1 Departament de Genética, Facultat de Biologia, Universitat de
Barcelona, Avinguda Diagonal, 645, E-08028, Barcelona, Spain
2 Departament de Ciéncies Médiques Bàsiques, Facultat de
Medicina, Universitat de Lleida, Avinguda Rovira Roure 44, E-25198, Lleida,
Spain
Author for correspondence (e-mail:
jordigarcia{at}ub.edu)
Accepted 24 February 2005
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SUMMARY |
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Key words: Amphioxus, Exon shuffling, Vertebrate transition, Nervous system, Neurotrophic activity
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Introduction |
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A deep understanding of the basal role of the Nt/Trk system is hampered by
the presence of multiple members in vertebrates. The study of homologous gene
families in simpler systems, devoid of high genetic redundancy, often has led
to a better understanding of complex functions in vertebrates
(Chao, 2000). Nevertheless, the
neurotrophic field currently lacks non-vertebrate representatives
(Hallböök, 1999
).
Genome sequencing projects have shown that invertebrate model systems, such as
Drosophila or C. elegans, do not possess either Nt or Trk
homologues (Adams et al., 2000
;
The C. elegans Sequencing Consortium,
1998
). The formerly claimed Drosophila Trk receptor
(Pulido et al., 1992
) has
recently been discarded as a Trk homologue, and re-named off-track
(Winberg et al., 2001
). Until
now, the closest invertebrate Trk-related molecule was the molluscan LTrk
receptor, but its extracellular domain features a non-vertebrate structure
(van Kesteren et al., 1998
).
Furthermore, the absence of Nt and Trk in the genome of the ascidian Ciona
intestinalis has recently led to the contention that Nt/Trk signalling
system may well be a vertebrate innovation
(Dehal et al., 2002
).
The origin of vertebrates dates back 530-550 million years from a
lancelet-like relative (Zimmer,
2000). Present-day lancelets (amphioxus) are in the appropriate
place to illuminate the critical transition towards vertebrate complexity.
Amphioxus (Cephalochordata) possesses a vertebrate-like body plan, but lacks
many of the complex features of vertebrates, including a complex nervous
system. Remarkably, the simplicity of the amphioxus body plan is mirrored by
the simplicity of its genome: it escaped the extensive gene duplication events
that took place coincidentally with the origins of vertebrates and the early
stages of vertebrate evolution (Furlong
and Holland, 2002
).
Whether Nt/Trk signalling mechanisms are compulsory for evolving complex
nervous systems, and whether their evolutionary appearance coincided with the
origin of vertebrates, remains a subject of debate
(Jaaro et al., 2001). We show
that Trk receptors are not a vertebrate innovation, and originated prior to
the cephalochordate/vertebrate split. We report the isolation, molecular
characterisation and expression of the single amphioxus Trk receptor,
AmphiTrk. Its proorthology to vertebrate Trk receptors is strongly supported
by both phylogenetic analysis and full-length protein domain structure. Its
genomic structure suggests that the Trk gene emergence occurred by means of
exon shuffling. We also investigate the physiological response of AmphiTrk to
vertebrate neurotrophins. Developmental expression suggests an ancestral
function for the Trk family in the formation of an ectodermal peripheral
nervous system.
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Materials and methods |
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Sequence and phylogenetic analysis
Public domain sequence tools were used to characterise the
AmphiTrk gene and the peptide domains
(http://www.cbs.dtu.dk/services/SignalP,
http://www.expasy.ch/tools).
Putative vertebrate and invertebrate orthologue sequences were retrieved from
the GeneBank database. Alignment with AmphiTrk sequence was performed using
ClustalX and bootstrapped trees were calculated by neighbour-joining method
over 1000 replicas. Cladograms were visualised using TreeView V.1.5.3. For
tyrosine kinase domain phylogenetic analysis, human and mouse ROR1 receptors
were used as outgroups.
Functional assays
To express AmphiTrk in mammalian cultured cells, the complete
coding region was cloned into a pCDNA3 vector. The chimaeric receptor
rTrkA-AmphiTrk, including an HA epitope in its 5' end, was generated by
replacement of the intracellular domain of the rat TrkA (kindly provided by M.
