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Keywords:

  • aristaless;
  • Arx;
  • Xenopus laevis;
  • forebrain;
  • transcription factor

Abstract

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

Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx-injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development. Developmental Dynamics 232:313–324, 2005. © 2004 Wiley-Liss, Inc.


INTRODUCTION

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

The seat of higher human thought and learning resides in the cerebrum, particularly in the cerebral cortex. The cerebral cortex develops as part of the telencephalon, the rostral-most region of the developing brain. Although the cerebral neocortex is a mammalian structure, the telencephalic region is common to all vertebrate brains. Subcortical telencephalic structures, such as the pallial, subpallial (including lateral and medial ganglionic eminences), and striatal domains are conserved among vertebrates (Fernandez et al.,1998; Bachy et al.,2001,2002). These comparisons are also supported by comparison of conserved molecular markers such as dlx5, Nkx2.1, emx1, pax6, eomesodermin, and lhx 1-9 (Fernandez et al.,1998; Bachy et al.,2001,2002). Based on comparison of molecular markers such as dlx1 and emx1, the precortical early embryonic mouse brain has been likened to the brain of lower vertebrates (Fernandez et al.,1998).

A major component of the cortex consists of radially migrating neurons and tangentially migrating interneurons from the subcortical telencephalon (Kato and Dobyns,2003). Abnormalities in cortical development result in morphological brain abnormalities, such as lissencephaly (smooth brain), with debilitating consequences for human mental function. Several genes have been linked to lissencephaly (reviewed in Kato and Dobyns,2003), including platelet activating factor acetylhydrolase, isoform Ib, alpha subunit (PAFAHIB1 or LIS1), doublecortin (DCX), reelin (RELN), and aristaless-related homeobox (ARX; Kitamura et al.,2002; Kato et al.,2003,2004).

Arx is expressed in a dynamic pattern during vertebrate development that suggests it may be integrally involved in forebrain development. Arx is one of many aristaless-related proteins, a subgroup of paired-type homeobox genes, related to the Drosophila aristaless (al) gene (Schneitz et al.,1993). Aristaless-related homeobox genes are involved in a diverse array of developmental events, including development of the central and peripheral nervous system (Meijlink et al.,1999). Arx orthologs have been isolated from zebrafish, Xenopus, mice, and humans (Miura et al.,1997; Bienvenu et al.,2002; El-Hodiri et al.,2003). Arx is expressed in the developing central nervous system (CNS), primarily in the developing forebrain, beginning during neural plate stages and continues through adulthood, spanning a vast variety of brain developmental stages, from specification to differentiation. ARX mutations are found in human patients with a variety of X-linked mental retardation syndromes including West syndrome and Partington's syndrome (Bienvenu et al.,2002; Kitamura et al.,2002; Stromme et al.,2002; Kato et al.,2003,2004). These patients exhibit mental retardation, infantile spasms and epilepsy, and abnormal electroencephalograms. Additionally, ARX mutations also have been found in patients with X-linked lissencephaly with abnormal genitalia (XLAG; Kitamura et al.,2002; Kato et al.,2004). Two mouse Arx mutants have been generated (Kitamura et al.,2002; Collombat et al.,2003). Kitamura and coworkers generated an Arx mutation that results in an XLAG-like phenotype (Kitamura et al.,2002). These mice are deficient in interneuron migration. It has been suggested that this mutation may not be a null Arx mutation (Collombat et al.,2003). Collombat and coworkers have reported generation of Arx null mice that exhibited deficiencies in pancreatic development but did not report on brain abnormalities that these mice might have (Collombat et al.,2003). Thus, the normal function of Arx in forebrain development remains unknown.

We have reported previously the identification of a Xenopus Arx gene (El-Hodiri et al.,2003). We have used a combination of gain- and loss-of-function approaches to investigate the involvement of Arx in forebrain development. Here, we report results that indicate that Arx is involved in regulating growth and differentiation of the forebrain.

RESULTS

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

We previously observed that xArx is expressed in the prospective diencephalon of neural plate stage embryos (El-Hodiri et al.,2003). At tailbud stages, expression appears in the telencephalon and persists in the medial diencephalon and the floor plate of the remainder of the brain and spinal cord. To further analyze the xArx expression pattern during these phases of development, we compared expression of xArx and other markers of anterior development by double whole-mount in situ hybridization (Fig. 1). At neural plate stages, xArx is expressed posterior/caudal to the telencephalic marker FoxG1 (also known as BF-1; Bourguignon et al.,1998; Fig. 1E). At this stage, xArx expression is also posterior to but overlapping Rx expression at the anterior neural plate (Fig. 1A) (Mathers et al.,1997). As development progresses through neural tube stages, xArx expression remains caudal to FoxG1 expression (Fig. 1F,G) and rostral to en-2 expression (Fig. 1D), a marker of the midbrain–hindbrain boundary (Hemmati-Brivanlou et al.,1991). Of interest, xArx expression is rostral to and slightly overlapping with otx1 expression (Fig. 1C), a primarily diencephalic marker (Kablar et al.,1996). At tail bud stages, the rostral portion of the xArx expression domain overlaps the caudal portion of the FoxG1 expression domain in the developing telencephalon (Fig. 1H) and is between Rx expression domains in the developing eyes (Fig. 1B). These results demonstrate that xArx expression spreads into the developing telencephalon as it expands during tail bud stages. Finally, we compared expression of xArx with additional genes expressed in the telencephalon. Expression of xArx overlaps expression of dlx5 (also known as xdll-3; Papalopulu and Kintner,1993), emx1 (Pannese et al.,1998), Nkx2.1 (Small et al.,2000), and eomesodermin (eomes; Ryan et al.,1998; Fig. 1I–L). Expression of xArx overlaps but is not superimposable with these markers. Specifically, xArx expression is mostly dorsal to the dlx5 expression domain, with overlap between the ventral portion of the xArx and the dorsal portion of the dlx5 expression domain (Fig. 1I). The overlap between xArx and Nkx2.1 is similar, although the Nkx2.1 domain also extends posterior to xArx (Fig. 1K). Expression of xArx extends posterior and ventral to the emx1 and eomes expression domains (Fig. 1J and L, respectively). These results establish the telencephalic expression of xArx and demonstrate the dynamic nature of xArx expression in the developing forebrain.

