Expression of doublecortin (DCX) and doublecortin-like kinase (DCLK) within the developing chick brain

Authors


Abstract

Doublecortin (DCX) is a microtubule-associated protein widely expressed in the developing mammalian nervous system and important for neuronal migration. DCX is known to belong to a novel protein family defined by sequence homology and the presence of a conserved microtubule-binding domain, but the functions of other members of this family are still undefined. In this study, we describe the cloning of the chick ortholog of doublecortin-like kinase (DCLK), a member of this family, and assess the expression of DCX and DCLK in the layered regions of the developing chick brain. DCX and DCLK are widely expressed in pallial and subpallial structures, including the telencephalon, optic tectum, and cerebellum, in similar distribution patterns. In addition to their expression in migrating cells, both proteins were also detected in the ventricular zone and in postmigratory Purkinje cells. Finally, DCX and DCLK were found to be coexpressed in all areas examined. In postmigratory Purkinje cells, DCX and DCLK both colocalized to the cell membrane, although DCLK was also distributed more generally throughout the cell soma. These data are consistent with multiple roles for DCX and DCLK in the developing chicken brain and suggest that the chick cerebellum will be an intriguing system to explore the effects of DCX and DCLK on postmigratory neuronal function. Developmental Dynamics 232:457–467, 2005. © 2004 Wiley-Liss, Inc.

INTRODUCTION

Doublecortin (DCX) is a microtubule-associated protein that is widely expressed in the developing mammalian nervous system. The human DCX gene was discovered through its association with a neuronal migration disorder known as lissencephaly (des Portes et al.,1998; Gleeson et al.,1998). Mutations in this X-linked gene are responsible for approximately 20% of clinical cases (Dobyns et al.,1996) and cause a loss of function of the DCX protein, resulting in abnormal migration of affected neurons from the ventricular zone to the developing cortical plate (Feng and Walsh,2001). Other genes associated with lissencephaly include LIS1 and Reelin (Kato and Dobyns,2003). Males carrying a single copy of the mutated DCX gene develop full-blown lissencephaly with a severe reduction in cortical thickness that results in mental retardation and intractable epilepsy. Heterozygotic females develop a milder heterotopia, with some neurons migrating to the cortical plate and others halting below this region to give the appearance of a “double cortex,” hence, the name doublecortin (Gleeson,2000).

The DCX protein belongs to a newly discovered protein family, more members of which are being identified. The DCX family is defined by sequence homology and, in most cases, by the presence of a unique microtubule-binding domain that stabilizes microtubules and causes bundling (Horesh et al.,1999; Sapir et al.,2000; Taylor et al.,2000). Other members of this family include a protein with over 70% homology to DCX named doublecortin and Ca/CaM-dependent protein kinase-like protein 1 (DCAMKL1) or KIAA0369 (in humans) and doublecortin-like kinase, DCLK or DCK1 (in other species; Omori et al.,1998; Sossey-Aloui and Srivastava,1999; Matsumoto et al.,1999; Shang et al.,2003). Although DCLK exists in various alternately spliced isoforms, the full-length protein has active kinase activity and is present in the developing mammalian nervous system in the same regions as DCX, suggesting that these proteins are coexpressed and that they may either compete or act in concert in binding to microtubules (Mizuguchi et al.,1999; Vreugdenhil et al.,2001; Burgess and Reiner,2002). DCX is expressed predominantly in migrating neurons within the developing mammalian brain (Francis et al.,1999; Gleeson et al.,1999) and is frequently used as a marker of neuronal migration (Rao and Shetty,2004). However, several groups have also shown that DCX is expressed in differentiated neurons, suggesting a role for DCX in neuronal plasticity, axonal outgrowth, or synaptogenesis (Nacher et al.,2001; Brown et al.,2003; Yang et al.,2004).

In studying the locations and functions of DCX and DCLK, a variety of model systems can be used to focus on neuronal migration and differentiation. Some of these model systems use tissue from other species such as the chicken, particularly in xenograft studies, where neuronal migration can be readily assessed (Durbec and Rougon,2001). The chicken is useful for studying migration because certain neuronal regions, such as the olfactory bulb and cerebellum, closely resemble their mammalian equivalents during differentiation. Other structures develop differently but offer a unique insight into the development of the vertebrate brain during evolution (Medina and Reiner,2000; Aboitiz et al.,2002). We have described previously a chick ortholog of DCX that is expressed specifically in the nervous system and whose expression peaks during early neuronal development (Hannan et al.,1999). Here, we show that DCX and DCLK orthologs are both present during chick development in a variety of structures containing migrating neurons, including the forebrain, olfactory bulb, optic tectum, and cerebellum. However, both proteins are also expressed in proliferating and postmigratory cells, suggesting that DCX and DCLK play a role in other processes in addition to migration. DCLK is coexpressed in the majority of DCX-expressing cells, supporting the hypothesis that the two proteins functionally interact during neuronal development.

