Siavash Fazel Darbandi1, Sarah E Robinson Schwartz1, Emily Ling-Lin Pai1, Amanda Everitt2, Marc L Turner1, Benjamin N R Cheyette1, A Jeremy Willsey3, Matthew W State4, Vikaas S Sohal5, John L R Rubenstein6. 1. Department of Psychiatry and UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA. 2. Institute for Neurodegenerative Diseases, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA. 3. Department of Psychiatry and UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA; Institute for Neurodegenerative Diseases, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94143, USA. 4. Department of Psychiatry and UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94143, USA. 5. Department of Psychiatry and UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA; Kavli Institute for Fundamental Neuroscience and Sloan-Swartz Center for Theoretical Neurobiology, University of California, San Francisco, San Francisco, CA 94143, USA. 6. Department of Psychiatry and UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: john.rubenstein@ucsf.edu.
Abstract
Tbr1 is a high-confidence autism spectrum disorder (ASD) gene encoding a transcription factor with distinct pre- and postnatal functions. Postnatally, Tbr1 conditional knockout (CKO) mutants and constitutive heterozygotes have immature dendritic spines and reduced synaptic density. Tbr1 regulates expression of several genes that underlie synaptic defects, including a kinesin (Kif1a) and a WNT-signaling ligand (Wnt7b). Furthermore, Tbr1 mutant corticothalamic neurons have reduced thalamic axonal arborization. LiCl and a GSK3β inhibitor, two WNT-signaling agonists, robustly rescue the dendritic spines and the synaptic and axonal defects, suggesting that this could have relevance for therapeutic approaches in some forms of ASD.
Tbr1 is a high-confidence autism spectrum disorder (ASD) gene encoding a transcription factor with distinct pre- and postnatal functions. Postnatally, Tbr1 conditional knockout (CKO) mutants and constitutive heterozygotes have immature dendritic spines and reduced synaptic density. Tbr1 regulates expression of several genes that underlie synaptic defects, including a kinesin (Kif1a) and a WNT-signaling ligand (Wnt7b). Furthermore, Tbr1 mutant corticothalamic neurons have reduced thalamic axonal arborization. LiCl and a GSK3β inhibitor, two WNT-signaling agonists, robustly rescue the dendritic spines and the synaptic and axonal defects, suggesting that this could have relevance for therapeutic approaches in some forms of ASD.
Autism spectrum disorders (ASDs) are defined by deficits in social
interaction and abnormalities in language development and repetitive behavior.
Considerable genetic and phenotypic heterogeneity has complicated efforts to
understand the underlying biology of ASD. However, recent progress in the genomics
of ASD has revealed more than 65 high-confidence ASD (hcASD) risk genes (Sanders et al., 2015). Systems analyses suggest
that expression of ASD risk genes have important functions in mid-fetal deep-layer
cortical excitatory neurons and that disruption may contribute to ASD
pathophysiology (Willsey et al., 2013). Among
these ASD genes, analysis of the Tbr1 transcription factor (TF) is
attractive, as it opens the possibility of defining a transcriptional pathway that
includes other ASD genes.Tbr1 has a central role in the development of mouse
early-born excitatory cortical neurons. Tbr1expression, which
begins in newborn neurons, dictates layer 6 identity (Bedogni et al., 2010; Bulfone et al.,
1998; Hevner et al., 2001, 2003; McKenna
et al., 2011). Using Tbr1 conditional
knockouts (CKOs), we recently demonstrated that neonatal Tbr1
function in layer 6 is required for maintaining corticothalamic identity and
synaptogenesis (Fazel Darbandi et al.,
2018).Here, we delved deeper into Tbr1’s function in
synaptogenesis in several ways. First, we identified convergent synaptic pheno-types
in Tbr1 and Tbr1
CKOs and Tbr1
(Tbr1+/−) mutants, including a defect in the
formation of mature dendritic spines. Next, we used single-cell RNA sequencing
(scRNA-seq) of Tbr1 mutant medial prefrontal
cortex (mPFC) neurons and identified Tbr1-regulated genes that
impact synapse formation in layer 5, including a kinesin motor protein
(Kif1a) and genes in the WNT-signaling pathway
(Gsk3β, Ctnnb1, and
Wnt7b). We also identified a number of
Tbr1-regulated ASD genes in the layer 5 neurons of the mPFC,
including Ank2, Ap2s1, Ctnnb1,
Dpysl2, Map1a, Rorb,
Smarcc2, and Gsk3β. Finally, we found
that LiCl, a drug approved by the US Food and Drug Administration, and a
GSK3β inhibitor (SB216763; Sigma-Aldrich) that promotes WNT signaling rescue
the spine and synaptic defects in adult Tbr1,
Tbr1, and
Tbr1
(Tbr1+/−) mutants. Lastly,
Tbr1 mutants exhibit decreased social
interactions with young mice, a phenotype that is rescued with LiCl treatment. The
LiCl results suggest an important and novel biological mechanism underlying ASD that
may have implications for the treatment of patients with TBR1
mutations and, potentially, other individuals with ASD or related neurodevelopmental
disorders.
RESULTS
Tbr1 Regulates Genes Involved in Cytoskeletal Dynamics and
Synaptogenesis in Layer 5 Pyramidal Neurons of Neonatal mPFC
In the frontal and motor cortex, Tbr1 is expressed in
most excitatory neurons in layers 5 and 6, whereas layer 5 expression in other
cortical regions is limited to a minority of neurons (Bulfone et al., 1995). Here, using a floxed allele,
we selectively eliminated Tbr1 in cortical layer 5 pyramidal
neurons around postnatal day (P)0 using Rbp4-cre ~8 days
after Tbr1expression begins. We refer to these mice as
Tbr1 CKOs.We focused on Tbr1 function in the developing
prefrontal cortex (PFC), a region that is implicated in ASD (Willsey et al., 2013). To overcome the limitations
caused by cellular heterogeneity of batch RNA sequencing (RNA-seq)
(Tbr1 is expressed in ~60% of layer 5 pyramidal
neurons at P5 and ~85% at P21; Figures S1A and S1B), we generated scRNA-seq data
from fluorescence-activated cell sorting (FACS) of layer 5 neurons isolated from
P5 mPFC (Figure 1). We studied the
transcriptomic changes from
Tbr1,
Tbr1 heterozygous and homozygous CKO
cells using the 10X Genomics platform (GenBank GEO: GSE146298).
Figure 1.
Tbr1 Regulates Genes that Are Implicated in Controlling the
Development of Axons, Synapses, and Dendrites in Layer 5 Pyramidal Neurons of
the mPFC
(A) t-distributed stochastic neighbor embedding
(t-SNE) plot displaying 11,070 single neuronal cells from
Tbr1 WT (red) and from
Tbr1 heterozygous (HET; green), and
Tbr1 homozygous (NULL; blue) CKOs.
t-SNE was performed after quality control and removal of non-neuronal cell
subtypes.
(B) Heatmap of DEX genes (FDR < 0.05) shared between both
genotypes (x axis, n = 218) over a randomly selected 1,000 cells from each
genotype (y axis, n = 3,000). Genes are ordered by hierarchal clustering within
direction of regulation grouping, and the Z score of normalized
gene expression data is shown. The genotype for each cell is depicted at the
top, and genes with membership in selected enriched GO categories are
highlighted at the right.
See also Figures
S1-S3.
To identify genotype-dependent gene expression changes, we used a
t-distributed stochastic neighbor embedding (t-SNE) dimensionality reduction
followed by differential expression (DEX) analysis to identify neuronal cells
(Figures 1A and S2). We captured 11,070 cells and
7,174 genes from Tbr1 (n = 1,778 cells),
Tbr1 heterozygous (n = 5,357 cells),
and Tbr1 homozygous (n = 3,935 cells) mutant
mPFCs that were used for downstream analysis (Figure S2). We excluded 873 cells
classified as atypical neuronal cells, with lower expression levels of
Neurod6 and Nrgn (two excitatory neuronal
markers; Figures S3A
and S3D) and high
levels of housekeeping genes, from DEX analyses (gray cells in Figure 1A). The t-SNE plot demonstrated clear
separation between Tbr1 and
Tbr1 CKOs (Figure 1A). DEX analysis identified 470 DEX genes when
comparing Tbr1 homozygous mutants to
Tbr1 (Table S1) and 320 DEX genes when
comparing Tbr1ayer5 heterozygous mutants to
Tbr1 (Table S2), 218 of which occur in
both comparisons (false discovery rate [FDR] ≨ 0.05) (Figure 1B, Table S3). Feature plots showing
the expression of layer 5 markers in our scRNA-seq cell population are shown
(Figure S3i). Gene
Ontology (GO) analysis of DEX genes identified terms including
“axon,” “synapse,” “dendrite,”
“cell body,” and “neurogenesis” (Figure 1B; Table S4).To determine whether the changes in gene expression in
Tbr1 CKOs are due to direct regulation
by TBR1, we used data from TBR1 chromatin immunoprecipitation (ChIP-seq) from P2
wild-type (WT) cortex (Fazel Darbandi et al.,
2018). TBR1 binds to the promoters and distal regions of layer 5 DEX
genes (within 100 kb) (Figure
S1C). This suggests that TBR1 may be involved in controlling the
expression by activating or repressing the target genes.We used in situ hybridization (ISH) to validate the
expression of several DEX genes (Table S5). Our scRNA-seq analysis
in conjunction with ISH aided in discovering Mgst3, as a new
layer 5 marker of prefrontal cortex (Figure S1D). To provide a
histological context, we defined laminar boundaries in the prefrontal cortex at
P3 using the following probes: Cux2 (layers 2 and 3),
Rorb (layer 4), Etv1 (layer 5),
Tbr1 (layers 2–3, 5, 6, and 6b),
Nr4a2 (subplate; Figure
2i). Cortical layers 2–4 appear as a single layer at this
stage (Figures 2A–2F). Expression of Calm2,
Kif1a, Mgst3, and Wnt7b
was altered as suggested by the scRNA-seq analysis (Figure 2ii; Figure S3ii). Thus, neonatal
Tbr1expression in layer 5 pyramidal neurons directly
regulates the expression of genes involved in cytoskeletal dynamics and synapse
development.
Figure 2.
Tbr1 Regulates Expression in the mPFC
In situ hybridization defines rostral cortical
lamination and validates the changes in scRNA-seq expression levels.
(i) PFC lamination. Prefrontal cortical lamination was defined using
ISH on coronal sections of neonatal mPFC in WT mice at P3.
(A–O) ISH was performed on rostral, medial, and caudal areas,
respectively, using (A–C) Cux2 (layers 2–4);
(D–F) Rorb (layer 4); (G–I) Etv1
(layer 5); (J–L) Tbr1 (layers2/3, 5, and 6); and
(M–O) Nr4a2 (subplate or layer 6b). Cortical layers in
the medial and dorsal regions are labeled. MO, medial orbital cortex; PrL,
prelimbic cortex; FrA, frontal association cortex; OC, orbital cortex; Cg1,
cingulate cortex area 1; M, motor cortex; S1, primary somatosensory cortex; IC,
insular cortex; II-IV, layers 2–4; V, layer 5; VI, layer 6; VIb,
subplate. Scale bar, 300 μm.
