A procephalic territory in Drosophila exhibiting similarities and dissimilarities compared to the vertebrate midbrain/hindbrain boundary region
© Zembrzycki et al. 2007
Received: 11 July 2007
Accepted: 05 November 2007
Published: 05 November 2007
In vertebrates, the primordium of the brain is subdivided by the expression of Otx genes (forebrain/anterior midbrain), Hox genes (posterior hindbrain), and the genes Pax2, Pax5 and Pax8 (intervening region). The latter includes the midbrain/hindbrain boundary (MHB), which acts as a key organizer during brain patterning. Recent studies in Drosophila revealed that orthologous sets of genes are expressed in a similar tripartite pattern in the late embryonic brain, which suggested correspondence between the Drosophila deutocerebral/tritocerebral boundary region and the vertebrate MHB. To gain more insight into the evolution of brain regions, and particularly the MHB, I examined the expression of a comprehensive array of MHB-specific gene orthologs in the procephalic neuroectoderm and in individually identified neuroblasts during early embryonic stages 8–11, at which the segmental organization of the brain is most clearly displayed.
Results and conclusion
I show that the early embryonic brain exhibits an anterior Otx/otd domain and a posterior Hox1/lab domain, but that Pax2/5/8 orthologs are not expressed in the neuroectoderm and neuroblasts of the intervening territory. Furthermore, the expression domains of Otx/otd and Gbx/unpg exhibit a small common interface within the anterior deutocerebrum. In contrast to vertebrates, Fgf8-related genes are not expressed posterior to the otd/unpg interface. However, at the otd/unpg interface the early expression of other MHB-specific genes (including btd, wg, en), and of dorsoventral patterning genes, closely resembles the situation at the vertebrate MHB. Altogether, these results suggest the existence of an ancestral territory within the primordium of the deutocerebrum and adjacent protocerebrum, which might be the evolutionary equivalent of the region of the vertebrate MHB. However, lack of expression of Pax2/5/8 and Fgf8-related genes, and significant differences in the expression onset of other key regulators at the otd/unpg interface, imply that genetic interactions crucial for the vertebrate organizer activity are absent in the early embryonic brain of Drosophila.
In vertebrates, the primordium of the brain is subdivided along the anteroposterior (AP) axis into three basic regions, reflected by the restricted expression of a highly conserved set of developmental genes before these brain regions become morphologically distinct. Otx genes are expressed in the anterior region, which comprises the forebrain and anterior midbrain, Hox genes in the posterior region comprising the hindbrain, and the genes Pax2,Pax5 and Pax8 in the intervening region. The intervening region includes the territory of the midbrain/hindbrain boundary (MHB), which encompasses the posterior part of the midbrain and rhombomere 1 of the hindbrain. The position of the MHB is controlled by the interface between the expression domains of Otx2 and Gbx2. The MHB exerts organizer properties that play an essential role in patterning the midbrain and hindbrain [1, 2]. These organizer activities are mediated by fibroblast growth factor 8 (Fgf8) and Wnt1 proteins, which are secreted from the MHB neuroectoderm. The MHB (or isthmic) organizer arises in consecutive developmental steps that are mirrored by the ordered temporal sequence of MHB-specific gene expression. Its development initiates with the formation of an Otx2/Gbx2 interface, where in a second step btd/Sp-related 1 (Bts1), Pax2, Fgf8 and Wnt1 become expressed. In a third step, in addition to the already activated genes, their downstream targets are upregulated, among which are Pax5, Pax8, En1 and En2, whose pathways are mutually dependent with respect to maintaining the boundary [1–5].
The brains of deuterostomes (for example, tunicates and vertebrates) and protostomes (for example, arthropods and annelids) both seem to contain a rostral domain specified by the Otx/otd family, and a caudal domain specified by genes of the Hox family (for example, reviewed by [6–10]). Expression of Pax2/5/8 in the intervening neck region between the Otx and Hox1 domains has been observed in vertebrates and in the closely related ascidian tunicates, suggesting that this tripartite ground pattern of the brain is conserved during evolution within the chordate lineage . Moreover, in the ascidian Ciona, the expression and activation of other crucial MHB determinants in the neck region, such as of Fgf8/17/18 and Engrailed (En) orthologs, are reminiscent of those in vertebrates [12, 13], suggesting that the conserved pattern of their expression also pre-dates the splitting of the vertebrates from the chordate lineage. However, since the expression of Pax2/5/8 and En is absent in the intervening neck of appendicularian tunicates , and the neck region of another invertebrate chordate, amphioxus, lacks expression of Pax2/5/8, En and Wnt [15–17], this has raised doubt about the existence of a MHB territory in invertebrate chordates (irrespective of whether it includes organizer properties or not) [18, 19]. On the other hand, in the late embryonic brain of Drosophila, a tripartite pattern of Otx, Pax2/5/8, and Hox1 expression has been reported, with Pax2/5/8 expression located at the interface between the domains of Otx/otd and Gbx/unpg, and coinciding with the neuromeric border between deutocerebrum and tritocerebrum. These findings have led to the hypothesis that the Drosophila deutocerebral/tritocerebral boundary region and the vertebrate MHB are corresponding structures, and that a basic tripartite regionalization of the brain was existent already in the common ancestor of the bilaterians [20, 21].