ABBREVIATIONS

AP alkaline phosphatase DCAMKL1 doublecortin and Ca/CaM-dependent protein kinase-like protein 1 DCK1 doublecortin kinase-1 DCLK doublecortin-like kinase DCL doublecortin-like protein DCX doublecortin DIG digoxigenin ED embryonic day PCR polymerase chain reaction RhX rhodamine-X RT-PCR reverse transcriptase-polymerase chain reaction

RESULTS

DCX and DCLK Orthologs Are Both Found in Chick

The DCX protein contains two microtubule-binding domains and a serine/proline-rich carboxyl terminal region (Sapir et al.,2000; Taylor et al.,2000) bearing phosphorylation sites for cdk5 and JNK (Tanaka et al.,2004b; Graham et al.,2004; Gdalyahu et al.,2004). Chick and human orthologs are almost 97% identical, with the most variation occurring in the C-terminal region (Hannan et al.,1999; Christiansen et al.,2001). By contrast, human DCAMKL1 has a region of DCX homology containing both microtubule-binding domains (with up to 80% similarity to human DCX), coupled to a carboxyl domain with homology to calcium/calmodulin-dependent kinases (Burgess et al.,1999; Lin et al.,2000). Various DCLK isoforms have been described in mammals, including doublecortin-like protein (DCL), CPG16, and CARP, but all have at least some DCX homology (Burgess et al.,1999; Burgess and Reiner,2002). No chick DCLK homologs have previously been described. We therefore searched for chick RNA sequence with homology to human DCAMKL1 by performing reverse transcriptase-polymerase chain reaction (RT-PCR) on total RNA derived from embryonic chick cerebellum and using degenerate primers. Through this approach, we obtained three PCR products, two with maximal homology to DCX and one with homology to DCAMKL1. The latter product was cloned successfully and shown to be a 540-bp fragment with 83% homology to human DCAMKL1 (Fig. 1A, upper panel). When translated, the amino acid sequence from this fragment displayed 93% identity to human DCAMKL1 (Fig. 1A, lower panel) and 78% identity to human DCX, comparing favorably to mouse DCLK (95% identity to human DCAMKL1, 72% to human DCX). When aligned with murine DCLK, the fragment cloned displayed 94% identity to isoforms DCL and DCLK (Burgess et al.,1999). The region cloned included sequence from two conserved microtubule binding domains.

Figure 1.

Cloning of chick doublecortin-like kinase (DCLK) and characterization of doublecortin (DCX) -specific and DCLK-specific antibodies. A: Sequence of the chick DCLK fragment cloned in this study. Upper panel, nucleotide and amino acid sequence; nucleotides are numbered on the right and amino acids on the left. Lower panel, alignment of this translated chick fragment to human DCAMKL1 (accession no. AB002367). B: Northern blot using chick DCLK as a probe. Five micrograms of total RNA from pooled mouse embryos at embryonic day (ED) 10–12 (lane 1) and from chick cerebellum at ED9 (lane 2), 14 (lane 3), and 16 (lane 4) were run on an RNA gel, transferred to nylon membrane by Northern blotting, and hybridized to the chick DNA clone sequenced above. Three mRNA species were detected in chick (but not mouse) lanes at 3.8, 4.8, and 7.2 kb. The 28S and 18S ribosomal RNA are also visible. C: Western blots comparing DCX and DCLK antibodies. Sixty micrograms of total protein from adult human brain (Medley samples, Clontech; lanes 5, 8) and from chick forebrain at ED14 (lanes 6, 9) and ED16 (lanes 7, 10) were separated on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose membranes by Western blotting, and exposed to anti-DCX or anti-DCLK antisera. The expected positions of DCX (40 kDa) and DCLK (85 kDa) proteins are shown.

To confirm its identity as chick DCLK, the 540-bp cloned fragment was used to probe Northern blots containing RNA extracted from embryonic chicken brains. Three RNA species hybridized to this probe at 3.8, 4.8, and 7.2 kb in chick total RNA samples (Fig. 1B, lanes 2–4) but not in murine samples (lane 1), showing that this probe was species-specific. This pattern differs from chick DCX, which exists as two species of 10.2 and 3.2 kb (Hannan et al.,1999) but is consistent with murine DCLK, which has five species of 1.8, 3.2, 4.4, 5.8, and 7.0 kb (Burgess et al.,1999). The 1.8- and 5.8-kb species described in mouse are thought to correspond to CARP and cpg16, isoforms that contain 3′ sequence not present in our probe. Our chick DCLK fragment was also used for in situ hybridization to assess its expression in the developing brain; expression patterns were similar to those previously published for DCX (Hannan et al.,1999; and data not shown).