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Figure 1. XArx is expressed in a dynamic pattern in the developing diencephalon and telencephalon. A–L: Double whole-mount in situ hybridization (WISH) using antisense riboprobes specific for xArx (blue color) and other forebrain markers (in red): Rx2A (A,B), otx1 (C), en-2 (D), FoxG1 (E–H), dlx5 (I), mex1 (J), Nkx2.1 (K), and eomesodermin (L). Regions of overlap appear purple. At neural plate and neural tube stages, xArx expression is caudal/posterior to telencephalic markers such as Rx2A and FoxG1. XArx expression is rostral to otx1 and en-2. E–H: xArx is expressed caudal to FoxG1 at neural plate and neural tube stages. During tailbud stages, xArx expression spreads rostrally to overlap a portion of the FoxG1 expression domain (arrow in H). XArx expression also overlaps expression of dlx5, mex1, Nkx2.1, and eomesodermin. Anterior views are shown in A–I. Lateral views are shown in J–L.

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To begin investigating xArx function in forebrain development, we overexpressed xArx in developing embryos (Fig. 2). Bilateral injection of xArx RNA (100 pg/blastomere) in the prospective anterior CNS resulted in a striking phenotype, including an apparent anterior truncation, a thickening and shortening of the rostral forebrain and eye defects such as coloboma (incomplete development of the retinal pigmented epithelium) and cyclopia (Fig. 2A–F). Of 61 embryos injected with xArx, 52 (approximately 85%) exhibited the anterior truncation phenotype and 10% had no head (not shown) over five independent experiments. Abnormalities in eye development were more variable, ranging from cyclopia or lack of eyes (34%) to lack of one eye (15%) to slightly diminished eyes (51%). Upon immunostaining with the pan-neural antibody Xen-1 (Ruiz i Altaba,1992), we found that injected embryos exhibited a loss of midline structures in the anterior portion of the embryo (Fig. 2G–J). Xen-1 immunostaining also revealed that the nasal organ is present in xArx-overexpressing embryos (Fig. 2I,J), demonstrating that the xArx overexpression phenotype was not a simple anterior truncation. The nasal organ was adjacent to the brain and displaced dorsally. In some cases, the nasal organ was positioned dorsal to the eyes (Fig. 2I, arrowhead). Caudal portions of the brain were bilateral and the trigeminal ganglion appeared to develop normally (Fig. 2G–J), suggesting that defects were primarily limited to anterior/rostral structures. Unilateral injections of xArx RNA resulted in a similar phenotype, but only on the injected side of the embryo (not shown). Consistent with these findings, expression of the notochord and floor plate marker shh (Ekker et al.,1995) is absent from the anterior/rostral-most region of xArx-injected embryos (Fig. 2K,L). Shh is necessary for the development of the head midline. Absence of shh in the head results in the loss of midline structures, resulting in conditions involving loss of bilaterality of the brain and eyes, such as cyclopia and holoprosencephaly (Wallis and Muenke,2000).

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Figure 2. Overexpression of xArx results in shortened forebrain, loss of anterior midline structures, and dorsal displacement of the nasal organ. A,B: Lateral and dorsal views of a normal tadpole, respectively. C,E: Lateral view of xArx-injected tadpole shows apparent anterior truncation. D,F: Dorsal views of the same embryos show displacement of the eyes toward the midline. G–J: Visualization of neural structures in control and xArx-injected embryos by whole-mount immunostaining with the pan-neural antibody Xen-1. G,H: Lateral and dorsal views of uninjected tadpoles. I,J: lateral and dorsal views of two different xArx-injected tadpoles. Tadpole in I exhibits incomplete retinal development and dorsal displacement of nasal organ (arrowhead). Tadpole in J exhibits single nasal organ (arrowhead) and fused rostral brain. K,L: Whole-mount in situ hybridization (WISH) of control and xArx-injected embryos with sonic hedgehog (shh; blue staining). XArx-injected embryos lack anterior/rostral shh expression. LacZ RNA was used as a lineage tracer in the control and xArx-injected embryos (turquoise and red staining, respectively). M–O: Histological staining of transverse sections of control (M) and xArx-injected (N,O) embryos. Sections were taken at the level of the eye. N: Thickening of ventral brain in embryos injected with xArx RNA on both sides. O: Section through embryo with fused eyes. P–R: WISH of xArx-injected embryos using antisense probes for Xic1 (purple/blue staining). LacZ RNA was used as a lineage tracer in xArx-injected blastomeres (red staining). P: Anterior view of unilaterally injected neural tube stage embryo. Q,R: Lateral views of uninjected (Q) and injected (R) sides of unilaterally injected tail bud embryo. S–V: Anti–Isl-1 immunofluorescence of control (S–U) and xArx-injected (V) embryos to label ventral forebrain cells. Progressively caudal transverse sections from the telencephalon (Q) to the diencephalon (S) show gradual thickening and medial concentration of ganglion cells in the forebrain. T: Isl-1 –positive cells are arranged in a broad thick ventral layer and in an ectopic lateral dorsal patch (arrowhead) in xArx-injected embryo.