Zanca) with that of AmphiTrk through PCR amplification. Plasmids (3 µg)
were transiently transfected into cultured PC12 nnr5 cells
(Green et al., 1986), as
described by Egea et al. (Egea et al.,
1999
). rTrkA and pcDNA3 were used as controls. DMEM medium
containing 10% foetal calf serum (FCS) was changed 5 hours after transfection
and cultures were left overnight. Cells were then serum-deprived for 12 hours
before stimulation for 5 minutes with 10, 50 or 100 ng/ml of NGF (Sigma),
BDNF, NT3 or NT4 (Alomone Laboratories). Cultures were then rinsed with
ice-cold PBS and solubilised in lysis buffer (2% SDS, 125 mM Tris, pH 5.8).
Protein was quantified with a BioRad-DC assay system. Total cell lysates were
western blotted and immunodetected with anti-phospho-ERK or anti-phospho-Akt
(Cell Signalling Technology). To control the protein content in each lane,
membranes were stripped and re-probed with an anti-
-tubulin antibody
(Sigma). For PLC
immunoprecipitation, pc12nnr5 cells were stimulated as
above with 100 ng/ml of NGF. Total cell lysates (500 µg) were incubated
overnight with 1.5 µg of an anti-PLC
-conjugated antibody. After
immunoprecipitation with Protein G-sepharose beads, blots were immunodetected
with anti-phospho-Tyr 4G10 (Cell Signalling Technology), stripped and
re-probed with anti-PLC
(BD Transduction Laboratories) as a control for
immunoprecipitation efficiency. For neurite outgrowth assays, PC12nnr5 cells
were plated onto polyornithine- and collagen-precoated 35-mm plates (1 x
106 cells/plate). Twenty-four-hour-old cell cultures were
co-transfected with enhanced yellow fluorescent protein (EYFP) and the chimera
rTrkA/AmphiTrk, AmphiTrk, rTrkA or the pcDNA3 vector as described above.
Following overnight incubation, medium was changed and supplemented with NGF
(50 ng/ml), and then renewed after three days. After 6 days, transfected cells
were examined and those whose neurite lengths were twice the cell body
diameter were counted as differentiated.
In situ hybridisation
Ripe B. floridae adults were collected from Old Tampa Bay (FL,
USA) and induced to spawn by electric stimulation. Embryos and adults were
obtained and fixed as described in Holland and Holland
(Holland and Holland, 1993).
Sense and antisense DIG-labelled probes were generated by in vitro
transcription of the cDNA full-length coding region. Whole-mount in situ
hybridisation and subsequent sectioning was performed in accordance with
Benito-Gutiérrez et al.
(Benito-Gutiérrez et al.,
2005
).
A novel method for in situ hybridisation of adult sections was specially developed, whereby fixed adults were dehydrated through an ethanol series, cut in three pieces and then prepared for embedding in paraplast (Sigma) by xylene series; xylene:ethanol (1:1), xylene, xylene:paraplast (1:1). Blocks were solidified and settled at 4°C for 12 hours prior to sectioning. Slides were immersed in 10% HCl for 5 minutes, washed with DEPC-water and immersed in acetone for 2 minutes before drying. They were then immediately covered with 2% silane-acetone (Sigma), dried and autoclaved. Serial sections (6 µm) were deposited on silane-coated slides, and settled at 45°C for 6 hours. Tissue slides were immersed in xylene and sections were rehydrated through an ethanol series. Tissue was digested using 1 µg/ml proteinase K in PBS for 30 minutes at 37°C and the reaction stopped by immersion in 0.2% glycine. Sections were refixed in 4% PFA-PBS for 20 minutes, and then immersed in 0.1 M triethanolamine with a posterior addition of 0.25% acetic anhydride. Slides were washed with PBS and then pre-hybridised for 3 hours at 60°C in 100 µg/ml heparin, 5 x SSC, 0.1% Tween-20, 5 mM EDTA, 1 x Denhardt's. A DIG-labelled probe (100 ng/ml) in pre-hybridisation buffer was added and incubated at 60°C for 14 hours. After hybridisation, slides were washed in 50% formamide/5 x SSC/1% SDS (twice for 15 minutes each) and 50% formamide/2 x SSC/1% SDS (twice for 15 minutes each), treated with 2 mg/ml RNAseA and 100 U/ml RNAseT1 in 2 x SSC/0.1% Tween20 at 37°C for 30 minutes, and washed with 0.2 x SSC/0.1% Tween20 (twice for 20 minutes each). DIG staining was performed following supplier recommendations (Roche). Slides were refixed in 4% PFA-PBS, immersed in 0.1% sodium azide/PBS and mounted in Mowiol.
