BDNF promotes target innervation of Xenopus mandibular trigeminal axons in vivo
- Jeffrey K Huang†1Email author,
- Karel Dorey†1, 2,
- Shoko Ishibashi1, 2 and
- Enrique Amaya1, 2Email author
© Huang et al; licensee BioMed Central Ltd. 2007
Received: 26 September 2006
Accepted: 31 May 2007
Published: 31 May 2007
Trigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland.
We found that the cement gland is enriched in BDNF mRNA transcripts compared to the other neurotrophins NT3 and NT4 during mandibular trigeminal nerve innervation. BDNF knockdown in Xenopus embryos or specifically in cement glands resulted in the failure of mandibular trigeminal axons to arborise or grow into the cement gland. BDNF expressed ectodermal grafts, when positioned in place of the cement gland, promoted local trigeminal axon arborisation in vivo.
BDNF is necessary locally to promote end stage target innervation of trigeminal axons in vivo, suggesting that BDNF functions as a short-range signal that stimulates mandibular trigeminal axon arborisation and growth into the cement gland.
Peripheral axon targeting comprises at least 2 morphologically distinct growth states: directional elongation, followed by terminal arborisation at the targets . Target derived diffusible factors are known to control the outgrowth and branching of growing axons . In Xenopus, the mandibular trigeminal nerve extends as fasciculated neurites for a visibly long distance toward the anterior facial epithelium, where in the vicinity of the cement gland, trigeminal axons turn ventrally, arborise and grow into the ventral posterior domain of the cement gland . The cement gland is a transient embryonic tissue, made up of highly pigmented, mucous secreting cells. These cells transmit mechanosensory information via pressure sensitive receptors in trigeminal axon terminals to activate tonic inhibition response in the swimming tadpole [4, 5]. Honore and Brivanlou have previously demonstrated that the surgical deletion of cement glands from Xenopus embryos resulted in mandibular trigeminal nerve targeting error, and proposed that a cement gland-derived chemoattractive signal operates from a short distance to control the branching and growth of mandibular trigeminal axons to the cement gland .
It is well known in co-culture experiments that directed outgrowth of trigeminal axons could be stimulated by tissue derived chemoattractants, termed Maxillary Factor from target maxillary/mandibular tissues . It has since been demonstrated that Maxillary Factor comprises the neurotrophins BDNF and NT3 . Neurotrophins are a family of secreted ligands, including NGF, BDNF, NT3 and NT4 that bind to designate Trk receptors in the nervous system to promote neuronal survival [9, 10]. Previous studies showed that mice deficient in BDNF or NT3 displayed a profound loss of sensory neuron populations in the trigeminal ganglia and spinal cord, and die shortly after birth [9–12]. Furthermore, these mice did not exhibit any defect in the projection of trigeminal axons . However, it remained unclear whether target-derived neurotrophins might function locally to promote the end stage targeting of trigeminal axons in vivo. In in vitro growth cone turning assays, growth cones from isolated spinal cord neurons could orient toward a directional gradient of neurotrophins emanating from a micropipette . Moreover, regional overexpression of neurotrophins in the Xenopus central nervous system could promote localised growth and branching from neurotrophin responsive axons . Additionally, neurotrophins when applied ectopically in mammalian slice cultures can promote sensory axon outgrowth from the spinal cord into the periphery . Since the cement gland is a well-defined target for mandibular trigeminal axons, Xenopus might provide a useful model for the analysis of neurotrophin function in axon-target interaction in vivo. Recently it has been demonstrated that TrkB, the receptor for BDNF and NT3 is highly expressed by trigeminal and spinal sensory neurons in Xenopus embryos, which suggests that neurotrophins might play a role in peripheral sensory axon development . Here we determine whether the Xenopus cement gland expresses neurotrophins, and focus on the role of BDNF during mandibular trigeminal axon target innervation in vivo.