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To further characterize the xArx overexpression phenotype, we analyzed sectioned material from xArx-injected embryos. We observed an expansion of the ventral portion of the brain in bilaterally injected embryos (Fig. 2M,N). The dorsal forebrain appears relatively normal, suggesting that there is hyperplasia of the ventral forebrain. However, at this point, it is not possible to distinguish between this possibility and ventralization of the dorsal forebrain. As reported above, some of these embryos showed abnormal eye development, including partial or complete cyclopia. We also observed ventral brain expansion on the injected side of unilaterally injected embryos (not shown). Histological examination of an embryo with fused eyes also reveals impairment of neural retina differentiation as evidenced by an apparent lack of lamination (Fig. 2O). This finding is also consistent with perturbation of hedgehog signaling, since reduction or absence of hedgehog signaling has been reported to result in abnormalities in photoreceptor development and retinal lamination (Stenkamp et al.,2000). These results suggested that injection of xArx RNA results in neural tube hyperplasia. Consistent with neural tube hyperplasia, we observed a severe decrease in expression of the cyclin-dependent kinase (cdk) inhibitor Xic1 at neural tube stages (Fig. 2P). Xic1 expression correlates with withdrawal of differentiating neurons from the cell cycle (Carruthers et al.,2003; Vernon et al.,2003). XArx-injected embryos expressed Xic1 at tailbud stages, albeit in an abnormal pattern (Fig. 2Q,R). We observed two distinct domains of Xic1 expression in brains of xArx-injected embryos (Fig. 2Q), compared with the three domains observed in brains of control embryos (Fig. 2R). The rostral Xic1 expression domain may be absent or the middle expression domain may be absent or fused to the caudal expression domain, which appears to be broader than normal. Additionally, the eye, otic, and dorsal branchial arch Xic1 expression domains were severely reduced. The aberrant pattern of Xic1 expression in tail bud embryos may be partially due to the abnormal brain morphology observed in xArx-injected embryos.

We further analyzed the ventralization of xArx-injected embryo brains by using Isl-1 immunofluorescence (Fig. 2S–V). In uninjected tadpoles, Isl-1–positive cells were sparsely distributed in the anterior ventral forebrain (Fig. 2S) and progressively concentrated in a distinct medial ventral domain (Fig. 2T,U), as sections progressed posteriorly from the telencephalon. However, the Isl-1 staining pattern matched neither of these patterns in xArx-injected tadpoles. Isl-1–positive cells were distributed in a thick dense layer across the ventral forebrain (Fig. 2V). Additionally, ectopic Isl-1–positive cells were also observed in a dorsolateral region of the brain. Taken together, these results suggested that overexpression of xArx resulted in a shortened, ventralized rostral forebrain lacking midline structures.

We continued to analyze the molecular consequences of xArx overexpression by whole-mount in situ hybridization (WISH) using additional molecular markers of the developing brain (Fig. 4). Embryos were injected at the four-cell stage with xArx and/or lacZ RNA into the prospective anterior CNS region of one or both dorsal blastomeres, fixed at either neural tube or tailbud stages, and analyzed for expression of several markers of forebrain development. Markers used included markers of telencephalon (otx2, pax6, FoxG1, emx1, lhx2, vax2, Nkx2.1, Rx2A), diencephalon (otx1, otx2), and retina (pax6, otx2, Rx2A, lhx2). Expression of most of these markers was diminished or absent in neural tube stage embryos but was visible at tail bud stages. As observed above in the case of Xic1 WISH, expression patterns in tail bud stage embryos were abnormal, possibly as a secondary result of abnormal forebrain development. Interesting exceptions to this pattern included the lim-homeobox genes lhx1 (lim-1) and lhx9. These genes are expressed in a complex pattern, including several domains of the developing brain (Bachy et al.,2001). Expression of lhx1 and lhx9 was essentially absent from xArx-injected embryos. The absence of marker gene expression early in development suggests a lack or delay in brain specification and regionalization. The appearance of expression of most of these markers in tail bud embryos suggests that injection of xArx generally results in a delay in brain development.

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Figure 4. Expression of some forebrain markers enhanced by xArx expression. A–N: Whole-mount in situ hybridization (WISH) of control (A,C,E,G,K,M) or xArx-injected (B,D,F–J,L,N) embryos using antisense probes for wnt8b (A–D), dlx5 (E–J), pax2 (K,L), or otx1 (M,N). LacZ was used as a lineage tracer (blue in control embryos, red in xArx-injected embryos). I,J: Transverse sections of embryo shown in H. Endogenous and ectopic otic structures are indicated (arrows in IJ). NT, neural tube; OV, otic vesicle; 2° OV, secondary otic vesicle. Filled arrowheads in E–H,K,M indicate normal otic vesicles. Open arrowheads in F,H,L,N indicate secondary or duplicated otic structures.