To broaden the perspective on the evolution of brain regions, and in particular the MHB, I have undertaken a comprehensive analysis of orthologous factors of vertebrate MHB-specific regulatory genes in the Drosophila early embryonic brain. Since the specification of the MHB is one of the earliest decisions in the developing vertebrate brain, taking place before and during the formation of neuroblasts, I focussed on the early period of embryonic brain development. I describe the expression of MHB-specific marker genes at a resolution of identified neuroblasts (NBs) and in relation to the segmental architecture of the brain at stages when it is most clearly displayed. Based on the expression of orthodenticle (otd (oc, Flybase)) and labial (lab), the early brain principally exhibits a tripartite pattern with an anterior otd domain, a posterior Hox (that is, lab) domain, and a territory intervening between both domains. However, the Pax2/5/8 orthologs, D-pax2 (sv, Flybase) and pox neuro, are not expressed in the neuroectoderm and brain NBs of the intervening territory. Moreover, I identified a small interface between the complementary procephalic domains of otd and unplugged (unpg) that is located within the anterior deutocerebrum, corresponding to the anterior border of the intervening zone. The expression of these and further MHB-specific genes (such as the Wnt1 ortholog wingless, the En1,2 ortholog engrailed, and the zebrafish Bts1 ortholog buttonhead), and of dorsoventral (DV) patterning genes (the Msx ortholog muscle specific homeobox (msh (Dr, FlyBase)) and the Nkx2 ortholog ventral nervous system defective) in relation to the otd/unpg interface suggests that the neuroectoderm around this interface may represent an ancestral territory, evolutionarily equivalent to the neuroectodermal region at the MHB in vertebrates. However, in this part of the early embryonic brain, the expression of other MHB-specific markers (the Fgf8-related genes, branchless, pyramus, and thisbe) exhibits profound differences compared to the embryonic MHB domain in vertebrates. This suggests that, for the initial period of neurogenesis, the expression and regulatory interactions of genes, and the accompanying functional properties of the neuroectodermal territory around this interface, have changed during evolution.
In vertebrates, the specification of the MHB is one of the earliest steps in brain development, taking place before and during the formation of NBs . Therefore, in this comparative study in Drosophila I largely focussed on the early developmental period until embryonic stage 11, throughout which the pattern of NBs in the brain and ventral nerve cord (VNC) is fully established [22, 23]. Furthermore, stage 11 represents the phylotypic stage of development  at which the segmental organization of the brain is most clearly displayed , and to which the expression patterns of MHB-specific gene orthologs can most accurately be related.
Expression of Otd and Labial regionalizes the anlagen of the embryonic brain and demarcates an intervening zone
Thus, the early embryonic Drosophila brain discloses an anterior Otd domain, a posterior Hox domain (that is, of Lab expression), and an 'intervening zone' (IZ) encompassing a fraction of deutocerebral NBs in which neither gene is expressed (Figure 1c).
Expression of Pax2/5/8 orthologous genes is missing in the pNE and NBs of the intervening zone
Taken together, Poxn and D-pax2 are not expressed in brain NBs but in certain progeny cells at later embryonic stages. Despite this lack of early Poxn and D-pax2 in NBs of the IZ, I suggest the brain anlagen to be tripartite, in the sense of consisting of three spatially distinct regions: an anterior Otd domain, a posterior Lab domain and an IZ, where, at the level of the pNE and brain NBs, neither gene is expressed. In this regard it is worth noting that the D-pax2 expressing SOPs of the hypopharyngeal organ (Dv1,3) are localized immediately ventral, and the D-pax2 expressing SOPs of the dorsal organ (Dd9,11,12,13) immediately dorsal to the NBs of the IZ (Figure 2a,b,i). However, these SOPs do not contribute to the brain. Considering these findings, I propose the IZ to encompass about eight NBs of the DC (Dd1,4,5,7,8,10, Dv7,8; Figure 2i).
