistry on tissue sections could be contributed by the decrease in proliferation of ventricular zone cells that is observed in Otx2-CKO mutants at E12.5.
Transient requirement for Otx2 in regulating the position of the mid-hindbrain organizer and Shh expression
Otx2 has been shown previously to coordinate A-P and D-V patterning by regulating the expression of key morphogenetic signals such as Fgf8 and Shh, respectively. Inactivation of Otx2 before E10.5 in Otx1cre/+; Otx2flox/- (Puelles et al., 2003) and En1cre/+; Otx2flox/flox (Puelles et al., 2004) embryos led to shifts in the anterior boundary of Fgf8 and an expanded dorsal domain of Shh. However, complete inactivation of Otx2 by E12.5 in Otx2-CKO embryos did not affect the expression of Fgf8 and Shh in the MHB region. Although Otx1 is still expressed at the MHB boundary (Fig. 1D″), it is unlikely that Otx1 is compensating for Otx2 in maintaining the MHB boundary in its dorsal domain, because Otx1 cannot compensate for Otx2 in maintaining the MHB boundary dorsally when Otx2 is inactivated in En1cre/+; Otx2flox/flox embryos at E9.5 (Puelles et al., 2004). Altogether, these results demonstrate that Otx2 is no longer required to maintain the MHB organizer and the dorsal extent of Shh expression at E12.5.
Otx2 inhibits the expression of transcription factors regulating hindbrain fate
In this paper, we demonstrated a later role for Otx2 from E10.5 onward in regulating midbrain identity. In the midbrain of Otx2-CKO mutants, Nkx2.2 and Shh are abnormally coexpressed in some progenitors as in serotonergic hindbrain precursors, and ectopic 5-HT neurons are formed adjacent to these progenitors. Rescue of 5-HT neurons in Otx2-CKO mutants in an Nkx2.2 mutant background indicate that Nkx2.2 is required for the generation of the 5-HT neurons. These results therefore strongly suggest that Otx2 normally prevents serotonergic development in the midbrain by limiting the ventral expression of Nkx2.2. How does Otx2 regulate Nkx2.2 in the midbrain? Otx2 may directly or indirectly regulate the activity of the Nkx2.2 promoter. Whatever the mechanism, Ox2 must cooperate with different spatially restricted cofactors in regulating D-V region-specific gene expression. Shh has been shown previously to regulate Nkx2.2 expression (Briscoe et al., 1999). Shh signaling does not appear to be modified in Otx2-CKO embryos and hence is probably not responsible for the enlarged domain of Nkx2.2 expression. Stronger evidence that the ectopic expression of Nkx2.2 is independent of Shh signaling at E10.5 comes from previous work on Otx1-cre/+; Otx2flox/flox embryos. In these conditional mutants, Shh signaling is enhanced, leading to a reduction of Nkx6.1 expression, but no changes in Nkx2.2 expression was observed. Given these results, we favor the hypothesis that Otx2 is directly involved in the regulation of Nkx2.2. However, we cannot formally rule out a role for Shh in contributing to changes in gene expression in ventral midbrain progenitors in Otx2-CKO mutants.
The concomitant rescue of the serotonergic neurons and hypoplasia of the RN suggest a causal relationship between these two events. The progenitors adjacent to Pou4f1+ red nuclei neurons ectopically express Nkx2.2, and loss of Nkx2.2 activity in Otx2-CKO; Nkx2.2-/- embryos results in normal development of the red nuclei. These results strongly suggest a role for Nkx2.2 in modifying the identity of red nuclei progenitors.
Dorsally, midbrain progenitors also ectopically express a hindbrain progenitor determinant Math1. Ectopic expression of Math1 as early as E11.5 anticipates and is likely involved in the formation of the ectopic cerebellar granule cells in the midbrain of Otx2-CKO embryos. Altogether, the transformation of some dorsal and ventral midbrain progenitors into more caudal hindbrain-like progenitors in Otx2-CKO embryos suggest that Otx2 maintains mesencephalic identity in part by repressing alternative hindbrain fates. It would be interesting to determine whether the molecular mechanism involved in the repression of Math1 expression in midbrain progenitors is similar or distinct from that involved in the repression of Nkx2.2 expression.
Otx2 regulates neurogenesis of DA progenitors in the ventral midbrain
Although Otx2 negatively regulates serotonergic differentiation via repression of Nkx2.2 expression, it positively regulates the development of ventral midline DA neurons. Loss of DA neurons in the ventral midbrain of Otx2-CKO embryos is also not rescued in an Nkx2.2 mutant background, indicating that the ectopic Nkx2.2 expression in progenitors is not responsible for the partial loss of DA neurons. This result is somewhat expected because the ectopic expression of Nkx2.2 occurred only in a small subset of dorsal Lmx1b+ DA progenitors and is therefore unlikely to lead to the loss of the most ventral DA neurons. Instead, we suggest that loss of DA neurons is attributable to the absence of neurogenesis in ventral midline progenitors in the midbrain of Otx2-CKO embryos for the following reasons. (1) Ventral midline progenitors normally express Mash1 and Ngn2, which are sufficient and necessary to drive neurogenesis in other regions of the CNS (Bertrand et al., 2002). (2) These progenitors likely differentiate into the DA neurons that coexpress β-tubulin and TH directly underneath them. (3) In Otx2-CKO mutants, loss of Mash1 and Ngn2 expression in ventral midline progenitors precedes and correlates well spatially with the region in which DA neurons are lost. (4) Ngn2 is required in these progenitors for the differentiation of DA neurons because Ngn2-/- mutants lack almost all DA neurons (our unpublished results). Together, these facts suggest that hypoplasia of DA neurons is attributable to loss of neurogenesis in ventral midline progenitors of Otx2-CKO embryos. However, this may not be the only reason for the loss of DA neurons in Otx2-CKO embryos, because, more dorsally, Lmx1b+ progenitors can also generate DA neurons. It is noteworthy that ventral midline cells of the hindbrain normally do not undergo neurogenesis (Pattyn et al., 2004). Given our hypothesis that Otx2 regulates neurogenesis in the midline progenitors of the midbrain, it will be interesting in the future to determine whether Otx2 is able to positively activate neurogenesis when ectopically expressed in the ventral midline of the hindbrain.
