bmp signaling specifies the development of a large and fast cns … · 2013-06-25 · 1...
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Supplementary Information for
BMP signaling specifies the development of a large and fast CNS synapse
Le Xiao, Nicolas Michalski, Elin Kronander, Enida Gjoni, Christel Genoud, Graham Knott,
Ralf Schneggenburger
This document contains Supplementary Figures 1 - 5, and Supplementary Table 1. Two
additional Supplementary Videos in relation to Figure 5 can be found online.
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Supplementary Figure 1:
Spatio-temporal expression of potential marker genes of auditory brainstem nuclei
validates the cDNA-array based screening approach.
(a) The glycine transporter Slc6a5 was expressed strongest in MNTB, followed by LSO and
VCN, at both ages.
(b) Conversely, the vesicular glutamate transporter VGluT2 showed the opposite spatial
expression pattern (strongest in VCN, and weakest in MNTB). The opposing expression
patterns of a glycinergic and a glutamatergic marker gene are expected because VCN contains
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VGluT2-expressing excitatory neurons like bushy cells and multipolar cells, whereas MNTB
mainly consists of glycinergic projection neurons.
(c) The Ca2+-binding protein Calbindin was developmentally upregulated mainly in the
MNTB, in agreement with previous immunohistochemical studies (Friauf, E. 1993, J. Comp.
Neurol. 334, 59-74).
(d) The Ca2+-binding protein Calretinin (CR) was only weakly upregulated between the two
ages studied here, and it was downregulated in MNTB, in agreement with previous IHC
findings (Lohmann, C. & Friauf, E. 1996, J. Comp. Neurol. 367, 90-109). At P14, the
expression level of CR was highest in VCN, as expected since VCN contains some CR-
positive neurons like globular bushy cells and octopus cells (Pór, A. et al. 2005, Brain Res.
1039, 63-74; Lohmann & Friauf 1996), whereas MNTB is essentially devoid of CR-positive
neurons.
(e) Parvalbumin was strongly upregulated developmentally, as expected from previous
immunohistochemical studies (Felmy, F. & Schneggenburger, R. 2004, Eur. J. Neurosci. 20,
1473-1482; Lohmann & Friauf 1996).
In conclusion, analyzing the spatio-temporal expression pattern of several marker genes
validates the DNA-array analysis approach used here.
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Supplementary Figure 2:
Expression pattern of BMPR2 revealed by In-situ hybridization.
In-situ hybridization at P3 showing the expression of BMPR2 (a), and hybridization with the
BMP4 sense probe as a negative control (b). Note that the BMP receptors 1a, 1b and R2, and
also BMP4, show an overlapping expression pattern amongst brainstem nuclei, with strong
expression in the VCN, MNTB and also in the trigeminal motor nucleus. See also Fig. 1d - f.
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Supplementary Figure 3:
The MNTB and LSO nuclei can be well identified in the BMPR1a/1b cDKO mice, but
show a somewhat different anatomy.
Immunohistochemistry was performed in transverse brainstem sections to study the anatomy
of the MNTB and LSO in a BMPR1a/1b cDKO mouse, and in a littermate control mouse
(both at P12). An anti-calbindin (CB) antibody (green channel) was used as a marker for
MNTB neurons (Friauf 1993); the anti-Syt2 antibody (red channel) was used as a presynaptic
marker.
(a) Anterior part of the superior olivary complex. In the BMPR1a/1b cDKO mouse (right), a
major, more dorsal part of the MNTB, and a minor, more ventral part can be observed. The
recordings, and morphological analyses were made in cells from the major, dorsal part of the
nucleus (see also Online Methods). Note that despite the change in anatomy, marker proteins
such as CB, Syt-2 (see also Figs 2, 4), VGluT2 (Fig. 4) and PV (Fig. 2) were expressed in the
correct neuronal populations, arguing against broad cell fate specification deficits of auditory
neurons in the BMPR1a/1b cDKO mice.
(b) Posterior part of the superior olivary complex. Scale bar = 100 µm.