Common interface of otd and unpg domains corresponds to the anterior border of the intervening zone
Taken together, procephalic Otd and unpg (mRNA) are complementarily expressed, exhibiting a small common interface at the anterior border of the IZ, which is positioned within the anterior half of the deutocerebral anlagen. These data suggest that the IZ and the anterior adjacent pNE, which are separated by the otd/unpg interface, represent an ancestral ectodermal territory, evolutionarily equivalent to the early embryonic vertebrate midbrain/hindbrain domain (including the Otx2/Gbx2 border).
Expression of other vertebrate MHB-specific orthologs in the region around the Drosophila otd/unpg interface
Early in embryonic development, the region of the vertebrate MHB is characterised by the expression of several other genes, among which are En and Bts1, as well as the secreted factors Wnt1 and Fgf8. These factors have been shown to be involved in the patterning and differentiation of the evolving structures of the midbrain and anterior hindbrain [1, 3, 4, 32]. I was interested to explore how far the expression of orthologous genes is conserved in the pNE and NBs around the otd/unpg interface in Drosophila.
Taken together, btd, en, and wg are expressed in the immediate vicinity of the otd/unpg interface, corresponding to the expression of orthologous genes at the vertebrate Otx2/Gbx2 border. However, the Fgf8-related genes pyr and bnl are not expressed in the area of the otd/unpg interface, and ths is activated only transiently at low levels and at an improper position in relation to the otd/unpg interface and other MHB-specific marker genes, indicating crucial differences to the situation in the vertebrate embryo.
Discontinuous expression of DV patterning genes msh and vnd at the otd/unpg interface
In the vertebrate neural tube, the order of expression of DV patterning genes of the Nkx and Msx gene families along the DV axis is analogous to that of the orthologs ventral nervous system defective (vnd) and muscle specific homeobox in the Drosophila neuroectoderm: Nkx/vnd are expressed in ventral regions, and Msx/msh in dorsal regions [38, 39]. Along the AP axis, Nkx2.2 and Msx3 (which presumably represents the ancestral Msx/msh gene) have been reported to be discontinuously expressed at the MHB. Nkx2.2 exhibits a gap of expression specifically at the MHB . Moreover, the anteriomost sharp limit of Msx3 abuts exactly the MHB [41, 42]. In Drosophila, the AP expression patterns of Vnd and Msh exhibit striking similarities. Until stage 11, I observed a lack of Vnd expression specifically at the AP level of the otd/unpg interface (Figure 4l,m,n). In addition, Msh, which is expressed in the dorsal NBs of the TC and DC , exhibits an anterior limit that largely coincides with the AP level of the otd/unpg interface (Figure 4j,k,n). Thus, the discontinuous expression of Vnd and Msh at the otd/unpg interface is similar to Nkx2.2 and Msx3 at the Otx2/Gbx2 border. This lends further support to the proposed ancient evolutionary origin of the pNE anteriorly and posteriorly adjacent to the otd/unpg interface.
This comprehensive expression analysis of factors orthologous to key regulatory genes of the embryonic vertebrate MHB was aimed at clarifying whether the early embryonic brain anlagen in Drosophila reveal a tripartite regionalization, contain a conserved Otx/Gbx border and include an ectodermal territory that shares similarities with the anlagen of the vertebrate MHB. I have focussed my study mainly on the early phase of embryonic brain development, because positioning and establishment of the MHB region is a very early decision in vertebrate central nervous system (CNS) development (taking place in the neuroectoderm before and during the formation of NBs). In addition, in Drosophila, the segmental organization of the brain is most clearly displayed in this phase and the examination can be done at the highest resolution, at the level of individually identifiable NBs.
Is a tripartite regionalization of the anterior CNS, based on Otx, Pax2/5/8, and Hox1 orthologous domains, conserved in bilaterians?