Because a high degree of identity exists between chick and human proteins, we proceeded to raise antisera against human DCX and DCAMKL1 sequence, aiming to detect both human and chick homologs. Two epitopes were used for antibody synthesis from comparable regions of DCX and DCAMKL1, but with minimal sequence homology (see Experimental Procedures section). Both antisera detected specific bands on Western blots containing human and chick brain samples (Fig. 1C). The DCX antibody detected a single band at 40 kDa in the developing chick forebrain (lanes 6, 7), whereas the DCLK antibody detected a band at approximately 85 kDa (lanes 9, 10). Both results agree closely with other Western blots from mammalian species, although the level of resolution was insufficient to determine whether we were seeing DCLK-α (84.2 kDa, present in mouse) or DCLK-β (81.1 kDa, present in human; Francis et al.,1999; Gleeson et al.,1999; Burgess et al.,1999; Burgess and Reiner,2002). Several additional bands were seen with these antibodies in human brain samples (lanes 5, 8), including a common band at approximately 60 kDa, which was thought to correspond to either human DCL or proteolytic cleavage products (Burgess and Reiner,2001; Kruidering et al.,2001). However, none of these bands was seen in chicken brain samples during embryogenesis (data not shown). It should also be noted that the band at 90 kDa seen in some chick samples with DCX (Fig. 1C, lane 7) was not equivalent to chick DCLK; these bands did not overlap when Western blots were sequentially probed with both antibodies. Our results showed that chicken orthologs exist for both DCX and DCLK and confirmed that the antibodies raised against both proteins are suitable for further expression analysis in the developing chicken brain.

DCX and DCLK Are Expressed in the Ventricular Zone and Migrating Cells in the Chick Forebrain

We examined DCX and DCLK expression predominantly in the telencephalon and in layered regions of the developing brain, because these areas are particularly affected by loss of DCX function in the mammalian brain. Our terminology for various regions of the chick telencephalon is derived from Medina and Reiner (2000) and a composite figure of the relative expression of DCX and DCLK at ED12 with nomenclature for the avian brain regions is shown in Figure 2H. Commencing at embryonic day (ED) 8, at a time when the predominant DCX mRNA species has been shown to reach peak intensity (Hannan et al.,1999), the DCX protein was found in the ventricular zone of the dorsal pallium (Fig. 2A) and in the neostriatum and diencephalon (Table 1). DCX was still present in the ventricular zone at ED12 (Fig. 2C) but at a lower level, and staining declined in this region thereafter, reflecting both intensity of staining and reduced numbers (Table 1). In other pallial regions, DCX was expressed at low levels in the developing Wulst and more strongly in the dorsal ventricular ridge and regions contributing to this structure, including the neostriatum, ventral hyperstriatum, and paleostriatum (Medina and Reiner,2000). Expression appeared to peak in the latter regions at ED12, and the protein thereafter became increasingly diffuse in its distribution at the cellular level (Table 1). In other layered structures within the telencephalon, DCX could be detected in the mitral cells of the olfactory bulb and more faintly in granule cells from ED12 (Fig. 2F).

Figure 2.

Expression of doublecortin (DCX) and doublecortin-like kinase (DCLK) message and protein in the developing forebrain. A–D: DCX and DCLK protein expression within the ventricular zone and adjacent telencephalon. Coronal sections from the chick forebrain at embryonic day (ED) 8 (A,B) and 12 (C,D) were exposed to anti-DCX (A,C) or anti-DCLK (B,D) antisera and stained using an alkaline phosphatase (AP) -conjugated secondary antibody. E:DCX RNA expression within the ventricular zone of the telencephalon, as assessed by in situ hybridization. Coronal sections from the chick forebrain at ED8 were hybridized to digoxigenin (DIG) -labeled RNA transcribed from chick DCX (Hannan et al.,1999). Hybridization was detected through the addition of an AP-conjugated anti-DIG antibody. F,G: DCX and DCLK protein expression within the olfactory bulb. Sagittal sections at ED12 were exposed to antisera directed against DCX (F) or DCLK (G), followed by an AP-conjugated secondary antibody. H: Composite diagram of relative expression and distribution of DCX and DCLK in coronal section of ED12 chick brain. Nomenclature is from Medina and Reiner (2000). CP, choroid plexus; DVR, dorsal ventricular ridge; gc, granule cells; H, ventral hyperstriatum; LC, lateral cortex; mc, mitral cells; NS, neostriatum; OT, olfactory tubercle; S, septum; STR, paleostriatum; VZ, ventricular zone; W, Wulst. Scale bars = 50 μm in A–E, 100 μm in F,G.