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Not all brain development markers followed the pattern described above. For example, FoxG1 was expressed in a single expression domain (Fig. 3D4) instead of two distinct ones (Fig. 3D3). The loss of the medial nonexpressing domain may have resulted from expanded expression of FoxG1, a loss of midline structures, or both. Additionally, expression of wnt8b in the forebrain (Cui et al.,1996) is relatively unaffected in neural tube stage embryos (Fig. 4A,B) and expanded rostrocaudally in tail bud stage embryos (Fig. 4C,D). Remarkably, we observed ectopic expression of dlx5 in xArx-injected embryos. At neural plate and tube stages, dlx5 is expressed in the nasal and otic placodes and continues to be expressed in the developing forebrain and otic vesicles (Papalopulu and Kintner,1993). At neural tube stages, we observed ectopic dlx5 expression in the region between the anterior and otic dlx5 expression domains (Fig. 4F). We also observed ectopic dlx5 expression at tail bud stages (Fig. 4H). Strikingly, at these stages, we also observed circles of dlx5 staining resembling otic vesicles (Fig. 4H). These structures appeared similar to otic structures in sectioned embryos (Fig. 4J) and were positive for expression of additional otic markers, pax2 and otx1 (Fig. 4K–N). Overexpression of dlx5 results in a phenotype that is morphologically distinct from the Arx-overexpression phenotype and is not sufficient to induce the development of ectopic pax2-positive otic structures (data not shown). These observations suggest that up-regulation of dlx5 expression alone is not sufficient to induce ectopic otic structures observed in xArx overexpression.

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Figure 3. Molecular analysis of xArx-injected embryos. Expression of forebrain markers was analyzed in control or xArx-injected embryos by whole-mount in situ hybridization (WISH). Embryos were injected with xArx and lacZ RNA (A2–J2, A4–H4) or lacZ RNA alone (A1–J1, A3–H3). A1–J2: Embryos were fixed at neural tube stages (A1–H1, A2–H2) or tailbud stages (A3–H3, A4–H4, I, J), processed for β-galactosidase activity using X-gal (control embryos, turquoise color) or Red-gal (xArx-injected embryos, red color) and WISH (blue-purple color) using antisense riboprobes for otx2 (A), pax6 (B), Rx2A (C), FoxG1 (D), emx1 (E), lhx2 (F), Nkx2.1 (G), vax2 (H), lhx1 (I), or lhx9 (J). Anterior views are shown in A1–H1, A2–H2, D3, and D4. Lateral views are shown in A3, B3, F3–H3, J1, A4, B4, F4–H4, and J2. Dorsal views are shown in C3, E3, I1, C4, E4, and I2.

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To further analyze the involvement of xArx in forebrain development, we set out to knock down xArx expression using antisense morpholino oligonucleotides (ASMO). We verified the specificity of the xArx ASMO by testing its effects on xArx translation in vitro and in vivo. We found that the xArx but not the control ASMO specifically inhibited in vitro translation of xArx (not shown). To test the specificity of the xArx ASMO in vivo, we coinjected 10 nl of a 0.025 mM xArx ASMO solution with plasmids encoding either a xArx–green fluorescent protein (GFP) fusion or a control GFP plasmid under the control of the CMV promoter. RNA encoding the lineage tracer dsRedExpress was also included. We observed that the xArx ASMO inhibited expression of the xArx-GFP fusion but not the control GFP (Fig. 5B,D). The control ASMO did not inhibit expression of either GFP construct (Fig. 5A,C). All injected embryos expressed dsRedExpress, indicating they had been successfully injected (Fig. 5A′–D′). These results led us to conclude that the xArx was capable of specifically inhibiting expression of xArx. Injection of 10 nl/blastomere of 0.025 mM xArx ASMO resulted in a decrease in forebrain and eye size in approximately 50% of injected embryos (17 of 35), whereas injection of a similar dose of a control ASMO had no apparent effect on forebrain development (Fig. 5E–G). Injection of lower doses of the control or xArx ASMO did not affect development; injection of higher doses of the control MO resulted in abnormal development (not shown), again suggesting the effects of the xArx ASMO were specific. XArx ASMO-injected embryos exhibited reduced expression of FoxG1, Nkx2.1, and pax6 (Fig. 5H–P). The FoxG1 expression domain was generally reduced, either in the anteroposterior (Fig. 5J) or dorsoventral dimension (Fig. 5I). The ventral telencephalon expression of Nkx2.1 was reduced or absent (Fig. 5L,M). Pax6 expression was also generally reduced, most notably in the developing retina (Fig. 5O,P). The effects of xArx ASMO injection on head morphology and marker gene expression suggest that xArx plays an essential role in forebrain specification and development.

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Figure 5. Morphological and molecular analysis of embryos injected with xArx antisense morpholino oligonucleotides (ASMO). A–D: The xArx ASMO specifically inhibits translation of an xArx–green fluorescent protein fusion (Arx-GFP) but not GFP lacking the xArx ASMO target (myc-GFP) fusion. Embryos were injected with control (CO, A,C) or xARX ASMO (ARX, B,D), CMV::myc-GFP (A,B) or CMV::xArx-GFP (C,D) plasmid DNA and dsRedExpress RNA. Embryos were cultured to midneurula stage and analyzed for GFP fluorescence (A–D) or dsRedExpress fluorescence (A′–D′). E–G: Phenotype of embryos injected with control (CON) or xArx (ARX) ASMO. H–P: Whole-mount in situ hybridization of embryos injected with control (H,K,N) or xArx (I,J,L,M,O,P) ASMO using antisense probes for FoxG1 (E–G), Nkx2.1 (H–J), or pax6 (K–M).