The situation in amphioxus and Oikopleura is comparable to the findings made in Drosophila in this study (Figure 5). The early embryonic brain can be subdivided into an anterior domain of Otx/otd expression, encompassing most of the PC and an adjacent part of the DC, and a posterior domain of Hox1/lab expression, encompassing the TC. Both domains are separated by an IZ, covering part of the deutocerebral pNE and eight deutocerebral NBs, in which neither gene is expressed. The two Pax2 genes (D-pax2 and poxn) are not expressed in the early embryonic IZ, which is a significant difference to the situation in vertebrates and ascidian tunicates (Halocynthia and Ciona). However, both Pax2 genes are expressed during later embryonic brain development , when they are also expressed in segmentally repetitive neuronal clusters in the VNC. Importantly, at that time, AP patterning (that is, segmentation) and early neurogenesis (that is, formation/specification of NBs) are already completed. Thus, the later phase of expression of both genes is presumably involved in specification/differentiation of neural progeny cells, but is not compatible with an early function in patterning or specification of the pNE or NBs at the IZ. Accordingly, no obvious brain phenotype has been observed in poxn or D-pax2 mutants . On the other hand, early expression of poxn and D-pax2 is found in progenitors of the peripheral nervous system, some of which are placed in immediate vicinity of NBs of the IZ, suggesting an early function of both genes in the development of head sensory structures. This is in accordance with findings made in the trunk, where poxn and D-pax2 are first expressed in the precursors of the developing peripheral nervous system [28, 31] (Figure 2a,d), and later on in the ventral nerve cord.
Similarly, in the neuroectoderm of another protostomia, the polychaete annelid Platynereis dumerilii, an anterior Otx domain  seems to be spatially separated from a posterior Hox1 domain (Figure 5) ; although expression of a Pax2/5/8 ortholog has been reported in the trunk nerve cord , it has not yet been described for the brain. Hemichordates, distant deuterostomes, which do not have an internalized CNS but a body-encircling basiepithelial nerve net, reveal an anterior Otx and a posterior Hox1 expression domain, comparable to the situation in chordates, Platynereis, and Drosophila (Figure 5). A Poxn ortholog has been identified in the hemichordate Saccoglossus kowalevskii, but it is distinct from the Pax2/5/8 group of genes; expression of Pax2/5/8 orthologs has not yet been characterized . Lastly, the MHB-specific marker En is expressed in the intervening region between the Otx and Hox1 domains .
Taken together, the data suggest that a tripartite ground plan characterizing the development of the chordate (and perhaps polycheate and hemichordate) brain is basically also present in the insect brain, which is in agreement with Hirth et al. . However, in the early embryonic Drosophila brain expression of Pax2/5/8 orthologs is absent in the IZ. Nevertheless, the presence of a brain region that expresses neither otd nor labial indicates that a domain regionally homologous to the vertebrate MHB domain may also exist in Drosophila (see also the following discussion). Thus, it is tempting to speculate that a tripartite ground pattern (which lacks early Pax2/5/8 expression) was acquired already in the bilaterian ancestor.
Is the Otx/Gbx border an ancestral condition in bilaterians?
In vertebrates,Otx2 and Gbx2 are among the earliest genes expressed in the nervous system [33, 54]. The establishment of a border between the complementary neuroectodermal domains of Otx2 (anterior) and Gbx2 (posterior) is the initial step in MHB development. The border, which forms due to mutual repression of these genes, is crucial for the proper positioning of the MHB [1, 2]. In the early amphioxus embryo, Gbx and Otx domains abut the cerebral vesicle and hindbrain; therefore, this border is likely to be homologous to the vertebrate MHB (although it seems unlikely that this boundary has organizer properties) . Yet there are no Gbx genes in urochordates, suggesting that they have been lost secondarily in this lineage . In hemichordates by contrast, the domains of Otx and Gbx overlap considerably, indicating that these genes do not antagonize each other . This is reminiscent of the initial phase of Otx and Gbx expression in amphioxus, when the domains of both genes slightly overlap, although they sharply abut later on .
Similar to the situation in vertebrates and amphioxus, in Drosophila a common border can be recognized between the procephalic expression domains of otd and unpg (the Gbx2 ortholog). However, expression of unpg initiates significantly later (by stage 11) than that of otd (by stage 6), different to the situation in vertebrates (Additional file 1). As shown at the level of NBs, the common border between both expression domains is positioned in the anterior DC. Thus, the otd/unpg interface is located more anterior and does not coincide with the boundary between the TC and DC as supposed previously . Nevertheless, in the late embryonic brain, there is evidence that otd and unpg negatively regulate each other, similar to the genetic interaction of their vertebrate orthologs . In this regard it is important to note that in the anterior neuroectoderm of the polychaete Platynereis, a boundary between the anterior domain of Otx expression and a posterior domain of Gbx expression has also been reported recently , supporting its ancient origin.