(ii) scRNA-seq validation. ISH confirms the changes in the
transcriptome changes from DEX analysis of scRNA-seq in
Tbr1 homozygous mutants.
(P–W) The expression of Mgst3 (P and Q) and
Calm2 (R and S) are increased in layer 5upper (Q
and S). Tbr1 mutants exhibit reduced
expression of Wnt7b (T and U) and Kif1a (V and
W) in layer 5 of the mPFC at P3. Only one hemisphere is shown from the ISH
images from WT and Tbr1 homozygous CKOs, which
are presented as mirror images, to aid in evaluating the changes in laminar gene
expression. Color code: downregulated (red) and upregulated (green). Red box
shown in (P) and (Q) indicates the region that was dissected for scRNA-seq
analyses. Cortical layers 2–4, 5upper, 5lower, 6,
and 6b (subplate) are labeled. Scale bar, 100 μm.
See also Figure
S3.
Excitatory and Inhibitory Synapses Are Reduced in
Tbr1 Mutants
We assessed excitatory synapse numbers on apical dendrites of layer 5
neurons (within layer 2-3) in the mPFC by analyzing VGLUT1+
presynaptic terminals that are apposed to PSD95+ postsynaptic zones
at P56 (Figure 3A′) and P21 (Figures S4D-S4F) using
immunofluorescence (IF) and confocal microscopy. Inhibitory synaptic density was
assessed by counting the overlapping VGAT+ presynaptic inhibitory
terminals and Gephyrin+ dendritic postsynaptic zones on the apical
dendrites of layer 5 pyramidal neurons (n = 30 dendrites) at P56 (Figure 3D′) and P21 (Figures S4J-S4L). Excitatory and inhibitory
synapses were decreased 34% and 42% in Tbr1
heterozygous and 70% and 73% in Tbr1
homozygous mutants at P56, respectively (Figures
3A and 3D). A similar synaptic
deficit was also present at P21 (Figures S4G and S4M).
Figure 3.
Tbr1 Mutants Have Reduced Excitatory and Inhibitory Synaptic
Densities at P56
IF was used to detect excitatory (i) and inhibitory (ii) synapses onto
dendrites of (1) the mPFC of Tbr1
(Rbp4-cre∷tdTomato;
red), Tbr1 heterozygous
(Tbr1∷∷tdTomato;
green), and Tbr1 homozygous
(Tbr1∷∷tdTomato;
blue) mutants (n = 30 dendrites), and (2) dendrites of layer 5 neurons from the
mPFC of Tbr1,
Tbr1+/− and layer 6 neurons from the SSCx
of Tbr1,
Tbr1+/− (n = 15 dendrites).
(A–C′) Excitatory synapses were identified by
colocalization of VGLUT1+ boutons and PSD95+ clusters on
dendrites of layer 5 pyramidal neurons at P56 (A′ and C′).
(A) Quantification of excitatory synaptic density.
(B) Quantification of the sEPSC frequency from layer 5 neurons at P56
(n = 6/6/6, WT/heterozygous/homozygous cells from two different animals per
genotype).
(B′) Sample traces of sEPSC recordings at P56.
(C) Quantification of excitatory synaptic density of
Tbr1+/− mutants in cortical layers 5 and
6 at P56.
(D–F′) Inhibitory synapses were identified by
co-localizing VGAT+ boutons and Gephyrin+ clusters
(D′ and F′).
(D) Quantification of inhibitory synaptic density on dendrites of layer
5 pyramidal neurons at P56.
(E) Quantification of the sIPSC frequency from layer 5 neurons at P56
(n = 7/7/7, WT/heterozygous/homozygous cells from two different animals per
genotype). (E′) Sample traces of sIPSC recordings at P56.
(F) Quantification of inhibitory synapse numbers on dendrites of layer
5 and 6 pyramidal neurons of Tbr1+/− mutants
at P56.
(iii) In vitro rescue assay was conducted by
transfecting Kif1a, Mef2c,
Rac3, and Syt4 expression vectors into P0
primary cortical culture from Tbr1 (red)
and Tbr1 CKOs (blue) (n = 3 biological
replicates).
(G–H′) Excitatory (G) and inhibitory (H) synaptic density
was analyzed 14 days post-transfection. Quantification of excitatory (G′)
and inhibitory (H′) synaptic density in vitro is
indicated. Red box indicates a successful rescue of synaptic density. Two-way
ANOVA was used for the statistical analysis of the control, heterozygote, and
null. Two-tailed t test with Tukey correction was used for pairwise comparisons.
Floating bar graphs represent the minimum-to maximum (min-max) distribution of
synaptic density and/or EPSC/IPSC frequency measured from each genotype.
Horizontal line in each box denotes the average distribution. Average
distribution is numerically indicated in each box (**p < 0.01; ***p
< 0.001; ****p < 0.0001). ns, not significant.
See also Figures
S4 and S5.
To assess the physiological consequences of the decrease in excitatory
and inhibitory synaptic densities, we measured spontaneous excitatory and
inhibitory post-synaptic currents (sEPSCs and sIPSCs, respectively) using
whole-cell patch clamp on the tdTomato+ layer 5 pyramidal cells in
mPFC brain slices at P56 and P21 (Figures
3B′ and 3E′; Figures S4H and S4N). The sEPSC frequency
was reduced 25% in Tbr1 heterozygous and 75%
in Tbr1 homozygous mutants; furthermore, the
frequency of sIPSCs was reduced 30% in Tbr1
heterozygous and 50% in Tbr1 homozygous
mutants as compared to cells from Tbr1mice
at P56 (Figures 3B and 3E). Similar decreases were also present at P21 (Figures S4I and S4O). We did not observe
changes in the amplitude of sEPSCs and sIPSCs at P21 and P56 (data not
shown).Since most de novo ASD-risk genes are heterozygous,
loss-of-function, rare variants, we explored the consequence of constitutive
Tbr1haploinsufficiency on synapse numbers of layer 5 and
layer 6 pyramidal neurons using Tbr1+/− mice
(Bulfone et al., 1995). We counted
excitatory and inhibitory synapse numbers in the mPFC of
Tbr1+/−∷Rbp4-cre∷tdTomato
(layer 5 neurons) and the somatosensory cortex (SSCx) of
Tbr1+/−∷Ntsr1-cre∷tdTomato
(layer 6 neurons) at P56 (Figure 3, i2 and ii2). Layer 5 excitatory and inhibitory synapse numbers were reduced
~40% and ~35% in the mPFC of
Tbr1+/−∷Rbp4-cre∷tdTomato
at P56 (Figures 3C and 3F). Layer 6 neurons in the SSCx of
Tbr1+/−∷Ntsr1-cre∷tdTomato
showed ~37% and ~39% decreases in excitatory and inhibitory
synaptic densities, respectively (Figures
3C and 3F). Thus,
Tbr1haploinsufficiency results in reduced synaptic density
on the excitatory neurons of cortical layers 5 and 6.
Kif1a Expression Restores Normal Synapse Numbers in
Tbr1 Mutant Neurons In
Vitro
We sought to identify molecular mechanisms underlying the decrease in
the excitatory and inhibitory synaptic densities in
Tbr1 CKO neurons using the results
from the scRNA-seq analysis (Figure 1). We
assessed a subset of DEX genes that control synapse biology, including
Kif1a (Li et al.,
2016), Mef2c (Barbosa
et al., 2008), Rac3, and Syt4
(Barber et al., 2009). We examined
whether Kif1a, Mef2c, Rac3,
and Syt4 could rescue synapse density by expressing them in P0
primary cortical cultures derived from Tbr1
and Tbr1 mutant neurons (n = 3 biological
replicates).After 14 days in vitro, we analyzed the number of
excitatory (VGLUT+ presynaptic and PSD95+ postsynaptic)
and inhibitory (VGAT+ presynaptic and Gephyrin+
postsynaptic) terminals of Tbr1 and
Tbr1 homozygous mutant neurons (Figure 3, iii). The reduced excitatory and inhibitory synaptic densities onto
Tbr1 CKO neurons were recapitulated
in vitro (Figures 3G,
3G′, 3H, and 3H′). Only Kif1a rescued the reduction in
both excitatory (Figures 3G and 3G′) and inhibitory (Figures 3H and 3H′) synapse numbers. Kif1a, a kinesin motor
protein, is implicated in the transport of vesicles for synapse development
(Guedes-Dias et al., 2019) and thus
may contribute to Tbr1’s function in promoting synapse
formation.
Tbr1 CKOs Have Increased
Hyperpolarization-Activated Cation Currents (Ihs)
We next examined the intrinsic properties of layer 5 neurons in
Tbr1 WT and CKOs using whole-cell
patch clamp to measure intrinsic physiological properties of
Rbp4-cre∷tdTomato+
neurons of layer 5 in the mPFC (Figure S5A). Resting membrane
potential and input resistance were not different between
Tbr1,
Tbr1 heterozygotes and homozygotes at P56
(Figures S5B and
S5C).A prominent feature of many layer 5 pyramidal neurons is the presence of
an Ih (or h-current) mediated by hyperpolarization-activated cyclic
nucleotide–gated HCN channels (Shepherd,
2013). Ih causes a characteristic “sag” and
“rebound” in current clamp recordings of responses to steps of
hyperpolarizing current. We examined responses to a −200-pA step and
found that mPFC layer 5 pyramidal neurons from P56Tbr1 heterozygotes and homozygotes
exhibited a significantly increased “sag and rebound” compared to
Tbr1 controls, suggesting increased
Ih, while other intrinsic electrophysiological properties were
largely unaltered (Figure
S5D).In deep-layer neocortical pyramidal neurons, the presence of
Ih shifts the resonant frequency toward higher frequencies (Dembrow et al., 2010). Therefore, to
further characterize potential increases in Ih in
Tbr1 CKOs, we estimated the resonant
frequency by injecting constant current to hold
Rbp4-cre+ neurons in current clamp near
−70 mV and then introduced a sinusoidal current stimulus with constant
amplitude (100 pA, peak to peak) and a frequency that increased linearly from 0
to 20 Hz over 20 s (Figure
S5E). Tbr1 heterozygous and
homozygous CKOs exhibited an increase in their resonant frequency compared to
Tbr1 controls at P56 (Figure S5G).Lastly, we blocked Ih by bath applying the specific HCN
channel antagonist ZD7288 (25 μM; Figure S5F). The resonant frequency
was reduced by over 50% in the Tbr1
heterozygous and Tbr1 homozygous CKOs (Figure S5G). Thus, both
Tbr1 heterozygotes and homozygotes
have an increased Ih in layer 5 pyramidal neurons of the mPFC.