In vivo DiI labelling
Hatching neurulae, which are extremely motile, were anesthetised by placing
them in a reduced drop of seawater, causing oxygen deprivation. A glass
capillary tube was filled with Fast DiI oil (D-3899, Molecular Probes) and
immersed in seawater causing the dye to crystallise at the tip. Using a
micromanipulator the crystallised dye was applied to the ventral surface of
the immobilised hatching neurulae. Immediately after dye deposition, embryos
were transferred to seawater, regaining movement and developing normally. At
late neurula stage, embryos were fixed and photographed under a fluorescence
rhodamine filter.
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Results |
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On the cytoplasmic side, AmphiTrk includes a tyrosine kinase (TK) domain
that contains all the key residues necessary to carry out its function as a
catalytic receptor. Like its vertebrate counterparts, it contains the
signature pattern of class II tyrosine kinase receptors [DIYSTDYYR
(Fig. 1B, grey background)].
Within this short amino acid sequence, three tyrosine residues (Y676, Y680 and
Y681; Fig. 1B, black
background) constitute the putative auto-phosphorylation activation loop. A
presumptive ATP-binding region is located at the N-terminal pole of the TK
domain and contains a conserved lysine (K544,
Fig. 1B, black background)
responsible for binding ATP. In addition, two potential phosphorylation sites
for cAMP/cGMP-dependent kinase proteins, lie in positions comparable to those
in vertebrates: KIS at the juxtamembrane intracytoplasmic part preceding the
TK domain; and RKFT following the activation loop within the TK domain
(Fig. 1B, black background). In
vertebrates, the former motif is a binding site for SNT, a protein involved in
neuronal differentiation and neurite outgrowth pathways
(Peng et al., 1995). Also
present in amphioxus is the docking site for Shc, an adaptor protein which in
vertebrates activates the Ras-Raf-Erk and PI3kinase-AKT signalling pathways
involved in neuronal survival and differentiation events. It is identically
placed, preceding the tyrosine kinase domain (Y493,
Fig. 1B, black background). A
distinctive feature of AmphiTrk resides at the furthest C terminus of the
protein, outside the TK domain: a glutamine residue (Q792,
Fig. 1B, black background). It
occupies the position of a tyrosine (Y785), which in mammals serves as the
docking site for PLC
, whose transduction pathway leads to initiation
and maintenance of long-term potentiation events
(Huang and Reichardt,
2003
).
Genomic structure
The genomic structure of the AmphiTrk gene was elucidated by
screening a Branchiostoma floridae genomic library. Isolated genomic
clones yielded an overlapped region of 21.4 kb, containing the complete
transcriptional unit and flanking regions. The AmphiTrk
transcriptional unit consists of 13 coding exons. Analysis of exonic domain
distribution, and comparisons with the genomic structure of human TrkA, TrkB
and TrkC, revealed a similar, but appreciably less fragmented, structure
(Fig. 3A,B).
|
The TK domain and the intracytoplasmic C-terminus are distributed over four exons (10, 11, 12 and 13). Exons 11, 12 and 13 are indistinguishable between human and amphioxus in terms of structure and key residue distribution. However, exon 10 of AmphiTrk is split in two exons in humans (13 and 14), with a breakpoint centrally located in the ATP binding residue K544.
Although we cannot formally rule out the loss of some introns in the amphioxus lineage, it is more likely that four new introns were generated early in vertebrate evolution, because in AmphiTrk particular domains are encoded by fewer numbers of exons than occur in vertebrates. In summary, our data suggest that fragmentation occurred after the generation of a new modular protein, constructed through the shuffling of domain-encoding exons and predating the cephalochordate/vertebrate split.
Phylogenetic analysis
To establish AmphiTrk phylogenetic relationship with its vertebrate
relatives and invertebrate Trk-related receptors, three different trees were
generated. Trk receptors from rat, human and chicken were taken to avoid a
biased mammalian representation; no other vertebrate species were included
because of the incompleteness of available sequences. Drosophila Trk
and Limnaea LTrk were used as Trk-related invertebrate
representatives, as no other similar receptors have been reported in
non-vertebrate clades.
