Role of the cytoplasmic isoform of RBFOX1/A2BP1 in establishing the architecture of the developing cerebral cortex

Roles of Rbfox1-iso2 in neuronal positioning during corticogenesis

We first examined the role of cytoplasmic Rbfox1-iso2 in the migration of newly generated
excitatory neurons by acute knockdown in utero. We designed three RNAi vectors, pSuper-mRbfox1-iso1,
pSuper-mRbfox1-iso2, and pSuper-mRbfox1-iso1/2, against distinct regions in the mRbfox1 coding sequences. While Rbfox1-iso2 is highly homologous to Rbfox1-iso, we confirmed
that pSuper-mRbfox1-iso2 specifically silenced mRbfox1-iso2 (Fig. 1a). Notably, pSuper-mRbfox1-iso2 had only faint effects on mRbfox1-iso1 expression
under the conditions where pSuper-mRbfox1-iso1 silenced the isoform in COS7 cell transfection
experiments (Fig. 1a). On the other hand, pSuper-mRbfox1-iso1/2, which targets for a common sequence of
Rbfox1-iso1 and Rbfox1-iso2, also knocked down mRbfox1-iso2 under the conditions (Fig. 1a).

Fig. 1. Role of mRbfox1-iso2 in neuronal migration during mouse brain development. a Characterization of RNAi vectors. pCS-MT-mRbfox1-iso1 or pCS-MT-mRbfox1-iso2 (Myc-iso1
or Myc-iso2) was transfected into COS7 cells with pSuper control vector (vector),
pSuper-mRbfox1-iso1, pSuper-mRbfox1-iso2 or pSuper-mRbfox1-iso1/2. After 48 h, cells
were harvested and subjected to western blotting (20 ?g protein per lane) with anti-A2BP1(Rbfox1).
Anti-Sept11 was used for loading control. The values represent relative density of the bands normalized to Sept11 with ImageJ software.
The band intensity of the control experiments was defined as 1. b Migration defects of mRbfox1-iso2-deficient cortical neurons. pCAG-EGFP was electroporated
in utero with pSuper control vector, pSuper-mRbfox1-iso2 (iso2), or pSuper-mRbfox1-iso1/2
(iso1/2) into cerebral cortices at E14.5. Coronal sections were prepared at P3 and
immunostained for GFP (white) and DAPI (blue). Bar, 100 ?m. c Quantification of the distribution of transfected neurons in distinct parts of the
cerebral cortex (bin 1–5 and IZ) for each condition shown in b. Error bars indicate SD; control (n?=?5), iso2 (n?=?5), iso1/2 (n?=?3). **p??0.01 *p??0.05 by Tukey-Kramer LSD. d Characterization of an RNAi-resistant version, mRbfox1-iso2R. pCAG-Myc-mRbfox1-iso2
(Myc-iso2) or pCAG-Myc-mRbfox1-iso2R (Myc-iso2R) was transfected into COS7 cells with
pSuper control vector or pSuper-mRbfox1-iso2. After 48 h, cells were harvested and
subjected to western blotting with anti-Myc. The blot was reprobed with anti-Sept11.
e Rescue experiments of mRbfox1-iso2 knockdown. pCAG-EGFP was coelectroporated in utero
with pSuper control vector plus pCAG vector (control) or pSuper-mRbfox1-iso2 together
with pCAG vector (empty) or pCAG-Myc-mRbfox1-iso2R (iso2R) into cerebral cortices
at E14.5, followed by fixation at P3. Coronal sections were stained as in b. Bar, 100 ?m. f Quantification of each condition shown in e. Analyses were done as in c. Error bars indicate SD; control (n?=?5), iso2 (empty, n?=?5), iso2?+?iso2R (n?=?5). **p??0.01 by Tukey-Kramer LSD. g Positioning of mRbfox1-iso2-deficient neurons at E17.5, P7, and P16. In utero transfection
was done as in b at E14.5, followed by fixation at the indicated time points. Coronal sections of
control and knockdown samples were immunostained for GFP. Bars, 100 ?m