Expression of BDNF in the Xenopuscement gland during mandibular trigeminal axon development
Phenotype of Xenopusembryos after BDNF knockdown
First, we tested whether MO BDNFatg inhibited translation of an HA-tagged XtBDNF construct in X. laevis embryos. The HA tag was added to the C-terminus of XtBDNF (BDNF-HA), and the region surrounding the start of translation was not altered. When the BDNF-HA RNA was injected alone in X. laevis embryos, a specific band at 34 kDa was detected by Western blot analysis (Figure 3B, arrowhead). However, the expression of BDNF-HA was completely inhibited by co-injection of MO BDNFatg. As control, we used an oligonucleotide designed against the X. tropicalis Sprouty2 gene, containing 4 mismatch nucleotides (MOC), which has previously been shown not to affect Xenopus development . We found that MOC had no effect on the translational efficiency of the HA-tagged BDNF construct (Figure 3B). This showed that the BDNF MO was highly effective in knocking down BDNF translation.
Additional File 1: A movie showing uninjected X. tropicalis embryos (St.32) exhibiting normal mechanosensory response. (MOV 940 KB)
Additional File 2: A movie showing control morpholino injected X. tropicalis embryos (St.32) exhibiting normal mechanosensory response. (MOV 806 KB)
Additional File 3: A movie showing BDNF morpholino injected X. tropicalis embryos (St.32) exhibiting paralysis. (MOV 761 KB)
Analysis of mandibular trigeminal axon targeting after BDNF knockdown
Analysis of mandibular trigeminal axon arborisation after in vivocement gland swaps.
MOC CG/WT Emb
WT CG/MOC Emb
MO BDNFatg CG/WT Emb
WT CG/MO BDNFatg Emb
Local BDNF expression promotes mandibular trigeminal axon arborisation in vivo
Xenopus mandibular trigeminal nerve as a model for in vivoaxon-target interaction studies
Previous in vivo studies in mice determined BDNF as a trophic factor to promote sensory axon development and survival. But because BDNF null mice die soon after birth, it has not been possible to show in vivo whether BDNF functioned as a short range signal to stimulate axon target innervation [8–10]. There are several advantages in using Xenopus to study the roles of BDNF on axon targeting. The Xenopus mandibular trigeminal nerve and its target, the cement gland, provide a simple and useful in vivo system to examine the molecular basis of axon-target interaction in vertebrates. Xenopus is an excellent model for embryological studies due to the advantage of its rapid external development, and ability for micromanipulations, grafting and explant cultures. Additionally, it is amenable to genetic manipulations by germ-line transgenesis , as well as effective antisense morpholino oligonucleotide analysis [23, 24]. By using these techniques, we showed that BDNF expression in the Xenopus cement gland functioned to locally promote end stage target innervation of mandibular trigeminal axons in vivo. Furthermore, because the developmental staging in Xenopus embryos is well characterised, we were able to precisely separate the effect of BDNF on target innervation from its effect on neuronal survival by analyzing distinct developmental time points.
BDNF is a short-range signal to locally stimulate mandibular trigeminal axon arborisation and target innervation
We found that BDNF knockdown did not affect the overall projection of mandibular trigeminal axons, but prevented their arborised growth into the target cement gland. Our data suggest that BDNF expressed at and near the cement gland functions to promote changes in axonal morphology necessary for trigeminal axon arborisation and growth into the cement gland. In the absence of BDNF, trigeminal neurons fail to arborise and enter the cement gland. By performing cement gland swapping experiments between control and experimental knockdown embryos, we were able to show that BDNF expression is required locally within the cement gland for terminal arborisation of the trigeminal nerves into the cement gland. Finally, transplantation of tissue overexpressing BDNF near the trigeminal nerve results in excessive branching and growth of the nerves into the BDNF expressing transplant, suggesting that BDNF is an important component of the local trigeminal nerve targeting signal, proposed by Honore and Brivanlou . This finding is consistent with the findings of Nosrat and colleagues  and Albers and colleagues , who have shown that ectopic overexpression of BDNF in mice can lead to aberrant branching and sprouting of axons at the mammalian tongue.