Tbr1 Mutants Have Reduced Mature Dendritic Spine
Density
The synaptic deficits described earlier prompted us to investigate the
state of dendritic spines in Tbr1 CKOs,
Tbr1 CKOs (Fazel Darbandi et al., 2018), and
Tbr1+/− mutants. We visualized
tdTomato+ spines using Airyscan confocal microscopy to capture
120×-magnification z stack images (using 2× optical zoom) from the
dendrites of layer 6 and layer 5 neurons of WT,
Tbr1 (Figure
4), Tbr1, and
Tbr1+/− mutant neurons at P5, P21, and
P60 (Figure S6). We
used Imaris software (v.9.2.1) to analyze dendritic spine morphology, density,
and distribution.
Figure 4.
LiCl Rescues Dendritic Spine Density of
Tbr1 CKOs
(A–L″) In (A)–(L),
∷tdTomato
allele was used to label the dendrites of layer 5 neurons. Imaris software was
used to analyze the dendritic spine density on the apical dendrites of
Tbr1 WT and
Tbr1 CKO neurons located within layers
2–4 of the mPFC (A′–L′). Changes in the dendritic
spine density of layer 5 neurons were examined at P5 (A–D), P21
(E–G), and P60 (H–L). (A″–L″) Merged
images.
(M) Quantification of dendritic spine density at P5 and P21. Spine
density was improved 24 hr after LiCl treatment at P5 in (C) and (D) and P60 in
(K) and (L), compared to the saline-injected control animals in (A) and (B) and
in (H) and (J).
(N) Quantification of mature dendritic spines of
Tbr1 WT and mutant neurons at P60, 24
h after injection with saline (control) or LiCl. Floating bar graphs represent
min-max distribution of the dendritic spine density of layer 5 neurons within
layers 2–4 of the mPFC. Horizontal line in each box denotes the average
spine density. Average mature dendritic spine density is numerically indicated
in each box.
****p < 0.0001. ns, not significant. Scale bar, 8 μm.
See also Figure
S6.
There were reductions in the density of mature dendritic spine density
in Tbr1 heterozygotes and homozygotes in
Tbr1 and
Tbr1 CKOs (Figures 4 and S6). Additionally, Tbr1+/−
mutants have reduced mature spine density on the dendrites of layer 5 and layer
6 pyramidal neurons (Figure
S6). Furthermore, Tbr1 mutant neurons had an
increased filamentous spine density (Figure S6). Thus, this defect in
mature dendritic spine density may underlie the reduction in synapse numbers in
Tbr1 mutants.
Restoring Reduced WNT Signaling in Tbr1 CKOs Rescues
Synaptic Deficits
We demonstrated that Tbr1 promotes synaptogenesis onto
layer 6 neurons in part via WNT signaling through Wnt7b (Fazel Darbandi et al., 2018). WNT signaling
promotes dendrite maturation and synapse formation (Ciani and Salinas, 2005). Here, we found several
lines of evidence to further support the role of Tbr1-dependent
WNT signaling in synapse development. First, Wnt7b and
Ctnnb1expression was reduced in the mPFC of
Tbr1 CKOs (Figures 1 and 2;
Tables S1 and S2).
Ctnnb1 encodes β-catenin, the critical intracellular
transducer of canonical WNT signaling (Budnik and
Salinas, 2011). Second, Tbr1 CKOs
had increased Gsk3β RNA expression (Figure 1); GSK3β negatively regulates WNT
signaling through increasing the destruction of β-catenin (van Noort et al., 2002).Thus, we tested whether promoting WNT signaling could rescue dendritic
spine and synapse phenotypes. Among its several pharmacological effects, there
is evidence that LiCl, a WNT-signaling agonist, promotes synapse development
(Farooq et al., 2017; Lenox and Wang, 2003; Martin et al., 2018). Thus, we administered LiCl and a GSK3β
inhibitor (SB216763, Sigma-Aldrich) to Tbr1 mutants.
LiCl Treatment of Tbr1 Mutants Restores Dendritic Spine
Density and Synapse Development
As noted earlier, Tbr1 mutants have a reduced density
of mature dendritic spines (Figures 4 and
S6). We tested
whether promoting WNT signaling by administering LiCl at P5 and P59 could rescue
the reduction in mature spine density and synaptogenesis in
Tbr1 mutants. We gave a single intraperitoneal (i.p.)
injection of 400 mg/kg LiCl; control animals received a single i.p. injection of
4 mL/kg saline. Impressively, LiCl treatment rescued the density of mature
dendritic spines within 24 h in Tbr1 mutants; LiCl did not have
a clear effect on the density of WT dendritic spines (Figures 4 and S6). These results, in combination
with the previously reported evidence that Wnt7b restores
synapse numbers on Tbr1 mutant neurons (Fazel Darbandi et al., 2018), led us to
test whether LiCl can rescue synapse numbers on adult Tbr1
mutant layer 5 and layer 6 neurons.We administered LiCl to Tbr1 WT and
homozygous CKOs (Figures 5A and 5E), Tbr1
WT and homozygous CKOs (Figures 5B and
5F), and
Tbr1+/− mutants (Figure 5). Layer 5 and layer 6 projection neurons were
labeled with
Rbp4-cre∷tdTomato
and
Ntsr1-cre∷tdTomato,
respectively. The control and LiCl-treated brains were harvested either 24 h or
4 weeks after injection at P60 (Figures 5
and S7). Confocal
images of IF from the mPFC (layer 5) and SSCx (layer 6) showed a nearly complete
rescue of synaptic densities, 24 h and 4 weeks after treatment (Figures 5 and S7). LiCl treatment also rescued
synaptic densities in the mPFC (layer 5) and SSCx (layer 6) of the constitutive
Tbr1+/− mutants (Figure 5).
Figure 5.
LiCl Treatment Restores Synapse Numbers and Corticothalamic Axonal
Arborization of Tbr1 Mutant Mice
Excitatory (i) and inhibitory (ii) synaptic densities were quantified
at P60 from: (1) apical dendrites of Tbr1
and Tbr1 mice 4 weeks after P30 injection
with saline or LiCl (n = 15 dendrites) and (2) dendrites of layer 5 neurons from
the mPFC of Tbr1,
Tbr1+/− and layer 6 neurons from the SSCx
of Tbr1,
Tbr1+/− mice 24 h after injection with
saline or LiCl at P59 (n = 15 dendrites).
(A and B) Excitatory synapses were quantified from (A) layer 5 neurons
of the mPFC of Tbr1 (green) and
Tbr1 (orange) mice and (B) layer 6
neurons from the SSCx of Tbr1 (red) and
Tbr1 (blue) mice at P60, 4 weeks
after saline and/or LiCl was administered.
(C and D) Quantification of excitatory synaptic density of (C) layer 5
neurons of the mPFC of Tbr1 (green) and
Tbr1+/− (orange) mice and (D) layer 6
neurons from the SSCx of Tbr1 (red) and
Tbr1+/− (blue) mice at P60, 24 h after
injection with saline or LiCl.
(E and F) Inhibitory synapses were quantified from (E) the mPFC of
Tbr1 and
Tbr1 and (F) the SSCx of
Tbr1 and
Tbr1 mice 4 weeks after saline
and/or LiCl was administered at P30.
(G and H) Inhibitory synapses were quantified from (G) layer 5 neurons
of the mPFC of Tbr1 and
Tbr1+/− and (H) layer 6 neurons of the
SSCx of Tbr1 and
Tbr1+/− mice at P60 24 h after injection
with saline or LiCl. Floating bar graphs represent the min-max distribution of
all excitatory and inhibitory synapse numbers measured from each genotype.
Horizontal line in each box denotes the average distribution. Average
distribution is numerically indicated in each box. Two-tailed t test with Tukey
correction was used for pairwise comparisons (***p < 0.001; ****p
< 0.0001). ns, not significant.
(I–P) In section iii, corticothalamic axonal arborization in the
thalamus is indicated by tdTomato’s endogenous fluorescence of
Tbr1 WT (I–L) and
Tbr1 homozygous CKO (M–P) mice.
The monochrome tdTomato signal (white) is indicated from saline-injected (I and
M) mice, 24 h after LiCl injection (J and N), 24 h after GSK3β-inhibitor
injection (K and O), and 4 weeks after LiCl injection (L and P). White
arrowheads in (M) indicate thalamic regions that have reduced corticothalamic
axonal arborization in Tbr1 CKO. Yellow
arrowheads in (N)–(P) correspond to improved corticothalamic axonal
arborization in Tbr1 CKO at P60 following LiCl
treatment after 24 h (N), GSK3β-inhibitor (GSK3βi) treatment after
24 h (O), and LiCl treatment after 4 weeks (P). Yellow box depicts a high
magnification of the SSCx, demonstrating that LiCl and GSK3β-inhibitor
treatments did not rescue the layer 6 apical dendrite morphogenesis in
Tbr1 CKOs. Thalamus, cortex (Cx), and
corticothalamic axons (CTAs) are labeled. Scale bars: white, 1 mm; blue, 50
μm.
(iv) Quantification of the tdTomato pixel intensity in the boxed
regions in (I) and (M) from saline-injected
Tbr1 (WT-Saline) and
Tbr1 homozygous mutants (Null-Saline)
at P60. tdTomato signal intensity is improved in the thalamus of the
Tbr1 homozygous CKO 24 h and 4 weeks
after treatment compared to treatment of
Tbr1 at 24 h and 4 weeks. Two-tailed t
test with Tukey correction was used for pairwise comparisons. Floating bar
graphs represent the min-max distribution of tdTomato pixel density measured
from region 1 of all genotypes and treatments. Horizontal line in each box
denotes the average distribution. Average distribution is numerically indicated
in each box (***p < 0.001; ****p < 0.0001).
See also Figures
S7, S8, and
S9.
Thus, LiCl treatment of Tbr1,
Tbr1, and
Tbr1+/− mutant mice at P60 rescues both
excitatory and inhibitory synaptic deficit in Tbr1 mutant
neurons of cortical layers 5 and 6 (Figures
5A-5H). Here, we postulate that
Tbr1 mutant neurons are in a “poised” state
but are not able to form synapses due to a defect in WNT signaling. Thus, we
provide in vivo evidence that augmenting WNT signaling via LiCl
treatment is sufficient to restore normal synapse numbers.