Table 1. Expression of DCX and DCLK in the Developing Chick Forebraina
Forebrain structureDCXDCLK
ED8ED10ED12ED14ED16ED18ED8ED10ED12ED14ED16ED18
  • a

    Staining here and in subsequent tables is marked −, not detectable; +, weakly staining; ++, moderately staining; +++, strongly staining; and ++++, intensely staining. The dorsal ventricular ridge, which is marked in Figure 2, is an anatomical structure containing portions of the ventral hyperstriatum, neostriatum, and paleostriatum (Medina and Reiner,2000). DCX, doublecortin; DCLK, doublecortin-like kinase; ED, embryonic day.

Telencephalon            
 Ventricular zone+++++++++++++++
 Septum++++++++++++++++++++
 Hippocampus+++++++++++++
 Wulst+++++++++++++++++
 Hyperstriatum ventrale+++++++++++++++++++++++
 Neostriatum++++++++++++++++++++++++++++
 Paleostriatum++++++++++++++++++++++
 Lateral cortex++++++++++++
 Olfactory bulb++++++++++++++++++
 Diencephalon+++++++++++++++++++++++++

The presence of DCX in the ventricular zone was surprising, because most mammalian expression studies have failed to detect either RNA or protein in this region (Matsuo et al.,1998; Mizuguchi et al.,1999; Burgess and Reiner,2000). To confirm that our results were not due to a lack of specificity on the part of the anti-DCX antibody, we stained comparable sections with an RNA probe specific to chick DCX. In situ hybridization results (Fig. 2E) confirmed that DCX was indeed strongly expressed in the ventricular zone, at least in the developing chick telencephalon.

Chick DCLK was expressed in similar areas to DCX but with a wider distribution pattern (Table 1). The significance of this difference was uncertain, because the anti-DCLK antiserum was noticeably more sensitive than anti-DCX when used for immunohistochemistry. We found that DCLK was expressed in the ventricular zone and neostriatum from ED8 (Fig. 2B,D), as for DCX, DCLK was strongly expressed in other regions such as the Wulst, dorsal ventricular ridge, and septum, including the chick hippocampus (Table 1). In some areas, such as the olfactory bulb, expression patterns for DCX and DCLK were almost identical (Fig. 2F,G). As in all other regions of the forebrain, the level of DCLK expression became fainter and more diffuse from ED14 (data not shown).

DCX and DCLK Are Expressed in Similar Patterns in Optic Tectum and Cerebellum

DCX and DCLK were found together in the layers of the optic tectum (Table 2), classified according to the system of LaVail and Cowan (1971). Both proteins again were found in the ventricular zone (Fig. 3A–D), a result that we confirmed by in situ hybridization (data not shown). DCX and DCLK also localized to all cellular layers of the optic tectum, including layers iv (the future stratum griseum centrale) and layers vi and viii (which contribute to the adult stratum griseum et fibrosum superficiale). Little expression was initially seen in the fibrous layers, including the developing stratum album centrale (layer iii), which represents the main efferent pathway of the tectum. However, ongoing expression of both DCX and DCLK was seen in the stratum opticum, which contains the ingrowing axons of the optic tract. As in the developing forebrain, expression of DCX and DCLK peaked at ED12 and, thereafter, became more diffuse and difficult to see (data not shown), suggesting either protein down-regulation or movement from the cell soma into developing neurites.

Table 2. Expression of DCX and DCLK in the Developing Optic Tectuma
Tectal layerDCXDCLK
ED8ED10ED12ED14ED16ED18ED8ED10ED12ED14ED16ED18
  • a

    SFP, stratum fibrosum periventriculare; SGP, stratum griseum periventriculare; SAC, stratum album centrale; SGC, stratum griseum centrale; SGFS, stratum griseum et fibrosum superficiale; SO, stratum opticum. N/A, not appropriate (layer not present at this date); DCX, doublecortin; DCLK, doublecortin-like kinase; ED, embryonic day.

Ventricular zone+++++++++++++++++++++++
Layer i, later SFP++++++++
Layer ii, later SGP+++++++++++++++++++++
Layer iii, later SAC+++++++
Layer iv, later SGC+++++++++++++++++++++++++++++++
Layers v-xiv, later SGFSN/A++++++++++N/A+++++++++++
Layer xv, later SO++++++++++++++++++++++++++
Figure 3.