In Xenopus embryos, trigeminal and spinal sensory neurons express TrkB during development . BDNF is known to bind to the receptor tyrosine kinase TrkB, which activates several downstream signalling cascades, including the Ras-Mek-Erk and the PI3K/AKT pathways [27, 28]. Activation of TrkB signalling might trigger local changes in actin cytoskeleton polymerisation within growth cones to promote arborisation of trigeminal axons . BDNF can also bind to the low-affinity neurotrophin receptor p75NTR [30, 31]. p75NTR plays a very important role in sensory neuron survival, outgrowth and innervation [32, 33], and can function in conjunction with or independently of Trk receptors [31, 34]. It is thought that the specificity of TrkB activation by BDNF increases in the presence of p75NTR . It is possible that at the cement gland, BDNF binds to both TrkB and p75NTR on the growth cone to promote mandibular trigeminal axon innervation and survival.
Possible involvement of other neurotrophins during mandibular trigeminal axon outgrowth
Additional File 5: A movie showing NT3 morpholino injected X. tropicalis embryos (St.32) exhibiting normal mechanosensory response. (MOV 1008 KB)
BDNF mediated target innervation might be necessary for mechanosensory function
We also found that all of the BDNF morphants appeared paralysed. This might be due to the loss of BDNF responsive neurons within the trigeminal ganglia and spinal cord, since BDNF expression is necessary for sensory neuron survival and maintenance [9, 10]. Neurotrophins and their receptors have been suggested to play a role in the development of cutaneous mechanoreceptors . Recently, BDNF expression in mouse was shown to regulate the development of slowly adapting mechanoreceptors in cutaneous sensory neurons, independently from its role in promoting cell survival . It is possible that local BDNF expression might provide a signal to stimulate mechanoreceptor differentiation in Xenopus sensory axons, but this remains to be determined.
BDNF is expressed in the cement gland during mandibular trigeminal axon development, and is required to directly stimulate the end stage targeting of Xenopus mandibular trigeminal axons in vivo. The disruption of BDNF signalling resulted in mandibular trigeminal axon targeting failure by preventing their arborisation and growth into the cement gland. BDNF's role in end stage axon targeting appeared to be independent from its role in cell survival.
Total RNA was extracted from embryos using TRIzol according to manufacturer's instructions (Invitrogen). Ten cement glands gave similar total RNA concentration as one whole embryo. After reverse transcription using Superscript II (Invitrogen), cDNAs were subjected to PCR using the following primers in 5' to 3' direction: BDNF forward: CAA GTA CCT TTG GAG CCA CC, BDNF reverse: CTA TCC ATG GTG AAA GCC CGC ACG; NT3 forward: ATG TGT CTA TTC TTA TCC ACC GCC, NT3 reverse: CAT CAA AGC ACC ATA CCA AAG CC; NT4 forward: CAT TGC TTT TTG TCT ACA CCT CGG, NT4 reverse: TCA TAC TGT TGT GCC ATC TGC; ODC . Samples were taken after 29 cycles of amplification for BDNF, NT3 and NT4 in the linear range of amplification.
Whole-mount in situhybridisation and immunohistochemistry
In situ hybridisation was performed following the protocol of Harland . Antisense probes were generated from pX112-13 containing partial Xenopus BDNF cDNA (S. Cohen-Cory, UC Irvine). Immunostaining was performed as previous described . Briefly, embryos were fixed in 4% paraformaldehyde and stored in methanol at -20°C until use. The embryos were then rehydrated and washed twice with BBT (1%BSA, 0.1%Tx-100 in 1 × PBS) for 1 hour, once with BBT+5% heat treated lamb serum for 1 hour, and then incubated overnight with mouse monoclonal antibodies against β-tubulin isotype I+II (Product No. T8535, Sigma) for peroxidase detection, or anti-acetylated α-tubulin (Product No. T-6793, Sigma) for immunoflourescence detection. The embryos were then washed four times with BBT for 1 hour, once with BBT+5% heat treated lamb serum for 1 hour, and then incubated overnight with secondary anti-mouse antibodies conjugated to HRP (Molecular Probes) or to Alexa Fluor 568 (Molecular Probes). Embryos then were washed once with BBT for 1 hour and 3 times with PBT (0.1%Tween in 1 × PBS) for 1 hour, then with PBT overnight. Then the embryos were transferred to methanol before peroxidase detection by ImmnunoHisto Peroxidase Detection kit (Pierce), or scanning confocal microscopy analysis. For viewing, the embryos were cleared in Murray's clearing solution (2:1, Benzyl Benzoate:Benzyl alcohol).