GSK3β Inhibitor Restores Defects in Dendritic Spine and Synaptic
Density of Tbr1 Mutants
Promoting WNT signaling via LiCl treatment of Tbr1
mutants rescued the defects in mature spine and synaptic density (Figures 5 and S7). Lithium’s best
validated mechanisms of action are inhibitory effects on IMP and INPP1, central
phosphatases in the phosphoinositide pathway, and on GSK3β, the central
kinase in the Wnt/β-catenin and AKT pathways (Lenox and Wang, 2003). To ascertain whether WNT
signaling is the main mechanism underlying the defects in dendritic spine and
synaptic density of Tbr1 mutants, we used a GSK3β
inhibitor (SB216763; Sigma-Aldrich).A single i.p. injection of GSK3β inhibitor (10 mg/kg) was given
to Tbr1 CKOs and WT at P59. Control animals received a single
i.p. injection of 4 mL/kg saline at P59. We studied the effects of these
treatments on Tbr1 WT and homozygous CKOs
(Figure S8i) and on
Tbr1 WT and homozygous CKOs (Figure S8ii). Layer 5 and
layer 6 projection neurons were labeled with
Rbp4-cre∷tdTomato
and
Ntsr1-cre∷tdTomato,
respectively. The control and GSK3β-inhibitor-treated brains were
harvested after 24 h (Figure
S8). GSK3β-inhibitor treatment rescued the decrease in mature
spine density in Tbr1 CKO mutants (Figures S8C and S8F). Furthermore, IF analysis of
excitatory and inhibitory synaptic densities from the
Tbr1 CKO mPFC (layer 5; Figures S8A and S8B) and from the
Tbr1 CKO SSCx (layer 6; Figures S8D and S8E) showed a nearly
complete rescue of synaptic density 24 h after treatment (Figure S8).Thus, GSK3β inhibitor treatment of
Tbr1 and
Tbr1 CKO mice at P60 rescues dendritic
spine density as well as excitatory and inhibitory synaptic deficit in
Tbr1 mutant neurons of cortical layers 5 and 6,
respectively (Figure
S8). This provides an additional line of evidence that augmenting WNT
signaling is a key mechanism in restoring mature dendritic spine and synaptic
density in Tbr1 mutants.
LiCl and GSK3β Inhibitor Treatment at P60 Improves Corticothalamic
Axonal Arborization in Tbr1 Mutant
Layer 6 corticothalamic neurons extend their axons to the thalamus where
they form synapses. Corticothalamic axons enter the thalamus in
Tbr1 CKOs; however, the
corticothalamic axonal arborization is reduced in the anteromedial thalamus of
Tbr1 CKOs (white arrowheads in Figure 5M) (Fazel Darbandi et al., 2018). Treatment with either LiCl or
GSK3β inhibitor rescued this defect after 24 h (yellow arrowheads in
Figures 5N and 5O) and 4 weeks
(yellow arrowheads in Figure 5P).
Quantification of tdTomato pixel intensity in the anteromedial thalamus (boxed
region in Figures 5I and 5M) showed a significant increase after treatment
(Figure 5, iv). We estimate that LiCl increased the
corticothalamic axonal arborization by ~250 μm in 24 h. Axon
growth rates in multiple regions of the nervous system and species have been
documented to range from 20 to 75 μm/h (equivalent to ~2,000
μm/24 h) (Goldberg, 2003; Lallemend et al., 2012). We postulate that
the rescue of the axonal arbors is through enhanced levels of WNT signaling as
result of the LiCl or GSK3β inhibitor treatment.
Evidence that WNT Signaling Promotes Synaptogenesis in Tbr1
CKOs through an Autocrine Mechanism
Previously, we demonstrated that restoring in vivo
Wnt7bexpression in layer 6 pyramidal neurons of
Tbr1 CKOs promoted synaptogenesis onto
layer 6 neurons (Fazel Darbandi et al.,
2018). Here, we have verified this finding and included additional
controls (Figures S9A
and S9B).Toward elucidating whether WNT7B functions through autocrine and/or
paracrine mechanisms, we used cortical transplantation of
Wnt7b-expressing cortical interneurons to study synaptogenesis
in Tbr1 CKO and control (WT) mice. We
introduced medial ganglionic eminence (MGE)-derived cortical interneurons (MGE
donor cells from
Nkx2.1-cre∷tdTomato
background) harboring either a Wnt7bexpression construct
(pLenti-DlxI12b-Wnt7b-GFP) or a control vector
(pLenti-DlxI12b-GFP) into deep cortical layers of
Tbr1 and
Tbr1 CKOs at P1; we analyzed
excitatory synaptic density in cortex at P30. We quantified excitatory synapses
on apical dendrites of WT and Tbr1 CKOs layer
6 neurons, adjacent to the MGE-transplanted cells
(tdTomato+-GFP+) within layer 5 (Figures S9C-S9F). We did not observe a rescue
of synapse numbers (Figure
S9E). Furthermore, we did not observe an increase of excitatory
synapses onto the soma of the transplanted Wnt7b-expressing
interneurons (Figure
S9F). Thus, this experiment provides evidence that WNT7B promotes
synaptogenesis in cortical excitatory neurons through a cell-autonomous
autocrine mechanism.
Tbr1 CKOs Exhibit Social Interaction
Defects that Are Rescued by LiCl Treatment
We studied motor function, anxiety, and social interaction of
Tbr1 mutant mice between P56 and P80.
Motor defects were not detected based on speed in an open field or performance
on a rotarod (data not shown). To assay social behavior, we measured the time
the experimental mouse spent exploring a novel juvenile WT mouse of the same
sex. Subsequently, we measured the amount of time the subject mouse spent
exploring a novel object. Tbr1 homozygous CKOs
exhibited social interaction deficit with a juvenile mouse; we did not observe a
social deficit between Tbr1 WT and
Tbr1 heterozygous CKOs (data not
shown). Loss of Tbr1 in layer 5 neurons did not affect the
amount of time Tbr1 CKOs spent exploring a
novel object compared to the WT.The improved synaptic density of Tbr1
CKOs due to LiCl treatment prompted us to assess the impact of LiCl treatment on
the social interaction of Tbr1 CKOs. We
performed the novel object exploration and social interaction assays at P60
using Tbr1 WT and CKOs that were treated with
a single i.p. injection of saline (control) and LiCl (experimental) 4 weeks
prior to the behavioral assays. LiCl treatment of
Tbr1 homozygous CKOs improved their social
interaction deficit with a juvenile mouse (Figure
6A), while LiCl treatment did not affect a novel object assay (Figure 6B). Thus, LiCl rescues defects in
dendritic spines, synapse density, and the social behavior of
Tbr1 CKOs.
Figure 6.
LiCl Treatment Rescues Social Interaction Deficit of
Tbr1 Mutants
(A) Tbr1 homozygous CKOs (blue)
showed reduced social interaction with a juvenile mouse at P56–P80. LiCl
treatment of Tbr1 CKOs rescued the social
deficit phenotype compared to the saline-treated mutants at P56–P80.
(B) LiCl treatment of Tbr1 CKOs did
not affect the time spent engaged in novel object exploration compared to the
saline-injected control. Floating bar graphs represent the min-max distribution
of interaction measured from all genotypes and treatments. Horizontal line in
each box denotes the average distribution. Average distribution is numerically
indicated in each box.
Two-tailed t test with Tukey correction was used for pairwise
comparisons (*p < 0.05).
DISCUSSION
Tbr1 Dosage in Layers 5 and 6 Is Essential for Promoting and
Maintaining Dendritic Spine and Synaptic Density
Tbr1 is expressed in post-mitotic excitatory neurons in
the neocortex, hippocampus, entorhinal cortex, pallial amygdala, piriform
cortex, olfactory bulb, Cajal-Retzius cells, and subplate neurons (Hevner et al., 2001, 2003). Tbr1 is best known for its
expression and function in layer 6, where it is required to initiate and then
maintain layer 6 identity by repressing markers of layer 5 identity (Fazel Darbandi et al., 2018; McKenna et al., 2011). There is also
prominent Tbr1expression in layer 5 of the rostral cortex,
where it is expressed in ~85% of pyramidal neurons (Bulfone et al., 1995).Here, by deleting Tbr1 late in gestation using a
layer-5-specific Cre (Rbp4-Cre), we have investigated the role
of Tbr1 in mPFC development. scRNA-seq from FACS-purified layer
5 neurons of Tbr1wild-type and
Tbr1 heterozygous and homozygous CKOs
demonstrated that Tbr1 deletion in mPFC layer 5 alters the
expression of a subset of genes that control synaptogenesis, synaptic
maturation, and microtubule assembly (Tables S1 and S2).The core phenotypes of the Tbr1 CKOs are: (1) reduction
in the density of mature dendritic spine density (Figures 4 and S6); (2) increased density of immature filamentous (thin) spines
(Figure S6); and
(3) reduced density of excitatory and inhibitory synapses (Figures 3 and S4). The dendritic spine defect is
apparent at the beginning of synaptogenesis (P5) and is maintained through
adolescence and into adulthood (Figures 4
and S6). Notably, the
Tbr1 CKOs neurons have an increased Ih. There is
evidence that HCN channels, the mediator of Ih, localize to thin spines (Paspalas et al., 2013). Thus, we
hypothesize that the increased Ih in Tbr1 CKOs may
be attributed to the increased filamentous spine density in
Tbr1 CKOs. Support for this notion comes from the
observation that layer 5 neurons have an ~2-fold increased density of
filamentous spines compared to that of layer 6 neurons (Figures S6E and S6F), which correlates with higher
Ih in layer 5 neurons (Shepherd,
2013).We postulate that the reduced mature spine density is central to the
reduction of excitatory synapses and synaptic activity observed in adolescent
(P21) and adult (P56) Tbr1 CKOs. In addition,
Tbr1 CKOs (Fazel Darbandi et al., 2018), as well as
Tbr1+/− constitutive mutants, show
defects in dendritic spines and synapses. The fact that we observed defects in
dendritic spine and synapse density in Tbr1 heterozygous CKOs
and Tbr1+/− constitutive mutants implies that
this phenotype could contribute to the behavioral phenotypes in neuropsychiatric
disorders such as ASD. This hypothesis is further strengthened by the dendritic
spine and synaptic phenotypes in the mPFC, a cortical region with critical
functions in cognitive and affective processing.
Molecular Mechanisms Downstream of Tbr1 that Promote Synapse
Development
We have evidence that TBR1 controls synaptic development by promoting
spine maturation and synaptogenesis through several mechanisms.
Tbr1 promotes WNT signaling (discussed more extensively
later), and TBR1 directly drives the expression of Cyp26b1,
Foxp2, Mef2c, and Wnt7b
in layer 6 (Fazel Darbandi et al., 2018),
as well as Kif1a and Wnt7b in layer 5. We
integrated these findings into a molecular model (Figure 7). The model also postulates how LiCl and GSK3β
inhibitor treatments, through promoting WNT signaling, rescues synaptic and
axonal phenotypes in Tbr1 mutants (Figure 7).
Figure 7.
Model of How Tbr1 Controls Spine Maturation and
Synaptogenesis through Promoting WNT Signaling: Links to ASD
Pathogenesis
Schematic representation of how Tbr1 controls spine
maturation and synaptogenesis in cortical layers 5 and 6. Tbr1
regulates WNT signaling by promoting Wnt7b and
Ctnnb1 and represses Gsk3β
expression. LiCl (blue) and GSK3β inhibitor (green) rescues
Tbr1 phenotypes through stimulating WNT signaling by
inhibiting GSK3β activity. WNT inhibition of GSK3β results in
phosphorylation of MAP1A and MAP1B, which promotes microtubule assembly and
axonal outgrowth. Tbr1 activates Kif1a, a
kinesin motor protein involved in synaptic vesicle trafficking. Furthermore,
Tbr1 activates Foxp2 and
Mef2c in layer 6 pyramidal neurons. Mef2c
promotes the development of excitatory synapses. Lastly, TBR1 promotes
expression of Cyp26b1 in layer 6 pyramidal neurons, which
controls RA levels and impacts synaptic development. Asterisks indicate hcASD
(red, reduced in Tbr1 mutants) and pASD (green, increased in
Tbr1 mutants) genes that are involved in these pathways.
