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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