Antisense morpholino oligonucleotides (MOs) were purchased from Gene Tools LLC. Genomic sequence encompassing the start ATG region of X. tropicalis BDNF was obtained from Ensembl : 5'-ATGGTCATCACTCTTCTCACCTGA-3'. As a control, a morpholino designed against the ATG of Xt Sprouty 2 containing 4 mismatch was used: 5'-GCCTTTTAGTACTCTCGTGTCCTTC-3' . X. tropicalis embryos were injected with 5–8 ng total MOs along with Micro-ruby fluorescent dextran (Invitrogen) at the one cell stage. X. laevis embryos were injected with 20 ng total MOs together with in vitro transcribed GFP mRNA into both blastomeres at the 2 cell stage. Embryos that were positive for fluorescence were further analysed for trigeminal axon targeting by whole mount immunostaining.
pCS2 5'UTR+XtBDNF was generated by PCR on genomic DNA using the following primers 5' CTG GAT CCA GAT GTT CCT AAT TCC TGT 3' and 5' GAC CAT TAA AAG GGG AAG ATA TCC ATA CGA TGT TCC AGA TTA CGC TTG AGA ATT CAG 3'. After digestion with EcoRI and BamHI, the PCR product was subcloned into pCS2 and verified by sequencing. Synthetic mRNA (250 pg) derived from this plasmid was injected in 1-cell stage X. laevis embryos alone or with 20 ng of MO BDNFatg or MOC. Embryos were harvested at St 12 and homogenized in Lysate Buffer (150 mM NaCl, 20 mM Tris pH7.5, 5 mM EDTA/EGTA and Proteases Inhibitors (Roche)). The equivalent of one embryo was loaded and fractionated on an SDS-PAGE gel. After electrophoresis, proteins were transferred to PDVF membrane (Millipore) and membranes were probed with anti-HA HRP conjugated (clone 3F10, 1667475, Roche) followed by ECL (Amersham).
Whole mount TUNEL staining
Fixed embryos were washed twice with PBTw (0.2% in 1 × PBS) and twice with 1 × PBS. For end labelling, the embryos were washed with terminal deoxynucleotidyl transferase (TdT) buffer (GibcoBRL) for 30 minutes and then incubated with 0.5 μM digoxigenin-UTP and 150 U/ml TdT in TdT buffer overnight. The embryos were then washed twice with 1 × PBS containing 1 mM EDTA for 1 hour at 65°C, then four times with 1 × PBS for 1 hour at room temperature. For detection and chromogenic reaction, embryos were washed with PBT, blocked with PBT+20% goat serum, and then incubated overnight with anti-digoxigenin antibody coupled to alkaline phosphatase in PBT+20% goat serum. The embryos were then washed extensively before performing an alkaline phosphatase reaction.