Cell membrane and nuclear membrane (blue) are indicated. Pathways unique to
layer 5 and layer 6 are shown in orange and yellow, respectively. Convergent
pathways between layers 5 and 6 are highlighted in blue.
Tbr1 promotes the expression of Foxp2
(a hcASD gene) and Mef2c transcription factors (TFs) in layer 6
(Fazel Darbandi et al., 2018).
Mef2c promotes the development of excitatory synapses
(Harrington et al., 2016). However,
restoring Mef2cexpression in Tbr1 mutant
neurons failed to rescue their synaptic deficit, suggesting that decreased
expression of this TF alone does not underlie the synaptic deficits in
Tbr1 mutants.Tbr1 also promotes the expression of
Cyp26b1, a gene encoding a retinoic-acid (RA)-degrading
enzyme, in layer 6 pyramidal neurons. Restoring Cyp26b1
expression in primary cortical cultures from
Tbr1 CKOs rescued synaptic deficit
in vitro (Figures S9E and S9F). RA acts via RARα in synapses to promote protein
synthesis (Chen et al., 2014; Chen and Napoli, 2008). This suggests that
Tbr1’s control of RA levels, via
Cyp26b1, can impact synaptic development (Figure 7).While these three mechanisms appear to contribute to
Tbr1’s orchestration of synapse development, we
believe that Tbr1’s control of WNT signaling may be the
overriding Tbr1-dependent mechanism (Figure 7).
Tbr1 Promotion of WNT Signaling Drives Dendritic Spine
Maturation and Synaptogenesis on Layer 5 and Layer 6 Pyramidal Neurons
WNT signaling is essential in postsynaptic differentiation of excitatory
synapses by recruiting NMDA receptors via promoting PSD95 clustering and local
activation of CaMKII within dendritic spines (Ciani et al., 2011). Furthermore, CaMKII is required for
WNT-mediated spine growth and increased synaptic strength, thus promoting
postsynaptic maturation and differentiation (Ciani et al., 2011). Moreover, WNTexpression increases microtubule
unbundling and stability by signaling through the canonical pathways downstream
of GSK3β (Ciani et al., 2004). WNT
inhibition of GSK3β results in phosphorylation of microtubule-associated
proteins such as MAP1B. This interaction is essential for microtubule assembly,
axonal arborization and outgrowth (Ciani et al.,
2004).Transcriptomic and ISH analyses demonstrate that Tbr1
promotes expression of Wnt7b and Ctnnb1
(β-catenin) and represses expression of Gsk3β.
Wnt7b encodes a WNT ligand of the canonical WNT signaling
pathway (Rosso et al., 2005).
Ctnnb1 encodes β-catenin, the central intracellular
signaling protein of the canonical WNT signaling pathway (Ciani and Salinas, 2005). GSK3β is a
ubiquitously expressed kinase that represses the canonical WNT pathway by
targeting β-catenin for ubiquitin-mediated proteasomal degradation (van Noort et al., 2002). Restoring
Wnt7bexpression rescued the synaptic deficit in
Tbr1 mutant neurons in
vitro and in vivo (Fazel Darbandi et al., 2018). To test whether Wnt7b
is acting through an autocrine or paracrine mechanism, we introduced cortical
interneurons ectopically expressing Wnt7b into the deep layers
of Tbr1 CKOs. We measured their effect on
synapse density onto apical dendrites of WT and
Tbr1 CKO layer 6 neurons (Figure S9). Because we did not find
a statistically significant increase in synapse density, we surmise that WNT7B
primarily promotes synaptogenesis cell autonomously onto layer 6 pyramidal
neurons.Furthermore, restoring Kif1aexpression in layer 5
pyramidal neurons rescued synapses in the Tbr1
CKOs in primary cultures of the neonatal cortex. Kif1a is a
member of the kinesin family and functions as an anterograde motor protein that
controls vesicle delivery in the assembly and function of synapses (Guedes-Dias et al., 2019). GSK3β
phosphorylation of kinesins inhibits their activity and thereby reduces
anterograde dendritic transport (Gottschalk et
al., 2017; Morfini et al.,
2002). De novo
KIF1A mutations in human have been associated with intellectual
disability (Ohba et al., 2015; Yoshikawa et al., 2018) and hereditary
spastic paraplegia (Pennings et al.,
2020). In Drosophila, loss-of-function mutations in
KIF1A homolog Unc-104 causes defects in
synaptic transmission by disrupting the formation of mature boutons (Zhang et al., 2017). Thus, the rescue of
the dendritic spine and synaptic deficits in Tbr1 mutants via
LiCl and GSK3β-in-hibitor treatments could be, in part, attributed to the
enhanced activity of KIF1A proteins as a result of reduced GSK3β
activity.
LiCl and GSK3β Inhibitor Rescue Defects in Dendritic Spine and
Synaptic Density in Tbr1 Mutants
To further explore the hypothesis that reduced WNT signaling in
Tbr1 mutants underlies the reduction in synapses, we tested
whether a canonical WNT-signaling pathway agonist, LiCl or GSK3β
inhibitor, could rescue dendritic spine and synapse defects. Among LiCl’s
best validated mechanisms of action is inhibition of GSK3β, a central
kinase in the WNT/β-catenin and AKT pathways (Lenox and Wang, 2003).LiCl or GSK3β-inhibitor treatment (within 24 h) rescued the
dendritic spine density of Tbr1 mutant neurons in cortical
layers 5 and 6. Furthermore, either LiCl or GSK3β-inhibitor treatment
rescued excitatory and inhibitory synapse numbers within 24 h. Remarkably, a
single dose of LiCl at P30 led to a sustained rescue of synaptic density,
measured 4 weeks after treatment. These results suggest that the
Tbr1 mutant’s dendrites have most of the machinery
needed to make synapses but have a deficit of the essential signal(s) to
initiate synaptogenesis. Once the LiCl- or GSk3β-inhibitor-induced
synapses are formed, they appear to be relatively stable.Corticothalamic axons in the Tbr1
mutants fail to fully arborize within the anterior and anteromedial regions of
the thalamus (Fazel Darbandi et al.,
2018). This phenotype was also rescued within 24 h of LiCl or
GSK3β-inhibitor treatment, suggesting that the reduced WNT signaling
underlies the defect of axonal elongation and/or arborization in
Tbr1 mutants.In sum, we postulate that Tbr1 mutant layer 5 and layer
6 cortical neurons have reduced WNT signaling that underlies their defects in
dendritic spines, synapses, and axonal arborization. LiCl or GSK3β
inhibitor rescues each of these defects, perhaps through promoting WNT
signaling.
LiCl Treatment Rescues Social Interaction Deficit in
Tbr1 CKOs
We eliminated Tbr1’s function in cortical layer
5 pyramidal neurons. In most cortical areas, a minority of layer 5 neurons
express TBR1, whereas in rostral areas, including the PFC, TBR1 is expressed in
~85% of layer 5 excitatory neurons (Figure S1). The PFC has a central
function in distributed circuits that control higher cognitive and emotional
functions that are disrupted in neuropsychiatric disorders such as ASD.
Tbr1 CKOs are viable and fertile,
allowing us to study the impact of Tbr1 deletion on the
behavior of heterozygous and homozygous CKOs. The
Tbr1 CKOs showed no deficit in their motor
functions (rotarod and open field) and interest in novel objects. However,
Tbr1 homozygous CKOs showed a
reduction in social interaction with a juvenile mouse. This phenotype had
previously been demonstrated in mice with Tbr1haploinsufficiency (Huang et al.,
2014).Importantly, treating Tbr1 CKOs with
LiCl at P30 rescued the social deficit of Tbr1
CKOs (measured at P56–P80). Thus, perhaps the LiCl-mediated rescue of
synaptogenesis may underlie the rescue of the social behavior phenotype. In
studies of multiple neuropsychiatric phenotypes, face-valid rodent behavior has,
so far, not proven to be a reliable assay for therapeutics development in humans
(Sestan and State, 2018). However,
the observation here is notable in that it links a risk-specific mutation to an
identifiable molecular mechanism and circuit level behavior, offering important
traction for future investigations of ASD pathophysiology.
Insights into How Tbr1 May Contribute to ASD
Pathogenesis
Co-expression network analysis suggests that the de
novo mutations of ASD-risk genes are enriched in excitatory
projection neurons of cortical layers 5 and 6 in the PFCs during human mid-fetal
development (Willsey et al., 2013), cell
types that also express Tbr1. The functions of many ASD-risk
genes converge on pathways that control synaptogenesis, synaptic development,
and plasticity (Sanders et al., 2015).
Thus, in this study, we deleted Tbr1 in the excitatory neurons
of mouse layer 5 of the mPFC at a stage similar to human mid-fetal
development.Our single-cell transcriptomic analysis of FACS-purified layer 5 neurons
from the mPFC revealed that Tbr1 regulates other ASD genes,
including Ank2, Ap2s1,
Ctnnb1, Dpysl2, Map1a,
Rorb, Smarcc2 (orthologs of
high-confidence ASD [hcASD] genes), and Gsk3β (ortholog
of a probable ASD [pASD] gene) in either Tbr1
heterozygous or homozygous CKOs. Tbr1
heterozygous and homozygous CKOs demonstrated a decrease in dendritic spines and
excitatory and inhibitory synaptic densities and reduced sEPSCs and sIPSCs,
phenotypes that are convergent with Tbr1
CKOs, and constitutive Tbr1+/−. This suggests
that decreased TBR1 dosage in human may also impair synaptic development and
thereby increase the risk for ASD. While some of the other phenotypes detected
in Tbr1 mutants were only present in the
homozygotes, including defects in social interaction, these observations could
have relevance for ASD, as they denote biological processes that could be
altered in Tbr1 heterozygotes.
Tbr1 and Shank3 Mutants Convergently
Present Synaptic and Physiological Defects
The complex genetic variation underlying ASD has complicated efforts to
understand the mechanism associated with ASD pathology and therapies. A possible
solution for such complex diversity is to identify core mechanisms, in which
ASD-risk proteins may act convergently on a common pathway (State and Sestan, 2012). Many mutations are thought
to predispose to idiopathic ASDs by causing primary impairments in synaptic
transmission (Rosti et al., 2014; Sanders et al., 2015).Reduced or increased Shank expression in
Drosophila reduces WNT signaling and excitatory synapses
(Harris et al., 2016). In mouse,
reduced Shank3 impairs synaptic function by reduction in
dendritic arborization, excitatory synaptic density, synaptic transmission, and
Ih current (Yi et al.,
2016). Similarly, Tbr1 CKOs have evidence for
reduced WNT signaling and have reduced mature spine density and excitatory
synaptic density (Fazel Darbandi et al.,
2018). Likewise, Tbr1 CKOs have abnormal
Ih currents in cortical layer 5 (Figure S5) and layer 6 (Fazel Darbandi et al., 2018), although, in
Tbr1 CKOs, Ih is increased. TBR1 binds to the
Shank1, −2, and
−3 loci (P2 TBR1 ChIP-seq data; GEO: GSE119362)
(Fazel Darbandi et al., 2018),
although there are only subtle changes in Shank RNA expression
in the Tbr1 mutants. Thus, synaptic dysfunction and, perhaps,
reduced WNT signaling are common features of mouseTbr1 and
Shank3 mutants; these defects may be the core
pathophysiology of some forms of ASD.