All trees had similar topology and positioned AmphiTrk, with high bootstrap values at the base of, and equally related to, all three vertebrate Trk receptors (Fig. 4). When full-length proteins were used, a clade formed by AmphiTrk and vertebrate TrkA, TrkB and TrkC excluded LTrk and DTrk (Fig. 4A). In the tree generated using only the extracellular domain, a similar topology was obtained (Fig. 4B). Although minor regions of the extracellular domains of DTrk and LTrk were conserved, these fell outside the group formed by AmphiTrk and vertebrate Trks. We also generated a tree using only the tyrosine kinase domain (Fig. 4C), wherein human and mouse ROR1 TK domains were taken as outgroups. In all analyses, AmphiTrk position was consistent with it being a direct descendant of the pre-duplicative, vertebrate-like, Trk, strengthening its condition as a primitive neurotrophic receptor.
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In addition, we studied the ability of AmphiTrk and the chimaeric rTrkA-AmphiTrk receptor to induce neurite outgrowth through NGF activation in cultured PC12 nnr5 cells (Fig. 5H,K). rTrkA-AmphiTrk was found to induce neurite outgrowth following stimulation with NGF at comparable levels with those measured in rTrkA. However, although native AmphiTrk was able to induce neurite outgrowth as well, no clear increase under NGF incubation was observed (Fig. 5L).
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Developmental expression of AmphiTrk |
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In larval stages, AmphiTrk is expressed asymmetrically in the left
dorsolateral quadrant of the developing Hatschek's pit
(Fig. 6P, arrow), a
neurosecretory structure thought to be homologous to the vertebrate
adenohypophysis (Gorbman et al.,
1999). In early larva, weak labelling is also visible at the most
anterior tip of the embryo (Fig.
6P, arrowhead). This probably corresponds to the sensory cell
clusters marking the future corpuscles of the Quatrefags, whose axonal
processes contribute to rostral nerves.
Expression in adults
AmphiTrk expression in adults was tested by in situ hybridisation
of sections (Fig. 7).
AmphiTrk transcripts were detected along the nerve cord and diffuse
labelling was still visible in the Hatschek's pit
(Fig. 7B, asterisk). The
anterior limit of AmphiTrk expression was located at the beginning of
the proper nerve cord, caudal to the Joseph cells group, and next to the
posteriormost dorsal part of the cerebral vesicle.
|
In the dorsal part of the nerve cord, only a few labelled cells were
intermittently visible on the right or left side of the central canal
(Fig. 7A,D). Such a segmented
pattern is consistent with the paired motoneurons of the dorsal compartment
(Lacalli, 2002). They are
positioned near the somitic boundaries, and as do the dorsal
AmphiTrk-expressing cells, they show a slight offset, that parallels
the left/right asymmetry of the somites. These iterative patterns through the
serial sections are concurrent with the weak labelling found in Hatschek's
pit-including sections (Fig.
7B). Large columnar cells in the dorsalmost part of the Hatschek's
pit are diffusely labelled in only the most posterior portions
(Fig. 7B, asterisk). Based on
previous examinations of this structure, AmphiTrk transcripts may
localise in the infundibulum, which is composed of axonal, endocrine and other
cell types (Gorbman et al.,
1999
).
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Discussion |
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Recent advances in comparative genomics suggest that the increase in genome
size is paralleled by a general decrease in genome compactness and an increase
in the number and size of introns (Patthy,
1999). AmphiTrk genomic structure reveals a more
compacted domain distribution when compared with that of human Trks.
Noticeably, the central core of the molecule may have been a target for the
generation of four new introns, present in humans but not in amphioxus. These
introns split domains that are coded by a single exon or by fewer exons in the
amphioxus gene. This finding suggests that exon shuffling was the crucial
mechanism involved in the generation of a ProtoTrk modular protein prior to
the cephalochordate/vertebrate split. Rearrangement of preexistent domains,
rather than creation of new ones, is a plausible mode for generating molecules
able to expand and subsequently evolve to complexity. When domains are
primordially encoded by single exons, the exchange between different genomic
regions may be facilitated. Thus, insertion of introns within single exons may
well be a secondary trait, result of the divergence from an original molecule
successfully selected by evolution.