Human placental alkaline phosphatase (PLAP) cDNA was removed from RISAP vector (C. Cepko, Harvard) by SalI and SpeI and subcloned into XhoI and XbaI site of pCS2+ plasmid to generate CS2-PLAP. CMV promoter was then replaced by the N-β-tubulin (NBT) promoter by SalI and HindIII ligation to generate N-PLAP. Generation of transgenic X. tropicalis embryos by restriction enzyme mediated integration (REMI) was carried out according to Kroll and Amaya , with the following modifications; 8 × 105 nuclei of a reaction was diluted in 130 μl of MOH, and injected into eggs using nuclear transplantation needles with an inner diameter between 40–60 μm and a flow rate of 0.2 μl/min; injection buffer was 0.1 × MMR in 6% Ficoll. In each reaction, 100 ng of NotI linearised DNA was used. For generation of the N-PLAP line, REMI transgenesis was performed on, using linearised N-PLAP and γ-crystallin-dsRed, which was modified from? γ-crystallin-GFP . Embryos were grown to the larval stage (St. 47) and assessed for transgenesis by RFP expression in the lens. Those with RFP expression were grown until adulthood. For morpholino experiments, sibling matings between transgenic male and female frogs were performed, and the fertilised embryos were used for injection. We found at least 65% of the F1 embryos expressed PLAP.
Whole mount alkaline phosphatase reaction
Xenopus embryos were fixed with 4% paraformaldehyde in 1 × PBS for 1 hour at room temperature, washed 3 times in 1 × PBS, then endogenous alkaline phosphatase was inactivated by incubation at 65°C for up to 1 hour in 1 × PBS. The embryos were then incubated in AP buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 15 minutes followed by AP reaction (NBT/BCIP tablet (Roche)) for 15–30 minutes in the dark. Once staining was detected, the embryos were fixed in MEMFA for 1 hour, and transferred to methanol for storage. For whole mount imaging, embryos were bleached in 1%H2O2, 5% Formamide and 0.5 × SSC, transferred to methanol, then viewed with Murray's clearing solution (2:1, Benzyl Benzoate:Benzyl alcohol).
In vivocement gland substitution
Capped mRNAs were synthesized using the mMessage mMachine kit (Ambion) for the following cDNAs: mouse BDNF from BDNF-pGem4Z (K. Albers, U Pittsburgh) and GFP from pCXGFP3. Embryos were injected with approximately 250 pg RNAs in the animal pole of both blastomeres at the 2 cells stage according to established protocols. Developmental staging was assessed as described (Nieuwkoop and Faber, 1967). Cement gland deletion and substitution assay were performed as described with modifications . All embryos were transferred to 0.4 × MMR saline solution containing 2% Ficoll for microdissection. Injected embryos at blastula stage (St. 8) and uninjected embryos at neurula stage (St. 18/19) were placed adjacent to one another in small chambers made in agarose-plated petri dishes to keep the embryos in place. Animal caps were dissected from blastula embryos, followed by the removal of the pigmented cement gland from neurula embryos using fine forceps. The caps were then trimmed and fitted onto the cement gland-null neurula embryos. These embryos were kept undisturbed for at least 3 hours following microsurgery to heal, and then transferred to Petri dishes containing 0.1 × MMR until late tailbud stage.
In vivocement gland swaps
Antisense morpholino oligonucleotides (20 pg MO BDNFatg or MOC) together with 250 pg mRNA encoding GFP were injected in the animal pole of both blastomeres at the 2 cells stage in X. laevis according to established protocols. At the same time, uninjected embryos from the same fertilisation were raised in separate dishes containing 0.1 × MMR. Developmental staging was assessed as described (Nieuwkoop and Faber, 1967). MO injected embryos were confirmed by the detection of GFP fluorescence before the in vivo swap experiments. All embryos were transferred to 0.4 × MMR saline solution containing 2% Ficoll for microdissection. MO injected embryos and uninjected embryos at neurula stage (St. 18/19) were placed adjacent to one another in small chambers made in agarose-plated petri dishes to keep the embryos in place. Pigmented cement glands were dissected from both embryos using fine forceps and exchanged with each other. The embryos were then kept undisturbed for at least 3 hours following microsurgery to heal, and then transferred to Petri dishes containing 0.1 × MMR until St.29/30 before fixation and analysis.
We thank C. Cepko (Harvard), K. Albers (U Pittsburg), and S. Cohen-Cory (UC Irvine) for reagents. This work was supported by the National Institutes of Health (RR13221) and a Wellcome Trust Senior Research Fellowship to E.A.
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