LiCl as a Therapy for Neurodevelopmental Disorders that Have Reduced Synapse
Development
Currently, there are no treatments for ASD that address its core
biological defects. The ability to restore synapse numbers following lithium
administration in the Tbr1 mutant mice provides an insight to a
possible human therapy, especially given that LiCl has a long history of
clinical use.Our study suggests the value of future study of LiCl as a potential
treatment for ASDpatients with TBR1 mutations. If successful,
LiCl could also conceivably prove relevant for ASD syndromes beyond individuals
with TBR1 mutations, particularly where reduced synaptic development is a
central feature. In a clinical case report, LiCl was reported to reverse
clinical regression, stabilize behavioral abnormalities, and restore brain
functioning in two SHANK3patients with ASD (Serret et al., 2015). Additionally, it is plausible
that the mechanisms identified here could be relevant for patients with
Ank2, Ap2s1, Ctnnb1,
Dpysl2, Map1a, Rorb,
Smarcc2, and Gsk3β. We also showed
that Tbr1 CKOs had arborization defects of
their corticothalamic axons that were improved with LiCl, suggesting that LiCl
could also improve presynaptic defects. This is consistent with the evidence
that WNT signaling positively regulated presynaptic and postsynaptic development
(Ahmad-Annuar et al., 2006; Stamatakou and Salinas, 2014).It is critically important that any hypothesis regarding novel
treatments in ASD be subjected to rigorous blinded clinical testing. This is
particularly the case for an agent such as LiCl, which has well-known long-term
side effects and a narrow therapeutic window. Open-label trials of novel
compounds to treat core symptoms in ASD have repeatedly shown promising results
early (Choi et al., 2011), only to be
followed almost uniformly by negative well-controlled trials. The foregoing
consideration of potentially relevant biological mechanisms should not be
construed as an immediate clinical recommendation but rather as a justification
for additional in-depth and rigorous studies.Finally, it is remarkable that LiCl in Tbr1 mutant mice
restores dendritic spine density, synaptogenesis, and axon arborization. LiCl
has a rapid action (24 h); furthermore, the effect of a single dose lasts over 4
weeks. However, there were many features of the Tbr1 mutants
that did not appear to be rescued by LiCl, including increased layer 5 and layer
6 filamentous spine density and layer 6 dendritic morphogenesis. Thus, while
LiCl may have some promise as a therapy, it is improbable that it would fully
rescue normal brain function of ASDpatients with TBR1
mutations.
STAR★METHODS
LEAD CONTACT AND MATERIALS AVAILABILITY
All unique/stable reagents generated in this study are available from
the Lead Contact, Dr. John L. Rubenstein
(john.rubenstein@ucsf.edu), without restrictions.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals
All procedures and animal care were approved and performed in
accordance with the University of California San Francisco Laboratory Animal
Research Center (LARC) guidelines. All strains were maintained on a C57BL/6
background. Animals were housed in a vivarium with a 12hr light, 12hr dark
cycle. Postnatally, experimental animals were kept with their littermates.
For timed pregnancies, noon on the day of the vaginal plug was counted as
embryonic day 0.5.The Tbr1 allele was generated by
inGenious Targeting Laboratory (Ronkonkoma, NY). LoxP sites were inserted
into introns 1 and 3, flanking Tbr1 exons 2 and 3 (Fazel Darbandi et al., 2018). To enable
selection of homologous recombinants, the LoxP site in intron 3 was embedded
in a neo cassette that was flanked by Flp
sites. The neo cassette was removed by mating to a
Flp-expressing mouse to generate the
Tbr1 allele. Cre excision removes
exons 2 and 3, including the T-box DNA binding region, similar to the
constitutive null allele (Bulfone et al.,
1998). Rbp4-cre mice (Gensat KL100) were used to
delete Tbr1 in layer 5 projection neurons.
tdTomato
(Ai14) mice were crossed with
Tbr1mice and used as an endogenous
reporter. Tbr1 layer 5 knockout mice
(Tbr1 mutant) were generated by
crossing
Tbr1∷tdTomato
mice with
Tbr1∷Rpb4-cre+.
The specific gender and age of experimental animals can be found in the
Results section and corresponding
figure legends.
TRANSGENIC ANIMAL MODELS
The mouse strains used for this research project,
B6.FVB(Cg)-Tg(Ntsr1-cre)GN220Gsat/Mmucd, RRID:MMRRC_030648-UCD and
B6.FVB(Cg)-Tg(Rbp4-cre)KL100Gsat/Mmucd,
RRID:MMRRC_037128-UCD, were obtained from the Mutant Mouse Resource and Research
Center (MMRRC) at University of California at Davis, an NIH-funded strain
repository, and was donated to the MMRRC by MMRRC at UCD, University of
California, Davis. Made from the original strain (MMRRC:032081) donated by
Nathaniel Heintz, Ph.D., The Rockefeller University, GENSAT http://gensat.org/index.html and Charles
Gerfen, Ph.D., National Institutes of Health, National Institute of Mental
Health.Information about the generation and genotyping of the transgenic lines
used in this study can be found in the corresponding original studies:
Rbp4-Cre (Gong et al.,
2007), lox-STOP-lox-tdTomato (Ai14;(Madisen et al., 2010)). Mice were maintained on C57BL/6J
background.
METHOD DETAILS
Genomic DNA extraction and genotyping
Tissue samples were digested in a solution containing 1 mg/mL of
proteinase K, 50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM NaCl and 1% SDS.
Genomic DNA was extracted using a standard ethanol precipitation protocol.
Genotyping was performed with PCR-based assays using purified genomic DNA,
and primer-pair combinations flanking the deleted region and detecting
Cre and tdTomato alleles.
RNA extraction and cDNA synthesis
Total RNA was extracted from the cortices of wild-type mice at P0
using RNeasy Plus® Mini Kit (QIAGEN) following the
manufacturer’s protocol. First strand cDNA was synthesized using
Superscript reverse transcriptase II following manufacturer’s
protocol (Thermofisher). cDNA library was used as template to clone and
generate in situ probes.
Single-Cell RNA-sequencing (scRNA-seq) on FAC-Sorted Cells
Layer specific transcriptome profiling was conducted by using 10X
Chromium scRNA-seq on FAC-Sorted cells from medial prefrontal cortex of
Tbr1 and
Tbr1 heterozygous and homozygous
mutants at P5. The medial prefrontal cortex was dissected in HBSS from P5
mice. Cortices were dissociated using a Papain Dissociation System
(Worthington Biochemical Corporation) following manufacturer’s
protocol. tdTomato+ cells were sorted using BD FACS Aria II Cell
Sorter at Center for Advanced Technology (UCSF). Approximately 20,000
tdTomato+ cells were collected from each
sample. Following FAC-sorting, the cell suspensions were centrifuged at 300
× g for 5 min. Cells were washed for a total of 3 times with 1 mL 1X
PBS supplemented with 0.04% BSA. Following the final wash, the cell pellet
was resuspended with 25 μL of 1X PBS supplemented with 0.04% BSA.
Cell concentration for each sample was determined using trypan blue and a
hemocytometer. We targeted to capture approximately 5000 cells per each
genotype to generate scRNA-seq libraries. Single cell RNA-seq was performed
using 10X Chromium Single Cell 3′ Reagent Kit v2 following
manufacturer’s protocol. Library concentration was assessed with
Qubit dsDNA HS Assay Kit following manufacturer’s protocol
(Thermofisher). Library fragment size distribution was examined on the
Agilent Bioanalyzer 2100 (Agilent Technologies) and Agilent High Sensitivity
DNA Kit (Agilent Technologies) following manufacturer’s protocol.
Libraries were sequenced on Hiseq4000 at Center for Advanced Technology
(UCSF).
Computational Analysis of FAC-Sorted Layer 5 scRNA-Seq data
Read pre-processing
Single cell RNA-sequencing libraries were sequenced on Illumina
Hiseq4000 to an average depth of 45K reads per cell. Read quality
control, UMI counting, barcode counting, and alignment to the mouse
reference genome (mm10) were performed using the “cell-ranger
2.0.1” pipeline provided by the manufacturer.
Filtering and Normalization
The initial dataset contained 17,823 cells with an average of
892 genes per cell. Cells with greater than 30% of mitochondrial genes
were removed as this is indicative of poor-quality cells (n = 82). Cells
with fewer than 500 or more than 10,000 unique-molecular-identifier
(UMI) counts were removed as this often represents sequencing errors (n
= 163). Cells with fewer than 500 or more than 3,000 genes were removed
based on the distribution (n = 182). Genes which occurred in less than
0.01% of cells were also removed (n = 13065). The remaining 17,396 cells
and 14,933 genes were used for downstream analysis. No experimental
factors were determined to explain a disproportionate of expression
variance using the Single Cell Analysis Toolkit for gene Expression in R
(scater; v 1.9.15).Using the R package Seurat (v 2.3.4), the data was log
normalized for each cell by the total expression and scaled to 10,000
transcripts per cell. Variable genes were identified using the
FindVariabieGenes() function which calculates the
average expression and dispersion for each gene, then bins genes and
calculates a z-score for dispersion within each bin. The data was
scaled, centered, and regressed on the percent of mitochondrial gene
content, number of UMI counts, and the number of genes.
Cell-type Identification and Clustering
TSNE was generated using all principal components accounting for
more than 2% of the variance and a clustering resolution of 0.3 which
resulted in 12 clusters (average silhouette width 0.16). Three clusters
were identified as neuronal cells using known markers
Nrgn, Rorb, and
Cnih2.The raw data from the three identified neuronal clusters was
retained and filtered again based on the distribution of UMI counts and
the number of genes per cell (N = 11,943). We applied more stringent
filtering to genes by removing mitochondrial genes, ribosomal genes,
pseudogenes, genes that did not occur in 1% of neuronal cells, and genes
with a variation below the median variation across all genes (N =
7,174). The data normalized as described above and TSNE was generated
using all principal components accounting for more than 4% of the
variance and a clustering resolution of 0.3 which resulted in 6 clusters
(average silhouette width 0.15). Two clusters were identified as
atypical cells due to a reduced expression in excitatory neuronal
markers and subsequently removed from downstream DEX analysis.