In summary, the border between the Otx/otd and Gbx/unpg domains found in chordates, polychaetes, and Drosophila (but not in hemichordates) suggests a homologous use of both genes in AP patterning. The Otx2/Gbx2 border in vertebrates determines the position of the MHB. Considering that the otd/unpg border can be identified in the early embryonic brain, and that the genetic interactions of otd and unpg appear to be conserved , it seems likely that at least the machinery for positioning the MHB might already be existent in Drosophila. Therefore, the border between the Otx/otd and Gbx/unpg domains may represent an ancestral condition in bilaterians.
Does the pNE surrounding the otd/unpg interface share homology with the vertebrate MHB organizer?
As shown above, the expression data for other conserved key factors, such as wg,btd, and the DV patterning genes msh and vnd, suggest that the pNE surrounding the otd/unpg interface may represent an ancestral territory that shares molecular similarities with the region around the vertebrate MHB. Important differences are that, in Drosophila, expression of Fgf8-related genes, bnl and pyr, and early expression of Pax2/5/8 is not found at the otd/unpg interface. In agreement with that, no obvious brain phenotype has been observed in bnl mutant embryos . Only ths expression is detected, but it is very faint, transient, and anterior to the otd/unpg interface. Since ths seems to be colocalized with wg expression in this region, it is unlikely to serve the same function as Fgf8 at the vertebrate MHB. These differences in the expression of Fgf8-like genes in Drosophila are most significant, as Fgf8 is the key player at the vertebrate MHB, eliciting the expression of other organizer genes and exerting most of the organizer functions .
In addition, the temporal gene expression profile of this brain territory and the vertebrate MHB domain reveals distinctions (Additional file 1); for example, unpg is expressed significantly later than otd. Altogether, the spatial and temporal dissimilarities indicate that during the early period of neurogenesis, basic regulatory interactions among these genes, crucial to exert organizer properties, do not exist or are modified. In the light of these data, it is therefore very unlikely that the pNE surrounding the otd/unpg interface represents a functional homolog of the MHB organizer. This supports the assumption (see work on Amphioxus ) that during evolution the Otx/Gbx border was established before it became equipped with organizer function. Since there is also no convincing evidence for an MHB organizer in any tunicate, it presumably first evolved in the vertebrate lineage.
Equivalents of 'hindbrain' and 'spinal cord' in Drosophila?
In the vertebrate neural tube, the border between hindbrain and spinal cord is supposed to be approximately indicated by the anterior limit of Hoxb5 expression [57–59]. This molecular regionalization may be conserved among chordates, since in the tunicates Ciona and Oikopleura the restricted expression of a Hox5 ortholog is also suggested to coincide with the anterior border of the spinal chord [14, 19, 60, 61] (Figure 5). However, recent data in vertebrates [59, 62–64] show that the Hoxb5 domain, although initially confined to the spinal cord, extends rostrally into the developing posterior hindbrain. Unlike Hoxb5, the rostral borders of Hoxb6 and Hoxb7 domains finally come to lie close to the transition between hindbrain and spinal cord , and might, therefore, be more suitable indicators to distinguish between both CNS domains. In Drosophila, the anterior border of the Scr/Hoxb5 domain maps within the maxillary neuromere, those of Antp/Hoxb6 and Ubx/Hoxb7 domains within the labial and third thoracic neuromere, respectively (Additional file 2). Assuming that, in analogy to vertebrates , the posterior border of otd expression (within the anterior DC) sets the anterior limit, the hindbrain equivalent would comprise four neuromeres, when considering the anterior border of Scr expression as its posterior limit, or, what is more likely, up to eight neuromeres, when considering the anterior Ubx expression border as the posterior limit. Interestingly, a comparable number of seven to eight rhombomeres comprises the vertebrate hindbrain . In this perspective, the CNS region posterior to the anterior limit of the Scr or Ubx domain would be equivalent to the vertebrate spinal cord.
The vertebrate hindbrain is clearly subdivided into segmental units , as opposed to the mid- and forebrain in which the underlying metamerism is unclear [40, 68, 69]. Accordingly, the segmented part of the anterior CNS is separated from the less overt segmented part by the Otx2/Gbx2 border. The situation in Drosophila exhibits similarities. Whereas the segmental characteristics of DC, TC and ventral nerve cord are obvious, they are cryptic in the PC [26, 25]. Considering the position of the otd/unpg border within the anterior DC, this border, as in vertebrates, separates the segmented part of the CNS (including the posterior DC, the TC, the gnathal, thoracic and abdominal CNS) from an anterior part (the PC) in which the metameric identity is less obvious.