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Supplementary Figure 4:
Intrinsic firing properties and membrane properties of MNTB neurons were unchanged
in BMPR1a/1b cDKO mice.
(a) Example traces of membrane potential (Vm) recordings in current clamp experiments from
MNTB neurons; Vm traces in response to both negative and positive current steps are shown.
The traces in response to higher positive steps which, in these example cells, caused the firing
of several APs, are shown separately for clarity. The membrane time constant was estimated
by single-exponential fits to Vm traces in response to negative current steps (blue fit lines, and
indicated time constants). Black traces (left) are from a control mouse, red traces (right) are
from a BMPR1a/1b cDKO mouse.
(b) Quantifications of input resistance (left), membrane time constant (middle) and membrane
capacitance (right), both for control cells (black data points; n = 13 cells), and for cDKO cells
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(red; n = 13 cells). None of these parameters was significantly changed in BMPR1a/1b cDKO
mice.
(c) Plots of the number of APs versus step current amplitude, both averaged over all cells
from each genotype (top), and shown as data from individual cells (bottom). Note that some
cells in each genotype fired more than one AP in response to larger current injections, but
most cells showed firing of 1-2 APs at the onset of the current injection, typical for these
auditory neurons (Forsythe, I.D. & Barnes-Davies, M. 1993. Proc. R. Soc. Lond. B 251, 143-
150). Thus, the intrinsic firing properties of MNTB neurons were unchanged in BMPR1a/1b
cDKO mice.
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Supplementary Figure 5:
IPSCs at the MNTB to LSO synapse were unchanged in BMPR1a/1b cDKO mice
To further verify the specificity of BMP action in the development of the calyx of Held, we
investigated the inhibitory synapse made between MNTB and LSO. This synapse has been
shown to undergo a vigorous strengthening of unitary IPSC amplitudes in the age range
between P2 - P12 in rats, together with an elimination of inappropriate inhibitory synapses
(Kim, G. & Kandler, K. 2003, Nat. Neurosci. 6, 282-290).
(a) IPSC example traces for three selected stimulus intensities, both for an example cell from
a control mouse (left), and for a BMPR1a/1b cDKO mouse. The bottom shows a plot of IPSC
amplitudes versus stimulus intensity. Note that large jumps in the IPSC amplitude can be
observed in both genotypes. The amplitude difference between each discernible level, as
indicated by horizontal lines, was taken as unitary IPSC amplitude.
(b) Unitary IPSC amplitude was unchanged between control mice (black symbols) and
BMPR1a/1b cDKO mice (red). N = 5 and 8 cells in control and BMPR1a/1b cDKO,
respectively, at an age range of P8 – P10.
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Gene symbol Accession number
M‐value fold differ. (MNTB/LSO)
Gene name
Hapln1 NM_013500 3.47 11.05 Hyaluronan and proteoglycan link protein 1
Hapln1 AF098460 3.13 8.73 Hyaluronan and proteoglycan link protein 1
Hapln1 AF098460 3.10 8.57 Hyaluronan and proteoglycan link protein 1
Hapln1 BB036951 3.01 8.08 Hyaluronan and proteoglycan link protein 1
Pp11r AI528824 2.50 5.66 Placental protein 11 related
Tph2 BG071575 2.24 4.73 Tryptophan hydroxylase 2
Esrrb AV333667 2.09 4.24 Estrogen related receptor, beta