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In hopes of creating an antimorphic form of xArx, we prepared fusions of the xArx coding region with either the engrailed repression domain or the VP16 transcriptional activation domain (Fig. 6). We found that overexpression of xArx-enR essentially phenocopied overexpression of xArx, including anterior truncation, shortening and thickening of the brain, and abnormalities in eye development (Fig. 6C), consistent with the prediction that Arx proteins function as transcriptional repressors (Stromme et al.,2002). Surprisingly, overexpression of xArx-VP16 resulted in a rostral expansion of the forebrain (Fig. 6D). We verified that xArx-VP16 fusion acts as a transcriptional activator in a GAL4-UAS–based gene reporter system (data not shown). This phenotype was different from that of ASMO-injected embryos (Fig. 5) and reported mouse Arx mutant phenotypes (Kitamura et al.,2002; Collombat et al.,2003), suggesting that xArx-VP16 does not represent a true antimorphic form of Arx.

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Figure 6. Morphological and molecular analysis of obligate transcriptional repressor and activator forms of xArx. A–D: Phenotypes of control (CON) and injected embryos. E–H: Expression of xArx and xArx-enR but not xArx-VP16 results in expansion of wnt8b expression. I–L: Injection of xArx or xArx-VP16 but not xArx-enR results in ectopic expression of dlx5 and ectopic otic structures (arrowheads). Control embryos (A,E,I) embryos injected with 100 pg/blastomere of xArx (B,F,J), xArx-enR (C,G,K), or xArx-VP16 (D,H,L). LacZ was used as a lineage tracer in E–L. A–D,I–L show lateral views, and E–H show dorsal views of control and injected tadpoles.

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We next used the xArx-enR and -VP16 fusions to probe the molecular involvement of xArx in activation of wnt8b and dlx5 expression. Embryos injected with Arx-enR RNA exhibited expanded wnt8b expression, as observed in Arx-overexpressing embryos (Fig. 6F,G). This result is consistent with the similarity in phenotype between Arx and the Arx-enR fusion and the observation the idea that Arx can function as a transcriptional repressor. Injection of xArx-VP16 resulted in diminished wnt8b expression (Fig. 6H). Remarkably, injection of xArx-VP16 RNA resulted in ectopic expression of dlx5 and the appearance of ectopic otic structures, as observed in embryos overexpressing wild-type xArx (Fig. 6L). Overexpression of xArx-enR resulted in the anterior truncation phenotype but not ectopic expression of dlx5 (Fig. 6K). These results suggest that not all the effects of Arx overexpression can be explained by repression of Arx target genes. One explanation for these results is that xArx may be a bifunctional transcription factor, capable of functioning as either an activator or repressor of transcription.

DISCUSSION

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

The results presented here indicate that Arx plays an important role in the program of forebrain development. Overexpression of Arx results in abnormal brain morphology. Most of the forebrain markers we tested were expressed in tailbud embryos, albeit in an abnormal pattern, probably due to the severe forebrain morphological abnormalities observed in these embryos. In most cases, the onset of marker expression was reduced or absent at neural tube stages. We interpret these data to suggest that forebrain patterning is delayed in xArx-injected embryos. Included in these markers is the cdk inhibitor Xic1. Xic1 expression was also reduced or absent in neural tube embryos and present in an abnormal pattern in tailbud embryos. Exit from the cell cycle is necessary for terminal cellular differentiation. More specifically, expression of cdk inhibitors is required for neural differentiation (Durand et al.,1998; Carruthers et al.,2003; Vernon et al.,2003). One interpretation of these results is that xArx overexpression leads to loss of structures expressing Xic1 and other markers absent in neural tube stage embryos. This process seems unlikely since we observed expansion of wnt8b and dlx5 expression, suggesting that at least the rostral forebrain tissues that express these genes are present, although in a developmentally compromised form. Another interpretation is that Arx may be involved in the coordination between cell proliferation and differentiation, so that Arx overexpression results in prolonged proliferation of neural ectodermal cells while maintaining their developmental potency. A similar role has been proposed for a related gene, Rx, in the development of the neural retina (Casarosa et al.,2003), also a developmental derivative of the anterior neural plate. The proposed role of Rx in neural retina development is partly based on results of experiments where Rx was over- or misexpressed later in retinal development by in vivo lipofection. Similar late-expression experiments will have to be performed to distinguish between these possibilities and better understand the role of xArx in brain development.

Is inhibition of shh expression and disruption of shh signaling sufficient to result in the xArx overexpression phenotype? Certainly, disruption of shh signaling would be expected to result in loss of midline structures. Additionally, perturbation of shh signaling has been reported to result in the development of ectopic otic structures (Koebernick et al.,2003). Furthermore, shh is necessary for the dramatic expansion of the telencephalon that occurs during forebrain development (Britto et al.,2002). Thus, it is possible that the shortened forebrain, loss of midline structures, and ectopic otic phenotypes observed in xArx-injected embryos all result from perturbation of shh signaling. Furthermore, Arx genes are expressed in the ventral hindbrain and spinal cord (Miura et al.,1997; El-Hodiri et al.,2003), overlapping a region of shh expression. Cyclops mutant zebrafish lack Arx expression in the ventral forebrain and spinal cord, while retaining expression in dorsal forebrain and somites (Miura et al.,1997). Cyclops mutants lack a floor plate and midline shh expression (Krauss et al.,1993) due to a mutation in the nodal-related gene, Ndr2 (Rebagliati et al.,1998). Taken together, these results suggest a possible relationship between shh signaling and xArx expression or function. Further investigations are necessary to determine the homeostatic relationship between Arx and shh.