We believe that AmphiTrk novelty resides in its new combination of protein
modules already present in lower invertebrate genomes. Certainly, LTrk
possesses leucine-rich motifs sandwiched between clustered cysteines.
Similarly, the tyrosine kinase domain itself arose before the divergence of
animals from other higher eukaryotes (King
and Carrol, 2001). Nevertheless, assembly of the central core most
probably occurred just predating the cephalochordate/vertebrate split, as the
genome of the lower chordate Ciona intestinalis does not contains a
Trk-like gene (Dehal et al.,
2002
). Conversely, we cannot totally rule out secondarily loss of
Trk in tunicates, even though the lack of Trk receptors in the nearly
completed genome of a sea urchin species
(http://sugp.caltech.edu)
strongly strengthens the argument for a nascent Trk gene close to the
cephalochordate/vertebrate split.
Acquisition of functional complexity
Neurotrophin signalling through Trk receptors elicits many biological
effects. Beyond its crucial role in neuronal survival and differentiation
during development, neurotrophin signalling is critically involved in axonal
regulation and dendritic outgrowth, synapse formation and function, and cell
migration. Among the numerous ways of controlling these processes,
differential splicing and distinct preferences for ligand binding seem to be
two decisive mechanisms (Segal,
2003).
We have no evidence for the presence of AmphiTrk splicing
variants. AmphiTrk does not possess the separated miniexon, which in
vertebrates confers a loose Trk-binding ability to the distinct neurotrophins.
Therefore, promiscuous ligand-receptor relationships may be absent in
amphioxus, or regulated by other means. The latter, however, seems unlikely as
the preduplicative nature of the amphioxus genome suggests the AmphiTrk ligand
to be a single amphioxus neurotrophin. AmphiTrk peptide characterisation
reveals all the features defining a functional vertebrate Trk receptor. The
sequence analysis was further confirmed by experiments in cultured rat cells,
where AmphiTrk activated the Ras-Raf-Erk and PI3kinase-AKT signalling pathways
in response to neurotrophins. AmphiTrk responded similarly to NGF, BDNF, NT3
and NT4. This indicates that after 500 million years of divergence, the
extracellular domain of AmphiTrk can still molecularly bind and recognise
post-duplicative vertebrate neurotrophins. Our functional data suggests that
the biological functions of AmphiTrk may include neuronal survival and
differentiation during development, as well as a likely role in axonal
elongation and dendritic outgrowth, processes that are associated with the
signalling pathways mentioned above
(Patapoutian and Reichardt,
2001). Remarkably, the inability of AmphiTrk to activate the
PLC
pathway underscores its primitive nature as a Trk receptor, because
this pathway is believed to drive the most complex responses generated by
Trk-Nt interactions in mammalians (Koponen
et al., 2004
).
The increase in the numbers of neurotrophin and Trk receptors does not
always imply the increase in nervous system complexity, as exemplified by the
lineage-specific duplications in teleost fishes
(Hallböök, 1999).
However, as we suggest here, vertebrate Trk receptors were originated from a
single ProtoTrk gene through the wide genome duplication events that
were linked to the invertebrate/vertebrate transition. Consistent with
amphioxus position in the transition from simple to complex nervous systems,
our results suggest that a ProtoTrk, AmphiTrk-like, ancestral gene
may well have provided the genetic basis for acquisition of functional
complexity. The recruitment of the PLC
pathway, the expansion of the
gene family, the appearance of alternative splicing, the co-evolution of
ligands and receptors, and the finely graded control of promiscuity may have
been instrumental in the development of the vertebrate complex nervous system
and in the acquisition of higher neuronal functions.
Evolutionary developmental insights from expression patterns
The variety of actions driven by the vertebrate Nt/Trk system are further
manifested by their complicated gene expression patterns during development
and adulthood. Furthermore, interpretation of gene knockout phenotypes in mice
is hampered by the partial overlapping and redundancy among the three Trk
receptors. The amphioxus simple nervous system offers an uncomplicated context
in which to study the expression of a single Trk receptor, and may serve to
provide insights into the basic function of this gene family in
vertebrates.