Differential Gene Expression (DEX) Analysis and Gene Ontology
Enrichment
To identify gene signatures of each genotype, we used MAST and
the zero-inflated regression (zlm) method to compare raw UMI counts
(i.e., non-normalized counts) per gene across the cells in the
population (FDR < 0.05). Genes that pass a 0.05 significant
threshold are considered as significantly differentially expressed (DEX)
genes. Gene Ontology enrichment analysis of common differentially
expressed genes was performed using the R package goseq (v 1.34.1) using
all expressed genes (N = 7,174) as background.
Data and Code Availability
The data used in this publication have been deposited in
NCBI’s Gene Expression Omnibus (GEO) under accession number
GSE146298 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146298).
In addition, all the scripts that were used for analyzing scRNA-seq data
as well as the result files are available on https://github.com/aseveritt/Darbandi_TBR1_L5scRNAseq.
Primary Cell Culture and in vitro Rescue Assay
Primary Cell Culture
Cortex was dissected from P0
Tbr1 and
Tbr1 homozygous mutants and
dissociated using papain dissociation kit following
manufacturer’s protocol (Worthington). A total of 300,000 cells
were seeded into tissue culture slides pre-coated with poly-L-lysine (10
mg/ml, Sigma) and then laminin (5 mg/ml, Sigma), and grown in
vitro with media containing DMEM-H21 with 5% fetal bovine
serum for 3 hr. After the cells recovered, DMEM-H21 media was replaced
by Neurobasal medium containing B27 supplement, 25% glucose, and
glutamax overnight.
In vitro Rescue Assay
Syt4, Mef2c,
Kif1a and Rac3 cDNA was cloned
into pcDNA3.1(−) (Thermofisher Scientific).
Tbr1 mutant cells were
transfected with Syt4, Mef2c,
Kif1a, Rac3expression vectors and
Tbr1 were transfected with
mock empty vector using Lipofectamine 3000 (Invitrogen) for 6 hr.
Following incubation, the media was replaced by Neurobasal medium
containing B27 supplement, Penicillin/Streptomycin, 25% glucose, and
glutamax. Cultures were grown for 14 days in vitro.
After 14 days, cultures were washed 3 times with 0.5 mL 1X PBSfor 5 min
each and fixed for 15 min with 4% PFA in 1X PBS at RT. Fixed cells were
washed 3 times with 0.5 mL 1X PBS and blocked in 1X PBS containing 10%
Normal Serum, 0.1% Triton X- 100 and 2% BSA for 1 hr at RT. Primary
antibodies including mouse anti-Vglut1 (1:200, Synaptic Systems) and
rabbit anti-PSD95 (1:200, Cell Signaling; excitatory synapses), rabbit
anti-Vgat (1:500, Synaptic Systems) and mouse anti-gephyrin (1:200,
Synaptic Systems; inhibitory synapses) were diluted 1:200 in blocking
solution. Cells were stained for excitatory and inhibitory synapses with
primary antibodies for 48 hr at 4°C with gentle shaking. On a
shaker, the cells were washed 3 times with 0.5 mL 1X PBSfor 5 min each
and incubated with the secondary antibody for 2 hr (room temperature),
washed 3X with 1X PBS, and mounted. This experiment was repeated twice
(n = 2).
In vivo Synapse Rescue Assays
We performed in vivo rescue assay of synaptic
deficit in Tbr1 mutant mice using three different
approaches. First, we directly injected a lentivirus harboring WNT7B.
Second, we utilized a transplantation assay to deliver the protein of
interest (WNT7B) by introducing MGE progenitor cells, following previously
published MGE transplantation assay (Vogt et
al., 2015). Lastly, we used a single intraperitoneal injection of
LiCl to rescue the decrease in synapse numbers in Tbr1
mutants.
Direct lentiviral injection
In vivo rescue assay was carried out by cloning
Wnt7b into a Cre-dependent
lentiviral backbone
(pLenti-CAG-Flex-IRES-GFP).
CAG-Flex-GFP (empty vector) and
Wnt7b-IRES-GFP expressing lentivirus
(pLenti-CAG-Flex-Wnt7b-IRES-GFP) were generated in
HEK293T cells as previously reported (Vogt et al., 2015) using Polyplus jetPRIMEH®
transfection reagent following manufacturer’s protocol.Lentivirus containing CAG-Flex-GFP or
Wnt7b-IRES-GFP were injected in the SSCx of
Tbr1 wild-type as well as
Tbr1 heterozygous and
homozygous CKO pups at P1. For injections, a glass micropipette of 50
μm diameter (with a beveled tip) was preloaded with sterile
mineral oil and viral suspension was front-loaded into the tip of the
needle using a plunger connected to a hydraulic drive (Narishige) that
was mounted to a stereotaxic frame. P1 pups from
Tbr1 wild-type and
Tbr1 heterozygous and
homozygous CKOs were anesthetized on ice for 1–2 min before
injections. Each pup received 2–3 viral injections (150 nL per
site) in the right hemisphere. These sites were about 1 mm apart along
the rostral to caudal axis. Viral suspensions were injected into layer 6
of the neonatal SSCx. After injections, pups were put back with the
mother to recover after they began to move around on their own. Mice
were sacrificed 21 days after injection and transcardially perfused with
PBS followed by 4% PFA.
MGE-Derived Interneuron Transplantation Assay
A detailed protocol for the MGE transplantation assay has been
previously described (Vogt et al.,
2015). First, E13.5 MGEs from
Nkx2.1-cre∷tdTomato
embryos were dissected in ice-cold HBSS. Next, cells were mechanically
dissociated by repeated pipetting (10–15 times) through a 1000
μL plastic pipette tip in DMEM media that contained 10% fetal
bovine serum. Cells were dissociated in DMEM with 10% FBS that was
preconditioned in a tissue culture incubator at 37°C and with 5%
CO2 to achieve a physiological pH. The cells were then
transfected with either DlxI12b-GFP (control) or
DlxI12b-Wnt7b-GFP (WNT7B-GFP expressing). Cells
were transfected for 30 min at 37°C then pelleted by
centrifugation (3 min, 700 × g), and resuspended in 2-3 μL
of DMEM, put on ice, and then remaining media containing the transfected
MGE cells was removed before loaded into the injection needle. For
injections, a glass micropipette of 50 μm diameter (with a
beveled tip) was preloaded with sterile mineral oil and cells were
front-loaded into the tip of the needle using a plunger connected to a
hydraulic drive (Narishige) that was mounted to a stereotaxic frame.
Tbr1 Wild-type and
Tbr1 homozygous CKO P1 pups
were anesthetized on ice for 1-2 min before being placed into a molded
surface (modeling clay) for injections. Each pup received 2-3 injections
of cells (~100 nL per site) in the right hemisphere. These sites
were about 1mm apart along the rostral to caudal axis; cells were
injected into layers 5/6 of the neocortex. After injections, pups were
put back with the mother to recover after they began to move around on
their own. Mice were sacrificed 28 days after transplantation and
transcardially perfused with PBS followed by 4% PFA.
Lithium chloride (LiCl) injection
P59 and P30mice were administered a single intraperitoneal (IP)
injection of 400 mg/kg LiCl or saline in a volume of 4 ml/kg (Martin et al., 2018). Treated mice
were anesthetized at P60, 24 hr or 4 weeks after LiCl injection with
intraperitoneal injection of 100 mg/kg Ketamine containing 15 mg/kg
Xylazine. A separate cohort of P58mice were administered a single IP
injection of 400 mg/kg LiCl or saline in a volume of 4 ml/kg. Treated
mice were anesthetized 24 hr after LiCl injection with intraperitoneal
injection 100 mg/kg Ketamine containing 15 mg/kg Xylazine. All brains
were processed at P60. Animals were perfused transcardially with
ice-cold 1X PBS and then with 4% PFA in 1X PBS, followed by brain
isolation, 1-2 hr post-fixation, cryoprotected in 30% sucrose in PBS,
and cut frozen (coronally or sagittally) on a sliding microtome at
40μm for immunohistochemistry.
Histology
For P0 and P3 experiments, neonatal animals were anesthetized on
ice. For P21 and P56 experiments, animals were anesthetized with
intraperitoneal injection of 100 mg/kg Ketamine containing 15 mg/kg
Xylazine. Animals were perfused transcardially with cold PBS and then with
4% PFA in PBS, followed by brain isolation, 1-2 hr post-fixation,
cryoprotected in 30% sucrose in PBS, and cut frozen (coronally or
sagittally) on a sliding microtome at 40μm for immunohistochemistry
or in situ hybridization. All primary and secondary
antibodies were diluted in PBS containing 10% Normal Serum, 0.25% Triton
X-100 and 2% BSA. The following primary antibodies were used: mouse
anti-Vglut1 (1:200, Synaptic Systems), rabbit anti-Vgat (1:500, Synaptic
Systems), rabbit anti-PSD95 (1:200, Cell Signaling), mouse anti-gephyrin
(1:200, Synaptic Systems). The secondary antibodies for immunofluorescence
were Alexa Fluor-conjugated and purchased from Thermofisher. For in
vivo synapse immunohistochemistry, a total of n = 30 apical
dendrites were counted from each of
Tbr1,
Tbr1 heterozygous and
Tbr1 homozygous mutants. The
coronal sections were pre-treated with pepsin to enhance the staining.
Immunofluorescence specimens were counterstained with 1% DAPI to assist the
delineation of cortical layers. For in situ hybridization a
rostro-caudal coronal series of at least ten sections from n = 2 brains from
Tbr1 and
Tbr1 heterozygous and homozygous
mutants were examined. Anti-sense riboprobes for Calm2,
Kif1a, Wnt7b, and
Mgst3 were prepared as previously described (Cobos et al., 2005; Fazel Darbandi et al., 2016). We also
investigated cortical lamination within rostral cortex including PFCx of
wild-type brain at P3 and P21 using anti-sense riboprobes for lamination
markers Cux2, Rorb, Etv1,
Tbr1 and Nr4a2. ISH was performed
using digoxigenin-labeled riboprobes.
Image Acquisition and Analysis
Fluorescent and bright-field images were taken using a Coolsnap
camera (Photometrics) mounted on a Nikon Eclipse 80i microscope using NIS
Elements acquisition software (Nikon). Confocal imaging experiments were
conducted at the Cancer Research Laboratory (CRL) Molecular Imaging Center,
supported by Helen Wills Neuroscience Institute at UC Berkeley. Confocal
images were acquired using Zeiss LSM 880 with Airyscan with a 63X objective
at 1,024 × 1,024 pixels resolution with 2.0X optical zoom using ZEN
2.0 software. Brightness and contrast were adjusted, and images merged using
Photoshop or ImageJ software. ImageJ software was used for image processing.
For synapse counting (presynaptic and postsynaptic boutons), confocal image
stacks (0.4μm step size) were processed with ImageJ software. In
brief, background subtraction and smooth filter were applied to each stack.
Using a threshold function, each stack was converted into a
‘masks’ image. Furthermore, the channels were co-localized
with the Image Calculator plugging. Lastly, the number of co-localizations
were counted, and the length of each dendrite was measured in each of the
focal plane. Staining for control and mutant were done in parallel as well
as the image capturing.