Otx2 is expressed in all Lmx1b+ DA progenitors, yet in Otx2-CKO embryos, neurogenesis is only affected in the most medial progenitors. Lack of neurogenesis defects in more dorsal DA progenitors may be attributable to functional compensation by Otx1 protein and/or the late timing of inactivation of Otx2 protein in these cells. Neurogenesis of DA progenitors initiates at E10.5 (Bayer et al., 1995), so neurogenesis at the earliest stages is likely not affected by loss of Otx2 expression in the conditional mutants. We also found that proliferation is reduced in Otx2-CKO embryos at E12.5 in the region of the ventral midbrain in which DA neurons originate. The reduction in cell proliferation likely also contributes to an additional reduction of DA neurons occurring between E12.5 and E15.5.
Comparison between Nestin-Cre/+; Otx2flox/flox and En1cre/+; Otxflox/flox embryos
While this work was in progress, we reported on the phenotype of En1cre/+; Otx2flox/flox embryos (Puelles et al., 2004). The phenotype in the RN and DA neurons in these two conditional mutants is similar except that En1cre/+; Otx2flox/flox embryos exhibit a much stronger phenotype, likely attributable to the earlier inactivation of the Otx2 protein in these embryos at E9.5. In En1cre/+; Otx2flox/flox embryos, the change of identity observed in the ventral midbrain is more severe because the ventral expansion of Nkx2.2 is likely earlier and more pronounced, whereas in Nestin-Cre/+; Otx2flox/flox mutants, the DA area is only slightly affected by the Nkx2.2 misexpression. Consistent with this finding, the correct number of DA neurons was not recovered in the Nkx2.2 null background. Rather, in Nestin-Cre/+; Otx2flox/flox embryos, loss of Otx2 revealed a specific and novel role for this gene in controlling the number of DA neurons by regulating proneural gene expression in their progenitors, as well as their proliferation.
Otx2-CKO mutants as a new tool for understanding cell fate specification in the cerebellum
We have so far identified two cerebellar cell types in the ectopic cerebellar-like structure located at the dorsal midline of the midbrain of Otx2-CKO mutants: granule neurons and Purkinje cells. The origin of the cerebellar granule neurons is relatively well understood, and several studies have identified the precursors (for review, see Wingate, 2001) as well some of the signals required for their formation (Alder et al., 1999). In contrast, molecular mechanisms controlling the generation of all other cerebellar cell types are poorly understood. Indeed, although their origin (ventricular zone), their time generation (from E10 to postnatal stages), their sequential generation (the deep nuclei, the Purkinje cells, and other classes of cerebellar neurons), and their migration pattern have been elucidated, the signals and transcription factors that regulate the induction and specification of these cerebellar cell types remain to be deciphered (Altman and Bayer, 1997). The formation of an ectopic cerebellum in the midbrain of Otx2-CKO embryos could provide a unique model for identifying genetic pathways involved in cell fate specification in the cerebellum. For example, comparison of gene expression profiles in the dorsal midbrain of wild-type and Otx2-CKO mutants may identify candidate genes involved in cerebellar development. In addition, although ectopic granule neurons are observed along the whole A-P axis of the midbrain, Purkinje cells are only formed ectopically in the caudal midbrain. This result suggests that the signal(s) required for Purkinje cell development may be localized near the MHB organizer. Otx2-CKO embryos could also be helpful to unravel interactions between granule and Purkinje neurons.
This project was supported by grants from the European Community Biotech Program and by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, and the Medical Research Council (MRC) (all to S.-L.A.). B.V. was supported by a predoctoral fellowship from the French Minister of Research, the Association pour la Recherche sur le Cancer, and the MRC. This work was also supported by MRC Grant G9900955, Wellcome Trust Grant QLG3-CT-2000-023109, and Fondo per gli Investimenti della Ricerca di Base Neuroscience grants (A.S.). We are grateful to Drs. P. Chambon, J. Ericson, A. Joyner, P. Gruss, F. Guillemot, R. Hill, R. Johnson, G. Martin, A. McMahon, T. Perlmann, M. Wassef, D. Wilkinson, and W. Wurst for providing probes for RNA in situ hybridization. We thank members of the laboratory for helpful discussion, Lan Chen for excellent technical assistance, and Andrée Dierich for help with generation of the Otx2flox mouse line. We also thank Dr. François Guillemot for critical reading of this manuscript.
Correspondence should be addressed to Dr. Siew-Lan Ang, Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK. E-mail:.
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