During embryogenesis, AmphiTrk expression is restricted to the
developing peripheral nervous system. Our results suggest that
AmphiTrk is involved in sensory neuronal fate commitment and
differentiation. AmphiTrk transcripts are detected earlier than
epidermal differentiated primary neurons are identified in SEM observations,
and also earlier than neurons are revealed by the pan-neural marker
AmphiElav (Satoh et al.,
2001; Mazet et al.,
2004
; Benito-Gutiérrez
et al., 2005
). As the number of AmphiElav-expressing
cells is higher than the number of positive cells shown by our whole-mount
experiments, we suspect that AmphiTrk is specifying only a subset of
the neurons later identified by AmphiElav. Conversely, the reduced
number of primary neurons shown by SEM observations suggests that
AmphiTrk also plays a role in differentiating other types of sensory
neurons, i.e. secondary neurons without an axonal process embedded in the
epidermal layer.
Interestingly, TrkB-/TrkC/
(Ntrk2/Ntrk3Mouse Genome Informatics) double-mutant mice show
severe sensory defects (Silos-Santiago et
al., 1997), and mice deprived of NGF/TrkA signalling during
embryogenesis exhibit reduced sensitivity to painful stimuli
(Fariñas, 1999
).
Neuronal losses of primary neurons in these null mice are localised in the
sensory ganglia, structures absent in amphioxus. Nevertheless, prevention of
TrkB and TrkC signalling in knockout mice for NT4, BDNF and NT3 leads to a
severe deficiency in cutaneous sensory neurons, e.g. D-hair receptors, slow
adapting mechanoreceptors and cutaneous mechanoreceptors, respectively
(Stucky et al., 1998
), all of
them secondary neurons. Considering that AmphiTrk may well function
in similar fashion to all the three vertebrate Trk receptors, these data
support the idea that AmphiTrk is expressed by both primary and
secondary neurons in the peripheral nervous system.
Some innovative vertebrate hallmarks can be correlated to the acquisition
of a complex nervous system. Neural crest and placodes are exclusive
vertebrate features clearly absent in amphioxus. It is widely accepted that
the genetic machinery of neural crests was already present in amphioxus and,
by extension, in the ancestor of vertebrates. However, the ability of neural
crest cells to migrate individually seem to have arisen during vertebrate
evolution (Trainor et al.,
2003). We have evidence to show the presence of individually
migrating cells in the amphioxus embryonic ectoderm (the first time this has
been reported), which appealingly mimics that of neural crest cells in
vertebrates. Interestingly, vertebrate Trk receptors are expressed in
migrating neural crest cells (Airaksen and
Meyer, 1996
). Moreover, many placode and neural crest derivatives
express Trk receptors in vertebrates (Baker
et al., 2002
; Huang and
Reichardt, 2001
). Among these derivatives, adenohypophysis, which
has its homologous in the amphioxus Hatscheck's pit, originates from
nonneurogenic placodes, and a wide variety of mechano- and chemosensory
structures, including the olfactory epithelium, from neurogenic placodes
(Holland and Holland, 2001
).
AmphiTrk is expressed during the formation of Hatscheck's pit
throughout larval stages, and into adulthood. Transcript locations in the
infundibulum are comparable with those of the vertebrate Trk receptors in the
anterior pituitary gland (Aguado et al.,
1998
). The corpuscles of Quatrefags are specialised organs,
presumably with sensory roles, whose structure has been likened to the
vertebrate olfactory placode. This structure, which lacks a counterpart in any
other chordate, expresses AmphiTrk for a short time during the early
larval stage.
Conspicuous labelling for AmphiTrk is evident in the adult central
nervous system. As the pathways activated by AmphiTrk seem to be
limited to survival and differentiation actions, as suggested by our
functional studies in cell cultures, we cannot discard more complex roles for
AmphiTrk in adults. Vertebrate neurotrophin signalling through Trk
receptors modulates not only dendritic growth, but also the number of synapses
as well as the efficacy of synaptic transmission
(Bibel and Barde, 2000). Thus,
AmphiTrk may have a similar role within the CNS of adults. Although
AmphiTrk is unable to drive PLC
-mediated actions, this does not prevent
AmphiTrk from having a role in regulating neurotransmitter release within the
neural tube.
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ACKNOWLEDGMENTS |
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Footnotes |
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