Of interest, we observed that the xArx injection phenotype is strikingly similar to mammalian holoprosencephaly (HPE). It is well established that mutations resulting in disruption of shh signaling result in HPE (Wallis and Muenke,2000; Roessler and Muenke,2003). The hallmarks of HPE include a loss of anterior midline structures resulting in a fused rostral forebrain and varying degrees of synophthalmia/anophthalmia. Severe HPE phenotypes also include the formation of a proboscis, a nasal rudiment developing apparently dorsal to the eyes. Tadpoles injected with xArx RNA exhibited fused rostral forebrain and eyes and a lack of the rostral shh expression domain. Furthermore, the nasal organ of these tadpoles was also fused and displaced dorsally beyond the level of the eyes. Based on gross morphology, xArx-injected tadpoles seem to have an HPE-like phenotype. However, disruption of shh signaling and HPE are also associated with a deficiency of ventral forebrain development. In contrast, xArx overexpression resulted in ventral brain hyperplasia, accompanied by reduced expression of some ventral forebrain markers (such as Nkx2.1 and vax2) and expanded expression of others (such as Isl-1 and wnt8b), suggesting that the hyperplastic ventral brain tissue we observed in xArx-injected embryos may not have been fully differentiated. Additional histological and molecular analysis is necessary to determine the extent of similarities between the xArx overexpression phenotype and HPE.

The ectopic expression of dlx5 and development of otic structures in Arx-overexpressing embryos was an unexpected result. Both were found to depend on Arx function as a transcriptional activator. Overexpression of xArx resulted in ectopic expression of dlx5, although it is not clear if dlx5 is a direct target of Arx. Dlx5 is necessary for murine otic development (Acampora et al.,1999; Ladher et al.,2000; Merlo et al.,2002), and otic-inducing signals also induce dlx5 expression (Ladher et al.,2000). However, we found that overexpression of dlx5 was not sufficient to induce the development of ectopic otic structures. Arx expression is not detected in the developing otic vesicle, suggesting that the development of ectopic otic structures develops as an indirect result of xArx overexpression. There are several possible explanations for the ectopic otic vesicle phenotype. One possibility is that xArx overexpression results in an expansion of the otic inducing region of the embryo. Otic induction requires interactions between the hindbrain and the overlying ectoderm (Gallagher et al.,1996; Noramly and Grainger,2002). In chick embryos, this interaction is also dependent on adjacent mesoderm (Ladher et al.,2000). It is possible that the brain development abnormalities that result from xArx overexpression result in a shift or expansion of the hindbrain region or overlying ectoderm involved in otic vesicle induction. A second possibility is that precocious xArx overexpression results in activation of targets of Arx-related genes that are normally involved in otic development. Examples of paired-type homeobox genes expressed in the developing murine hindbrain include Unc4.1 (Rovescalli et al.,1996) and Dmbx (Ohtoshi et al.,2002; Stromme et al.,2002; Takahashi et al.,2002). A third possibility is that xArx overexpression alters intercellular signaling events regulating otic development, such as the shh pathway. It has been reported that shh signaling plays a complex role in regulating otic development (Koebernick et al.,2003). Remarkably, either hyperactivation or inhibition of shh signaling resulted in development of ectopic otic structures. It is interesting to note that perturbation of shh signaling resulted in ectopic dlx5 expression, limited to the ectopic otic structures. It is not clear if perturbation of shh signaling resulted in ectopic dlx5 expression in neural tube stage embryos, as we observed in xArx-injected embryos. Thus, perturbation of shh signaling may not be sufficient to explain the development of extra otic structures by overexpression of xArx.

The penetrance of the xArx ASMO injection phenotype we observed was considerably lower than that of the xArx overexpression phenotype (50% vs. 82%). This finding is probably the result of at least partial compensation by a second Arx gene. As is the case for many genes in Xenopus laevis, a second Arx gene, xArx2, has been discovered (M. Crawford, personal communication). Furthermore, xArx2 does not encode the target sequence for the xArx ASMO used in these experiments. Therefore, it is not expected that injection of xArx ASMOs would affect the expression of the xArx2 gene product. These results suggest that Xenopus forebrain development is sensitive to changes in xArx dose, because decreasing the expression of one of the Arx gene products is sufficient to induce abnormalities in forebrain development. It remains to be seen if simultaneous knockdown of both xArx and xArx2 would result in a more penetrant and/or more severe forebrain development phenotype.

Our results indicate that the hypothesized function of Arx as a transcriptional repressor may not be sufficient to explain its role in normal brain development. First, Arx expression spans multiple distinct phases of forebrain development. Arx is expressed at neural plate stages when the forebrain is being specified and patterned, at neural tube stages when the forebrain (especially the telencephalon) is rapidly expanding, and at tadpole stages when brain maturation and differentiation occurs. It seems unlikely that the same set of target genes would be repressed or activated during all of these stages. It is interesting to note that the pan-telencephalic gene FoxG1 is also thought to be a bifunctional transcription factor (Bourguignon et al.,1998). Like Arx, FoxG1 is expressed early and late in forebrain patterning and development. It is interesting to speculate that there may be a set of bifunctional transcriptional regulators, including Arx and FoxG1, that regulate different aspects of forebrain development by activating or repressing transcription of target genes.