Molecular characterization of the neuroectoderm and of individually identified neural stem cells in the early Drosophila embryo, indicate the existence of a non-segmental Otx/Gbx orthologous interface located within the anterior DC. Furthermore, my data support the idea that the area surrounding this interface (encompassing the anterior DC/posterior PC) may represent an ancestral territory that shares molecular similarities with the region around the vertebrate MHB. Otherwise, lack of expression of Pax2/5/8 and Fgf8-related genes, and significant differences in the expression onset of other key regulators at the otd/unpg interface, imply that genetic interactions crucial to exert vertebrate organizer activity do not exist or are modified in the early embryonic brain of Drosophila.
Materials and methods
Staging and mounting of embryos
Staging of the embryos was done according to ; additionally, the trunk NB pattern  was used as a further reference for staging. Flat preparations of the head ectoderm of stained embryos and mounting were done as described previously .
Antibodies and immunohistochemistry
Embryos were dechorionated, fixed and immunostained according to previously published protocols . The following primary antibodies were used: mouse-anti-Antennapedia (1:20; DSHB, Iowa City, IA, USA), rabbit-anti-Atonal (1:5000; A Jarman, Edinburgh, UK), anti-DIG-AP (1:1,000; Roche, Mannheim, Germany), mouse-anti-Engrailed/Invected (1:4; DSHB), mouse-anti-β-galactosidase (1:500; Promega, Madison, WI, USA), rabbit-anti-β-galactosidase (1:2,500; Cappel, Costa Mesa, CA, USA), rat-anti-Labial (F Hirth, London, UK), rabbit-anti-Muscle specific homeobox (1:500; MP Scott, Palo Alto, CA, USA), rabbit-anti-Pax2 (1:50; M Noll, Zürich, Switzerland), mouse-anti-Pox-neuro (1:100; C Dambly-Chaudiere, Montpellier, France), rabbit-anti-Sex comb reduced (1:1,000, T Kaufman, Bloomington, IN, USA), mouse-anti-Ultrabithorax (1:20; DSHB), rabbit-anti-Ventral nervous system defective (1:500; CQ Doe, Eugene, OR, USA), mouse-anti-Wingless (1:10; DSHB). The secondary antibodies (all from Dianova, Hamburg, Germany) were either biotinylated (goat anti-mouse, goat anti-rabbit) or alkaline phosphatase-conjugated (goat anti-mouse, goat anti-rabbit, goat anti-rat) and diluted 1:500.
Whole mount in situ hybridization
Dioxigenin (DIG)-labelled buttonhead RNA probe was synthesized using HindIII linearised pBKS-btd  as a template with T7 polymerase. Other DIG-labelled RNA probes were synthesized using oligonucleotide primers amplified by PCR on wild-type genomic DNA: branchless (1,209 bp; forward primer CAGAACTACAACACTTACTCCTCC, reverse primer CTCGTAGCTCGCATCTTCTAGG); pyramus (2,020 bp, forward primer GGCAATCAGAACTTTAGTAGCG, reverse primer CAGACCACCATCGTTATGATTC); thisbe (2,284 bp, forward primer GCCCAATGTCAGCCACATCGG, reverse primer GTCGAGGTGGGCAGGAACC); unplugged (908 bp, forward primer GTGTCTGCTCGGGAACA-GAAACG, reverse primer GTCCATCTCGCCGTTGTAGTTCC). In all cases, the resulting PCR fragment was used as a template. All DIG-labelled RNA probes were prepared using a DIG-RNA-labelling mix (Roche) according to the manufacturer's protocol. The hybridization on embryos was performed as described previously [74, 75].
Embryos were viewed under a Zeiss Axioplan equipped with Nomarski optics using 40×, 63× and 100× oil immersion objectives. Pictures were digitized with a CCD camera (Contron progress 3012) and different focal planes were combined using Adobe Photoshop 7.0. Semi-schematic presentations are based on camera lucida drawings.
This project was carried out in the lab of GM Technau and I am grateful for the fruitful discussions and his general support. I also thank D Volland for excellent technical assistance, GM Technau and A Rogulja-Ortmann for critical reading of the manuscript, and C Dambly-Chaudiere, CQ Doe, F Hirth, A Jarman, T Kaufman, M Noll, MP Scott and E Wimmer for providing reagents. This work was supported by grants from the Deutsche Forschungsgemeinschaft to RU and GM Technau.
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