Expression of doublecortin (DCX) and doublecortin-like kinase (DCLK) in the optic tectum and cerebellum. A–D: DCX and DCLK protein expression within the optic tectum. Adjacent sections from the chick tectum at embryonic day (ED) 8 (A,B) and ED10 (C,D) were stained with antisera directed against DCX (A,C) or DCLK (B,D) and stained using an alkaline phosphatase (AP) -conjugated secondary antibody for the appropriate species (rabbit or sheep). Layers are identified according to the system of LaVail and Cowan (1971). E,F: DCX and DCLK protein expression within the cerebellum at ED18, with anti-DCX (E) or anti-DCLK (F) antisera stained using an AP-conjugated secondary. G–J: DCX and DCLK expression within the cerebellum at ED18, as assessed by confocal analysis. Sections were exposed concurrently to antisera directed against DCX and DCLK, and then to the appropriate species-specific secondary antibody conjugated to fluorescein isothiocyanate (FITC, green, DCX) or rhodamine-X (RhX, red, DCLK). G–I: Photomicrographs were taken using a confocal microscope with the appropriate filter sets for FITC (G) or RhX (H) and superimposed to allow analysis of both colors (I). J: Adjacent sections were also exposed to both secondary antibodies, but with no primary antibodies present, as a negative control. Arrows in I point to individual Purkinje cells. EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; PCL, Purkinje cell layer; so, stratum opticum; VZ, ventricular zone. Scale bars = 100 μm in A–D, 50 μm in E–H, in J (applies to I,J).

As for the optic tectum, DCX and DCLK were expressed very similarly in the developing chick cerebellum (Table 3), although DCLK antibody again stained more intensely in most areas examined. Both proteins were first seen in the deep cerebellar nuclei and in the Purkinje cell layer at ED12, with expression persisting in the latter to at least ED18 (Fig. 3E,F). DCX and DCLK were also strongly expressed in the molecular layer, which contains Purkinje cell dendrites and migrating granule cells. Granule cells were initially negative for DCX and DCLK while in the external granular layer but came to express both proteins during migration through the molecular layer into the internal granular layer (Fig. 3E,F). These data show that DCX and DCLK are expressed in both granule cells and Purkinje cells during migration but also that they are present in postmigratory cells, with Purkinje cells expressing both DCX and DCLK at ED18.

Table 3. Expression of DCX and DCLK in the Developing Chick Cerebelluma
Cerebellar layerDCXDCLK
ED12ED14ED16ED18ED12ED14ED16ED18
  • a

    N/A, not appropriate (structure not present at this date); N/S, not seen; ED, embryonic day; DCX doublecortin; DCLK, doublecortin-like kinase.

External granular layer++++++
Molecular layerN/AN/A++++N/AN/A+++
Purkinje cell layer++++++++++++++++++++++
Internal granular layerN/A+N/A++++
Axonal tractsN/AN/A+
Deep cerebellar nuclei++N/SN/SN/S++N/SN/SN/S

DCX and DCLK Are Coexpressed in All Areas Examined

To determine whether DCX and DCLK colocalized to the same cells as well as the same regions of the brain, we performed confocal microscopy using fluorescein isothiocyanate (FITC) -labeled and rhodamine X (RhX) -labeled secondary antibodies to detect DCX and DCLK, respectively (Fig. 3G–J). DCX and DCLK were found in the same cells, although in distinct subcellular locations, in all sections examined (from forebrain, tectum, and cerebellum) and at all embryonic stages (ED8–18, data not shown). DCX was predominantly seen near the cell membrane in association with DCLK, whereas DCLK was distributed more evenly throughout the cell cytoplasm. This finding was particularly obvious in larger cells such as postmigratory Purkinje cells (Fig. 3I, arrows). These data would suggest that DCX and DCLK have overlapping, but distinct, roles in both migratory and postmigratory neuronal cells.

DISCUSSION

DCLK Is Highly Conserved and Coexpressed With DCX During Chick Embryogenesis

We describe in this study the cloning and sequence from a chick ortholog of DCLK. The PCR fragment cloned is highly homologous to human DCAMKL1 (93% identity when translated) and to murine isoforms DCL and DCLK (94% identity). This degree of homology, a lower homology to DCX, and more than one mRNA species obtained when this fragment was used as a probe for Northern hybridization (Fig. 1A,B) all support the conclusion that this sequence comes from chick DCLK and is not derived from chick DCX. It should be noted that both DCX and DCLK are highly conserved between mammalian and avian species, suggesting that both proteins have important and possibly conserved roles in the developing nervous system.