Bmp5 AV032115 1.94 3.84 Bone morphogenetic protein 5
Tmem182 AK010233 1.86 3.64 Transmembrane protein 182
Tgfb3 BC014690 1.79 3.46 Transforming growth factor, beta 3
AU021034 BG069877 1.75 3.36 Expressed sequence AU021034
Il2rg L20048 1.67 3.19 Interleukin 2 receptor, gamma chain
A030001D16Rik BB149729 1.62 3.08 RIKEN cDNA A030001D16 gene
Slc6a5 AF411042 1.60 3.03 Solute carrier family 6 (neurotransmitter transporter, glycine), member 5
Slc32a1 AI845218 1.59 3.02 Solute carrier family 32 (GABA vesicular transporter), member 1 (Slc32a1), mRNA
B3galt2 BB254922 1.53 2.89 UDP‐Gal:betaGlcNAc beta 1,3‐galactosyltransferase, polypeptide 2
Ebf2 (probe1) U71189 1.51 2.86 Early B‐cell factor 2
Ebf2 (probe2) U71189 1.51 2.86 Early B‐cell factor 2
‐‐‐ AW456568 1.51 2.84 ‐‐‐
Insm1 NM_016889 1.48 2.78 Insulinoma‐associated 1
Olfm4 AV290148 1.47 2.77 Olfactomedin 4
Sstr3 BQ174132 1.43 2.69 Somatostatin receptor 3
Scn5a BB516098 1.41 2.65 Sodium channel, voltage‐gated, type V, alpha
Ass1 NM_007494 1.39 2.63 Argininosuccinate synthetase 1
4933417D19Rik AK016841 1.37 2.59 RIKEN cDNA 4933417D19 gene
Kank4 BB486252 1.37 2.59 KN motif and ankyrin repeat domains 4
Tinagl1 BC005738 1.35 2.55 Tubulointerstitial nephritis antigen‐like 1
B3galt2 BB223909 1.34 2.53 UDP‐Gal:betaGlcNAc beta 1,3‐Galactosyltransferase, polypeptide 2
‐‐‐ BM217698 1.31 2.48 ‐‐‐
Scrt2 BG807042 1.31 2.48 Scratch homolog 2, zinc finger protein (Drosophila)
Pcdh8 NM_021543 1.31 2.48 Protocadherin 8
Bmp4 NM_007554 1.30 2.46 Bone morphogenetic protein 4
2900046L07Rik AK013658 1.30 2.46 RIKEN cDNA 2900046L07 gene
‐‐‐ BB056248 1.29 2.45 ‐‐‐
Enpep NM_007934 1.29 2.45 Glutamyl aminopeptidase
Pp11r AI528824 1.28 2.43 Placental protein 11 related
En1 NM_010133 1.27 2.42 Engrailed 1
B3gnt9 BB547181 1.26 2.39 UDP‐GlcNAc:betaGal beta‐1,3‐N‐Acetylglucosaminyltransferase 9
Ebf2 U71189 1.25 2.38 Early B‐cell factor 2
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Supplementary Table 1:
The most differentially expressed genes between mouse MNTB and LSO at P3.
The list is sorted according to the most differentially expressed genes between MNTB and
LSO at P3, with the M value representing the difference in the absolute hybridization values
on a natural logarithmic scale. Candidate diffusible messengers which might be involved in
synaptogenesis are BMP5 (3.84-fold higher expression in MNTB), TGFβ-3 (3.46 fold),
Protocadherin-8 (2.48 fold) and BMP4 (2.46 fold). The differential expression of TGFβ-3
was, however, not confirmed by qPCR. Hyaluronan and proteoglycan link protein 1
(HAPLN1) was the most differentially expressed gene between MNTB and LSO at P3 (8 - 11
fold according to probe identity). HAPLN1 is a component of the proteoglycan aggregate of
the cartilage, and in brain localizes to perineuronal nets, an extracellular matrix structure
normally found around a sub-class of Parvalbumin-positive interneurons (Carulli, D. et al.
2006, J. Comp. Neurol. 494, 559-577). This suggests that MNTB may have a strongly
developed perineuronal net. Also note the higher expression of the neuronal glycine
transporter GlyT2 (Slc6a5; Liu, Q.R. et al. 1993, J. Biol. Chem. 268, 22802-22808) in MNTB
as compared to LSO. This is expected because MNTB contains mainly
GABAergic/glycinergic neurons. See also Supplementary Figure 1 for further analysis of
marker genes which can be expected to be differentially expressed between MNTB and LSO.
Nature Neuroscience: doi:10.1038/nn.3414