Several mechanisms can be envisioned for activation or derepression of Arx target genes in different spatio-temporal patterns in the developing forebrain. One possibility is that Arx may function as a bifunctional transcription factor, capable of functioning as both a transcriptional repressor and activator, perhaps depending on promoter context. Alternatively, Arx repression activity itself may be regulatable. Arx encodes an octapeptide or engrailed homology motif, known to mediate transcriptional repression through recruitment of groucho/TLE-related proteins (Fisher and Caudy,1998). The activities of these proteins may be regulated by posttranslational mechanisms, by interaction with AES, a putative negative regulator of the family, or promoter context. Arx and Xenopus groucho family orthologs are expressed in overlapping domains during brain development (Fisher and Caudy,1998; Qu et al.,1999; Molenaar et al.,2000), suggesting that Arx and groucho-related proteins may interact in several possible combinations with different implications for regulation of Arx-mediated transcriptional repression. Resolution of these possibilities depends on future investigations involving Arx targets and potential interactions between Arx and groucho-related proteins. Results involving other members of the aristaless-related gene family suggest there may be additional mechanisms of regulating Arx function. For example, the conserved OAR domain, a hallmark of the aristaless-related gene family, has been implicated in mediating regulation of prx1 activity, including transcriptional repression and activation activities, by an intramolecular mechanism (Norris and Kern,2001). The OAR domain could likewise play a role in controlling the activity of Arx as a transcriptional regulator. Additionally, the activities of paired-type homeobox proteins may be modulated through interactions with other transcription factors. For example, Alx4 physically interacts with CART-1 and other paired-type homeobox proteins to regulate gene expression (Qu et al.,1999; Tucker and Wisdom,1999). Also, several paired-type homeobox proteins, including Rx, are thought to be capable of physically interacting with paired proteins such as Pax6 (Mikkola et al.,2001). Recently, it has been demonstrated that Drosophila paired-type homeobox proteins, including aristaless, physically cooperate with chip and lim homeobox gene products to regulate development of the leg (Pueyo and Couso,2004). It is interesting to note that xArx is coexpressed with several lim homeobox genes during brain development (Bachy et al.,2001). The specificity or activity of Arx and its family members as transcriptional regulators may be controlled by the formation and composition of these complexes. The applicability of such mechanisms to the regulation of Arx function will be a focus of future research.

EXPERIMENTAL PROCEDURES

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

WISH, Double-WISH, and β-Galactosidase Staining

Embryos were staged according to Nieuwkoop and Faber (1994) and fixed, dehydrated, and subjected to WISH essentially as described previously (Sive et al.,2000). Hybridization and posthybridization washes were incubated at 65°C. After color reaction using BM Purple (Roche), embryos were fixed in modified Bouin's (picric acid was omitted) for at least 2 days. Embryos were then dehydrated in 70% ethanol, rehydrated stepwise, and bleached. Embryos were then stored in PBSE (phosphate-buffered saline + 1 mM ethylenediaminetetraacetic acid). Double-WISH was performed using digoxigenin- and fluorescein-labeled probes and corresponding alkaline phosphatase–conjugated antibodies essentially as described (Sive et al.,2000) using BCIP (Roche) alone in the first color reaction and 5-bromo-4-chloro-3-indolyl sulfate (Sigma) in the second reaction. The second color reaction often took over a week to develop, and the magenta color often appeared purple until bleaching. Embryos subjected to staining for β-galactosidase activity essentially as described (Sive et al.,2000), except that fixation was reduced to 30 min in MEMPFA. Embryos were stained using X-gal or Red-gal (Research Organics).

Histology, Whole-Mount Immunostaining, and Immunofluorescence

For histology, embryos were fixed and dehydrated as described above, transferred to 70% ethanol and subjected to processing and paraffinization by the CCRI Morphology Core Facility. Embryos were mounted, sectioned (10 μM thickness), dewaxed by using Citrisolv (Fisher Scientific), and stained by using Gill's hematoxylin 3 and eosin Y.

Whole-mount immunostaining was performed by using Xen-1 antibody (Developmental Studies Hybridoma Bank, University of Iowa) as described previously (Klymkowsky and Hanken,1991), except that TBTr (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.2% bovine serum albumin, 0.1% Triton X-100) was used for all wash steps. Color was developed using diaminobenzidine (FAST DAB, Sigma). Embryos were then fixed in modified Bouin's and dehydrated as described above.

For immunofluorescence with anti–Isl-1 antibodies, embryos were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde, incubated overnight at 4°C in 25% sucrose, cryopreserved in OCT medium (Tissue-Tek), frozen and sectioned (10 μM thickness). Sections were permeabilized with 0.5% (v/v) Triton X-100, blocked in 1% albumin, and further permeabilized with 1% saponin. Permeabilized cryosections were incubated with primary antibody, washed with PBS, incubated with secondary antibody, and mounted using Vectashield (Vector Labs). Antibodies used were anti–Isl-1 antibody 39.4D5 (DSHB) and Cy3-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories).