The DCX and DCLK antibodies used in this study were shown to be appropriate for further analysis of chick embryogenesis, because they each detected a single species of the appropriate size when tested using samples from embryonic chick brain (Fig. 1C). This finding was not the case for human brain, where at least three different additional species were seen after immunoblotting with each antibody. However, as none of these bands was seen in the chick brain during embryogenesis, they can have no significance for these studies. Antibodies specific to DCX and DCLK detected them during chick embryogenesis in a wide variety of forebrain structures and in overlapping expression patterns. Expression was seen at the earliest time tested, ED8 (Fig. 2A,B), and was still present close to hatching at ED18 (Fig. 3E,F). However, staining became fainter and more diffuse in individual cell soma after ED12, suggesting either that production of these proteins was declining or that the protein had moved from the cell soma to become localized in developing neurites, as described in other studies (Francis et al.,1999; Gleeson et al.,1999; Burgess and Reiner,2000).

The overlapping distribution patterns seen for DCX and DCLK raised the possibility that the majority of neuronal cells were coexpressing these proteins. This coexpression was tested by confocal analysis and confirmed (Fig. 3G,J). DCX was predominantly expressed at the cell membrane, whereas DCLK was strongly present in the cell cytoplasm. This finding agrees with other studies, where DCX and DCLK were thought to reside in the membrane of the cell body and at the extremities of developing neurites, with DCLK expressed more strongly in the cell cytoplasm (Francis et al.,1999; Gleeson et al.,1999; Lin et al.,2000; Friocourt et al.,2003). A more recent study would suggest that DCX in the cell body interacts primarily with microtubules and microtubule-associated proteins, coupling the nucleus to the centrosome during cell migration (Tanaka et al.,2004a). However, DCX is also known to interact with cytoplasmic proteins such as the μ subunits of AP-1 and AP-2, neurofascin, and neurabin II (Friocourt et al.,2001; Kizhatil et al.,2002; Tsukada et al.,2003). DCLK is also known to interact with microtubules, and its kinase portion can be released from the microtubule into the cytoplasm by proteolytic cleavage (Burgess and Reiner,2000,2001; Lin et al.,2000). Considering that the interaction between DCX and microtubules is kinase-dependent (Schaar et al.,2004) and that DCX and DCLK are coexpressed in migrating neurons in a variety of species (Mizuguchi et al.,1999; Gleeson et al.,1999; Lin et al.,2000), it is likely that DCX and DCLK are functioning in a common, evolutionarily conserved pathway and that the localization of each protein will prove to be of relevance to their function.

DCX and DCLK Are Expressed in Pallial and Subpallial Layered Structures

The broad distribution pattern of DCX and DCLK in the forebrain suggests that these proteins are important in chick for the development of both the pallium and subpallium. These two regions are of significance when comparing the evolution of various structures in the brains of birds, reptiles, and mammals. They have been assessed and debated using embryology, topology, neurochemistry, and, more recently, through extensive gene mapping (Medina and Reiner,2000; Abiotiz et al.,2001,2002). In birds, the pallium develops into the ventral hyperstriatum, a portion of the neostriatum and the lateral cortex beyond these structures. It also gives rise to more dorsal structures such as the hippocampal complex, which is present in the septum, and the Wulst. Subpallial structures include the paleostriatum and a portion of the septum. Both DCX and DCLK are expressed in structures derived from the pallium (ventral hyperstriatum, neostriatum, septum, Wulst) and subpallium (paleostriatum). This broad expression pattern suggests that DCX and DCLK are important for the development of a wide variety of structures within the avian brain but also makes it less likely that these proteins' expression patterns will be helpful in the ongoing evolutionary debate.

In examining pallial and subpallial expression patterns, it is interesting to note that the overlap in expression of DCX and DCLK is particularly prominent in layered structures. In the olfactory bulb (Fig. 2E,F), DCX and DCLK were expressed in precisely overlapping patterns in the developing mitral layer and, more faintly, in the adjacent granule cells. This result is similar to the expression pattern seen for Reelin, another protein associated with neuronal migration (Bernier et al.,2000). It also agrees with other studies in adult mammalian cortex, where DCX has been detected in cells migrating to the olfactory bulb and in differentiated granule cells (Nacher et al.,2001). Mice heterozygous for the LIS1 gene and showing clinical signs of lissencephaly display a disordered mitral cell layer (Royal et al.,2002).