Electrophysiology
Coronal brain slices (250 μm) including medial prefrontal
cortex were made from three mice (n = 3) at age p21-28 and at p56-p80.
Slicing solution was chilled to 4°C and contained (in mM): 234
sucrose, 26 NaHCO3, 11 glucose, 10 MgSO4, 2.5 KCl,
1.25 NaH2PO4, 0.5 CaCl2, bubbled with 5%
CO2/ 95% O2. Slices were incubated in artificial
cerebrospinal fluid (aCSF) at 32°C for 30 minutes and then at room
temperature until recording. aCSF contained (in mM): 123 NaCl, 26
NaHCO3, 11 glucose, 3 KCl, 2 CaCl2, 1.25
NaH2PO4, 1 MgCl2, also bubbled with 5%
CO2/ 95% O2. Neurons were visualized using
differential interference contrast or DODT contrast microscopy on an upright
microscope (Olympus). Rbp4-cre positive neurons were
identified by fluorescent visualization of cre-dependent tdTomato. We
obtained somatic whole-cell patch clamp recordings using a Multiclamp 700B
(Molecular Devices) amplifier and acquired with pClamp. Patch pipettes (2-5
MΩ tip resistance) were filled with the following (in mM): 130
KGluconate, 10 KCl, 10 HEPES, 10 EGTA, 2 MgCl2, 2 MgATP, 0.3
Na3GTP. All recordings were made at 32-34°C. Series
resistance was compensated in all current clamp experiments and monitored
throughout recordings. Recordings were discarded if Rs changed by >
25%. For spontaneous EPSC and IPSC recordings cells were held in voltage
clamp at −70 mV and +10mV, respectively. In both cases patch pipettes
were filled with the following (in mM): 135 Cesium Methanesulfonate, 8 NaCl,
10 HEPES, 0.3 EGTA, 5 QX314, 4 MgATP, 0.3 Na3GTP.
Behavioral Assays
Experiments were conducted during the light cycle (8am to 8pm). Mice
were habituated to investigator handling for 1-2min on three consecutive
days. On the testing day, mice were transferred to experimental room and
allowed to habituate for at least 45 minutes prior to testing. All behavior
assays were performed on mice age P56 to P80. We were blind to the genotypes
during scoring of videos.
Open-field test
An individual mouse was placed near the wall-side of 50 ×
50 cm open-field arena, and the movement of the mouse was recorded by a
video camera for 10 min. The recorded video file was analyzed with
Any-Maze software (San Diego Instruments). Time in the center of the
field (a 25 × 25 cm square) was measured. The open field arena
was cleaned with 70% ethanol and wiped with paper towels between each
trial.
Elevated plus maze test
An individual mouse was placed at the junction of the open and
closed arms, facing the arm opposite to the experimenter, of an
apparatus with two open arms without walls (30 × 5 × 0.5
cm) across from each other and perpendicular to two closed arms with
walls (30 × 5 × 15 cm) with a center platform (5 ×
5 cm), and at a height of 40 cm above the floor. The movement of the
mouse was recorded by a video camera for 10 min. The recorded video file
was analyzed with Any-Maze software and time in the open arms of the
apparatus was measured. The arms of the elevated plus maze apparatus was
cleaned with 70% ethanol and wiped with paper towels between each
trial.
Rotarod test
The assay consisted of four trials per day over the course of 2
days with the rotarod set to accelerate from 4rpm to 45rpm over 5
minutes. The trial started once five mice were placed on the rotarod
rotating at 4rpm in separate partitioned compartments. Each trial ended
when a mouse fell off, made three complete revolutions while hanging on,
or reached 300 s. Digital videos of the mice on the rotarod were
recorded from behind. The rotarod apparatus was cleaned with 70% ethanol
and wiped with paper towels between each trial.
Social interaction and novel object task
An individual mouse was allowed to habituate for 5 minutes in
their home cage prior to starting the trial. A juvenile (3-4 weeks old)
mouse of the same strain and sex was introduced to the home cage. After
5 minutes, the juvenile was removed from the home cage. After a 5 min
break a novel object (typically a plastic test tube cap) was introduced
into the home cage for five minutes. We scored videos offline, blind to
genotype. We measured the number of seconds the mouse spent with its
nose in direct contact with the novel object or engaged in social
interaction with the juvenile (defined as sniffing, close following, or
allo-grooming) in the 300 s following the time the juvenile or object
was introduced into the cage. In addition, we noted any
aggressive-appearing behaviors toward the juvenile, freezing, and
grooming behaviors. We repeated this behavioral assay on adult wild-type
and mutant mice that were treated with a single IP injection of LiCl and
compared to vehicle treated animals injected with saline.
QUANTIFICATION AND STATISTICAL ANALYSIS
All individual data points are shown as well as mean ± SEM. All
statistical analyses were performed using GraphPad Prism 7.0 software.
Statistical significance was accepted at the level p < 0.05. We used
Student’s t test to compare pairs of groups if data were normally
distributed (verified using Lillie test). If more than two groups were compared,
we used one-way ANOVA with post hoc tests between groups corrected for multiple
comparisons (Holm-Sidak or Tukey). Forthe ISH experiments reported in this paper
n = 2 represents two biological replicates for each of the reported genes. We
examined the changes in synapse numbers of n = 30 different dendrites from n = 2
animals for each genotype. Whole-cell patch clamp experiments at P21 and P56
were conducted from n = 3 different animals for each age and genotype. Lastly,
behavioral analysis was conducted from n = 11/8/9, wild-type/
heterozygous/homozygous animals. The specific n for each experiment as well as
the post hoc test, exact F and corrected p values can be found in the Results section.
DATA AND CODE AVAILABILITY
Data and MATLAB analysis scripts are available upon request from the
Lead Contact.
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Rabbit anti-Vglutl polyclonal antibody
Synaptic Systems
Cat# 135303
RRID: AB_887875
Mouse anti-PSD95 antibody
NeuroMab (UC Davis)
Cat# 75-028
RRID: AB_2307331
Rabbit anti-Vgat polyclonal antibody
Synaptic Systems
Cat# 131002
RRID: AB_887871
Mouse anti-Gephyrin polyclonal antibody
Synaptic Systems
Cat# 147011
RRID: AB_887717
Goat anti-Rabbit IgG Alexa Fluor 488
Thermofisher Scientific
Cat# A-11008
RRID: AB_143165
Goat anti-Mouse Alexa Fluor 647
Thermofisher Scientific
Cat# A32728
RRID:
AB_2633277
Bacterial and Virus Strains
pLenti- CAG- Flex-Wnt7b-IRES-GFP
This paper
N/A
pLenti-DlxI12b-Wnt7b-GFP
This Paper
N/A
pLenti-DlxI12b-GFP
This Paper
N/A
pLenti-CAG-Flex-IRES-GFP
This
Paper
N/A
Chemicals, Peptides, and Recombinant Proteins
Sucrose
Sigma Aldrich
Cat# S5016
Sodium bicarbonate (NaHCO3)
Sigma Aldrich
Cat# S6014
Glucose
Sigma Aldrich
Cat# G5767
Magnesium sulfate (MgSO4)
Sigma Aldrich
Cat# 230391
Sodium phosphate monobasic monohydrate
(NaH2PO4)
Sigma Aldrich
Cat# P9638
Potassium chloride (KCl)
Sigma Aldrich
Cat# P9333
Calcium chloride dehydrate
(CaCl2)
Sigma Aldrich
Cat# 223506
Magnesium chloride dexahydrate
(MgCl2)
Sigma Aldrich
Cat# M9272
Potassium gluconate (KGluconate)
Sigma Aldrich
Cat# P1847
HEPES
Sigma Aldrich
Cat# H3375
EGTA
Sigma Aldrich
Cat# E4378
Adenosine 5′-triphosphate magnesium
salt (Mg-ATP)
Sigma Aldrich
Cat# A9187
Guanosine 5′-triphosphate sodium salt
hydrate (Na3GTP)
Authors: Lorenza Ciani; Kieran A Boyle; Ellen Dickins; Macarena Sahores; Derek Anane; Douglas M Lopes; Alasdair J Gibb; Patricia C Salinas Journal: Proc Natl Acad Sci U S A Date: 2011-06-13 Impact factor: 11.205
Authors: William L McKenna; Jennifer Betancourt; Kathryn A Larkin; Benjamin Abrams; Chao Guo; John L R Rubenstein; Bin Chen Journal: J Neurosci Date: 2011-01-12 Impact factor: 6.167
Authors: Pedro Guedes-Dias; Jeffrey J Nirschl; Nohely Abreu; Mariko K Tokito; Carsten Janke; Maria M Magiera; Erika L F Holzbaur Journal: Curr Biol Date: 2019-01-03 Impact factor: 10.834
Authors: Stephan J Sanders; Xin He; A Jeremy Willsey; A Gulhan Ercan-Sencicek; Kaitlin E Samocha; A Ercument Cicek; Michael T Murtha; Vanessa H Bal; Somer L Bishop; Shan Dong; Arthur P Goldberg; Cai Jinlu; John F Keaney; Lambertus Klei; Jeffrey D Mandell; Daniel Moreno-De-Luca; Christopher S Poultney; Elise B Robinson; Louw Smith; Tor Solli-Nowlan; Mack Y Su; Nicole A Teran; Michael F Walker; Donna M Werling; Arthur L Beaudet; Rita M Cantor; Eric Fombonne; Daniel H Geschwind; Dorothy E Grice; Catherine Lord; Jennifer K Lowe; Shrikant M Mane; Donna M Martin; Eric M Morrow; Michael E Talkowski; James S Sutcliffe; Christopher A Walsh; Timothy W Yu; David H Ledbetter; Christa Lese Martin; Edwin H Cook; Joseph D Buxbaum; Mark J Daly; Bernie Devlin; Kathryn Roeder; Matthew W State Journal: Neuron Date: 2015-09-23 Impact factor: 17.173
Authors: P-M Martin; R E Stanley; A P Ross; A E Freitas; C E Moyer; A C Brumback; J Iafrati; K S Stapornwongkul; S Dominguez; S Kivimäe; K A Mulligan; M Pirooznia; W R McCombie; J B Potash; P P Zandi; S M Purcell; S J Sanders; Y Zuo; V S Sohal; B N R Cheyette Journal: Mol Psychiatry Date: 2016-10-18 Impact factor: 15.992
Authors: Marissa Co; Rebecca A Barnard; Jennifer N Jahncke; Sally Grindstaff; Lev M Fedorov; Andrew C Adey; Kevin M Wright; Brian J O'Roak Journal: J Neurosci Date: 2022-08-09 Impact factor: 6.709
Authors: Siavash Fazel Darbandi; Andrew D Nelson; Emily Ling-Lin Pai; Kevin J Bender; John L R Rubenstein Journal: J Neurodev Disord Date: 2022-02-05 Impact factor: 4.025
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.