Plasmid Construction, RNA Preparation, and Microinjection

The coding region of xArx was amplified from our original coding region clone (El-Hodiri et al.,2003) by polymerase chain reaction (PCR) using specific primers corresponding to the entire coding region. To minimize nonspecific amplification errors, we used a high fidelity polymerase (Platinum Pfx; Invitrogen) and minimized the number of amplification cycles (10 cycles), starting with 10 ng of plasmid DNA in a 50-μl reaction. After PCR, 1 U Taq (Fisher) was added to the reaction and incubated for an additional 10 min at 72°C to add T overhangs for TA cloning. The PCR product was cloned using the TopoTA system (Invitrogen) and sequenced. The Arx coding region was then liberated from the TA cloning vector by using EcoRI and inserted into pCS2 (Turner and Weintraub,1994). DNA sequencing was performed by the CCRI DNA Sequencing Core Facility.

Arx-GFP was prepared by ligating an xArx cDNA spanning nucleotides 99–1752 in frame with GFP in the SGP plasmid (pCS2 containing GFP; Rubenstein et al.,1997). The xArx region included a portion of the 5′ untranslated region, the translation start, and a portion of the coding region. The DsRedExpress coding sequence was isolated from pdsRedExpress (Clontech) and ligated with pCS2 containing three copies of the HA tag (N-3HA/pCS2+, a gift from P. McCrea), in frame with the HA tags.

Dlx5 (x-dll-3) and vax2 coding regions were amplified by high fidelity RT-PCR (Platinum Single Step RT-PCR Kit; Invitrogen) using RNA isolated from dissected heads of stage 28 embryos and primers spanning the translated region of each gene (Papalopulu and Kintner,1993; Schulte et al.,1999). Amplified cDNA fragments were gel-purified, cloned (Topo TA Cloning Kit; Invitrogen), and sequenced. After sequence confirmation, cDNA fragments were subcloned into pBlueScript II KS (Stratagene).

An expression plasmid containing the VP16 transcriptional activation domain, pCS2/VP16, was prepared by amplifying by amplifying base pairs 83–333 from pVP16 using a sense and antisense primers that included XbaI and SpeI sites, respectively. The amplified DNA was digested with XbaI and SpeI and inserted into the XbaI site of pCS2. An expression plasmid containing a portion of the engrailed repressor corresponding to amino acids 2–298, pCS2/enR was prepared similarly. Arx was inserted into these plasmids by amplifying Arx cDNA corresponding to amino acids 1– from pCS2/Arx using sense and antisense primers containing EcoRI and XbaI recognition sequences, respectively.

Capped RNA for microinjection was prepared from NsiI-linearized pCS2/Arx, pCS2/Arx-VP16, or pCS2/Arx-enR DNA templates by using the SP6 mMessage mMachine Kit (Ambion) as described previously (El-Hodiri et al.,1997). Digoxigenin- or fluorescein-labeled antisense RNA was prepared for use as probe in whole-mount in situ hybridization from DNA template linearized with an appropriate enzyme by using an appropriate bacteriophage RNA polymerase (El-Hodiri et al.,1997).

Embryos were injected with 100 pg Arx RNA + 50 pg lacZ RNA at the four-cell stage. Embryos were injected in either one or both dorsal blastomeres, near the animal pole and dorsal midline (presumptive anterior CNS). Embryos were cultured after injection at 16°C overnight and at various temperatures subsequently as described previously (El-Hodiri et al.,1997).

ASMOs

Arx and control ASMOs were obtained (Gene-Tools). The Arx ASMO, designed by the manufacturer, targeted nucleotides 108–132 of XArx, spanning the translation initiation codon. The control ASMO was designed to not target Xenopus transcripts. ASMOs were resuspended in water and diluted to working concentrations in 0.1× MMR. ASMOs were disaggregated by heating 10 min at 65°C, chilled on ice, and centrifuged briefly before injection. ASMOs were injected into the animal pole region of both blastomeres of two-celled embryos. Embryos were cultured as described above. To test specificity of the ASMOs in vivo, 10 nl of a mixture containing control or xArx ASMO (0.025 mM), xArx-GFP or SGP plasmid DNA (30 ng/μl), and dsRedExpress RNA (10 ng/μl) was injected into the animal pole region of one blastomere of a two-cell stage embryo.

Acknowledgements

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

We thank Srivamsi Nekkalapudi and Xiu-Lan Qi for technical assistance and Kay Lee for preparing the pCS2/VP16 and pCS2/enR plasmids. We also thank Christine Beattie and Katia del Rio-Tsonis for advice on anti–Isl-1 immunofluorescence and the following for providing plasmids: Jan Christian, Beatrice Durand, Steve Elledge, Rob Grainger, Milan Jamrich, Jim Jaynes, Paul Krieg, Pierre McCrea, Alicia Paulson, Anna Philpott, Sylvie Retaux, Ken Ryan, Amy Sater, Jonathon Slack, Robert Vignali, and Peter Vize. We thank Michael Crawford, Beatrice Durand, and Amy Sater for critical reading of the manuscript. We also thank Deb Stenkamp and members of the El-Hodiri, Robinson, and Herman labs, especially David Cunningham, at CCRI for helpful and critical discussions. Finally, we thank Michael Crawford and lab for sharing results before publication. The Xen-1 and 39.4D5 (anti–Isl-1) antibodies, developed by A. Ruiz i Altaba and T. Jessell (respectively), were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA).

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  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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