Another example of this precise colocalization comes from the optic tectum (Fig. 3A–D). Expression was primarily seen here in the cellular layers of the tectum, which include layers ii and iv (LaVail and Cowan,1971), and again resembled the expression pattern seen for Reelin (Bernier et al.,2000). Both DCX and DCLK were expressed to a lesser degree in most of the fibrous layers of the tectum but were abundant in the outermost fibrous layer of the tectum, the developing stratum opticum, which is the primary tract extending from the retina to the optic tectum. Our results are consistent with other studies showing that DCX and related proteins are expressed in specific populations in the developing mammalian retina and may play a role in retinitis pigmentosa (Sullivan et al.,1999; Lee et al.,2003). Further studies hopefully will expand on the role of DCX and DCLK in retinotectal development and their relation to other tectal markers.

DCX and DCLK Are Expressed in Early Migratory and Postmigratory Cells

DCX and DCLK were found also to colocalize in another layered structure, the chick cerebellum (Fig. 3E–J). Surprisingly, DCX and DCLK were found here not only in migrating granule cells, where DCX is well known to be a late marker of neuronal migration (Helms et al.,2001), but also in postmigratory Purkinje cells. DCX and DCLK were both strongly expressed in Purkinje cells from ED12 to ED18 and were present in the soma and the dendritic tree (Fig. 3E,F); this was particularly marked when compared with the faint staining seen in adjacent premigrating cells within the external granule layer. Migration-associated proteins such as DCX have an uncertain role in differentiated cells. Other studies of the adult mammalian telencephalon have shown that DCX and DCLK can be expressed in postmigratory neurons (Burgess and Reiner2000; Nacher et al.,2001; Yang et al.,2004) and have postulated a role for DCX in neuronal plasticity or synaptogenesis. The strong expression of both proteins in Purkinje cells would suggest that the chick cerebellum is an excellent model system for untangling the functions of DCX and other migration-associated proteins in neuronal development and plasticity (Jeffrey et al.,2003).

The presence of DCX and DCLK in the ventricular zone in both forebrain and optic tectum was unexpected; neither protein has been detected previously in the ventricular zones of mammalian brains. However, these findings agree with previous expression data showing that Reelin is also present in the ventricular zone within the developing chick forebrain (Bernier et al.,2000), particularly adjacent to the dorsal ventricular ridge, where DCX and DCLK are strongly expressed (e.g., Fig. 2A,B). The avian telencephalon is already known to develop differently from the mammalian neocortex, with neuronal migration occurring from outside to inside rather than the “inside-out” gradient seen in mammalian development (Tsai et al.,1981; Tissir et al.,2002). The presence of DCX, DCLK, and Reelin in the chick ventricular zone would support the hypothesis that migration pathways are activated earlier in birds than in other species. Exploring these pathways in an avian model may help us to further our understanding of the various pathways used in neuronal migration and the involvement of DCX-related proteins in this process.

EXPERIMENTAL PROCEDURES

Animal Handling and Preparation of Embryonic Samples

All animal procedures were carried out under approval of the Children's Medical Research Institute/Children's Hospital at Westmead Animal Care and Ethics Committee.

Tissue Fixation and Embedding

Embryos were fixed and prepared for sectioning as described (Sheppard et al.,1988). Briefly, after dissection, tissues were immersed in 5% (v/v) glacial acetic acid in absolute ethanol for 6 hr at 4°C, with changes, and then dehydrated and embedded in polyester or paraffin wax. For paraffin wax sections, tissues were prepared as described by Wilkinson and Nieto (1993). Coronal and sagittal sections described in this study were cut at 8- μm thickness.

RNA Isolation and Northern Blot Analysis

Tissues were obtained from White Leghorn × Australorp chickens (Gallus gallus), staged with reference to Hamburger and Hamilton (1951), and from ED10 mouse brain. Total RNA was isolated from a minimum of 2 g of tissue by the guanidinium thiocyanate method of Chomcyznski and Sacchi (1987).

Ten-microgram samples of total RNA were resolved on 1% agarose gels containing 4.3 M formaldehyde and transferred to Hybond-N nylon membranes (Amersham Biosciences) by Southern blotting. Radiolabeled DNA probes were prepared by removing the chick DCLK insert described below from pGEM-3Z through digestion with EcoRI and labeling the purified insert using the Gigaprime DNA random prime labeling kit (Bresatec). All probes were synthesized in a reaction mixture containing 50 μCi [α-32P]dCTP (NEN) and were passed through ProbeQuant G-50 microcolumns (Amersham Biosciences) before use to remove unincorporated nucleotides. Membranes were hybridized using 2 × 106 cpm/ml of probe, with overnight incubation at 50°C in 6× standard saline citrate (SSC, 1× SSC = 0.15 M sodium chloride, 0.015 M trisodium citrate, pH 7.0), 10× Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), and 1 μg/ml herring sperm DNA. Membranes were washed at 55°C in 2× SSC (20 min), 2× SSC/0.1% SDS (20 min) and 1× SSC/0.1% SDS (5 min). Hybridization levels were visualized using a STORM PhosphorImager and quantitated with the aid of ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).

RT-PCR and Cloning

Chick DCLK sequence was isolated by performing RT-PCR on total RNA extracted from chick cerebellar samples removed at ED14. Reverse transcription was performed using 1 μg of RNA, 20 μM primer, 1.5 mM dNTPs, 25 mM magnesium chloride, 20 mM Tris pH 8.4, 50 mM potassium chloride, 0.1 M dithiothreitol, 12 U of RNasin (Promega), and 40 U of Superscript II (Life Technologies). The primer used (F416, 5′-gactctgcattcattctcatcc-3′) was complementary to murine DCX and was known to have high homology to both DCX and DCLK. Samples were transcribed at 42°C for 40 min and then denatured at 95°C for 5 min, before being immediately chilled and used for PCR. One microliter of RT mix was used as the template for a 25-μl reaction containing 20 mM forward and reverse primers, 10 mM dNTPs, 25 mM magnesium chloride, 10 mM Tris pH 8.3, 50 mM potassium chloride, and 1.25 U of Taq polymerase. Forward (F415, 5′-ttaacctgcctcagggagtg-3′) and reverse (F416) primers were again complementary to murine DCX and were known to have high homology to both DCX and DCLK in at least two mammalian species. Samples were subjected to 40 cycles of amplification using a Robocycler (Stratagene), with each cycle comprising 1 min at 94°C, 1 min 30 sec at 56°C, and 1 min 30 sec at 72°C. The resulting PCR product was cloned into the vector pGEM-TE (Promega) according to the manufacturer's instructions and then subcloned into pGEM-3Z (Promega) by digestion with SalI and SphI.

Antibodies to Avian DCX and DCLK

Peptides for antibody production were designed from human DCX and DCLK sequence to identify comparable regions with minimal homology. In the case of DCLK, peptides were expected to identify only those isoforms containing the DCX-like domain. Amino acids are numbered according to GenPept accession nos. G2764748 (for DCX) and G4758127 (for DCLK): DCX, 153-171 SANMKAPQSLASSNSAQAR and 342-360 KDLYLPLSLDDSDSLGDSM; DCLK, 158-176 SASRAVSSLATAKGSPSEV and 343-361 KQRSSQHGGSSTSLASTKV. Peptides were synthesized by Chiron Technologies (Clayton, Victoria, Australia), linked to diphtheria toxoid and injected into sheep (DCX) or rabbit (DCLK). Conjugation of the peptides to the carrier diphtheria toxoid was by means of maleimide using cysteine residues incorporated into the synthetic peptide. N- and C-termini, thus, were exposed at the surface of the toxoid and had the flexibility to allow binding of antipeptide antibodies. Sera from two sheep and two rabbits were tested using an enzyme-linked immunosorbent assay for antibodies specific to the peptides and carrier protein. Microtiter plates were coated with saturating levels of the biotinylated form of each peptide immobilized on avidin. Antisera diluted at greater than 10,000-fold for each of the N- and C-terminal peptides for DCX and DCLK recognized the predicted protein species at 40 and 85 kDa, respectively, and did not cross-react with one another.

Gel Electrophoresis and Western Blot Analysis

Human brain samples (total brain protein Medley) were obtained from Clontech. Gel electrophoresis and Western blot analysis was carried out as previously described (Jeffrey et al.,2000).

Immunohistochemistry

Immunohistochemistry was performed as previously described (Sheppard et al.,1988), with modifications (Jeffrey et al.,2000).

In Situ Hybridization

Paraffin sections were hybridized to digoxigenin (DIG) -labeled RNA probes made using the Ampliscribe transcription kit (Epicentre Technologies, Madison, WI) and with DIG–11-UTP (Boehringer-Mannheim) according to the manufacturer's instructions. Sense and antisense riboprobes were transcribed from 300 bp of chick DCX sequence cloned into the vector pGEM-TE (Hannan et al.,1999).

Coexpression and Confocal Analysis

A confocal scanning microscope (Wild Leitz Instruments, Heidelberg, Germany) was used to analyze the intracellular locations of DCX and DCLK, by means of fluorescent label distribution (FITC, DCX; RhX, DCLK), in sections of ED18 chick cerebellum. Sections were optically sectioned in the x–y plane with minimum slice thickness of 0.5 μm with multiple scanning, and the image was stored on optical disc.

Acknowledgements

We thank Dr. P.J. Robinson for his assistance with epitope design in antibody production, Prof. P.B. Rowe and Dr. P. Ruma-Haynes for helpful discussions, and Ms. J. Meaney for immunohistochemical expertise.

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