pCAG-EGFP was electroporated in utero with pSuper-H1.shLuc control or pSuper-mRbfox1-RNAi
vectors into ventricular zone (VZ) progenitor cells of mice brains at E14.5, and localization
of the transfected cells and their progeny was visualized at postnatal day (P) 3.
While control neurons were positioned normally at the superficial layer (bin 1; layers
II~III) of cortical plate (CP), a considerable portion of cells transfected with pSuper-mRbfox1-iso2
or pSuper-mRbfox1-iso1/2 remained in the lower zone of CP and intermediate zone (IZ)
(Fig. 1b, c). To examine the specific role of mRbfox1-iso2, we hereafter used pSuper-mRbfox1-iso2
throughout the study. It should be noted that many mRbfox1-iso2-deficient neurons
reached the superficial layer (Fig. 1b, c). Transfection efficiency may explain the result; mRbfox1-iso2 was incompletely knocked
down in neurons incorporating low amount of the RNAi vector. Rescue experiments were
then performed to rule out off-target effects by the use of mRbfox1-iso2R that was
resistant to pSuper-mRbfox1-iso2 (Fig. 1d). When pSuper-mRbfox1-iso2 was coelectroporated with pCAG-Myc-mRbfox1-iso2R, the
positional defects were rescued at P3 (Fig. 1e, f), indicating that the abnormal positioning observed was indeed caused by reduced
expression of mRbfox1-iso2. When we analyzed the effects of mRbfox1-iso2-knockdown
on the neuronal migration at E17, positioning defects were detected at this stage;
while most control neurons were migrating in CP, many mRbfox1-iso2-deficient neurons
were still in IZ (Fig. 1g). We then examined the long-term effects at P7 and P16 and found that many mRbfox1-iso2-deficient
cells did not make it to the correct target destination (layers II–IV) at these time
points. These results suggest that efficient mRbfox1-iso2-silencing caused defects
in the radial migration of cortical neurons.

Since cell morphology is closely associated with migration, we looked into the shape
of mRbfox1-iso2-deficient neurons with abnormal positioning at E17.5 (Fig. 1g). Consequently, the deficient neurons transformed smoothly into bipolar status in
the upper IZ and had apparently normal bipolar morphology in CP (Fig. 1g; E17.5), suggesting that mRbfox1-iso2 is not directly involved in neuronal morphology
before and during migration. However, it should be noted that we here analyzed fixed
cells and the obtained results are considered as snapshots at the time point.

Time-lapse imaging of migration of mRbfox1-iso2-deficient neurons in cortical slices

Since we might miss the important morphological changes in the snapshot data, we next
examined the morphology of migrating mRbfox1-iso2-deficient neurons by the use of
time-lapse imaging. VZ progenitor cells were coelectroporated with pCAG-EGFP together
with the control vector or pSuper-mRbfox1-iso2 at E14.5. When time-lapse imaging was
started at E16.5, mRbfox1-iso2-deficient cells were multipolar and some cells were
transforming into bipolar neurons as in the case of control cells (Fig. 2a). However, during the observation, migration profile of the deficient cell became
abnormal. Control neurons smoothly moved into CP and migrated toward the pial surface
after multipolar-bipolar transition in the upper IZ (Fig. 2b, c, control panels, and Additional file 1: Video 1). On the other hand, although the deficient cells showed normal multipolar-bipolar
transition, they then frequently remained stranded in the upper IZ and subsequent
migration was significantly prevented during the imaging time period (~24 h) (Fig. 2b, c, iso2 panels, and Additional file 2: Video 2).

Fig. 2. Time-lapse imaging of migration of mRbfox1-iso2-deficient neurons. Analyses were repeated
three times for each case, and the migration pattern was observed for 10 cells in
each imaging. Representative results were shown in a–d and f. a Confocal images of cortical slices at the beginning of time-lapse imaging. E14.5
cortices were coelectroporated in utero with pCAG-EGFP together with pSuper control
vector or pSuper-mRbfox1-iso2 (iso2), followed by coronal section slice preparation
at E16.5 and time-lapse imaging (Additional file 1: Video 1 and Additional file 2: Video 2 for control and iso2, respectively). Note that there were no differences
in transfection efficiency between the experiments. Bars in a–d, 20 ?m. b Time-lapse imaging of control and mRbfox1-iso2-deficient neurons (iso2) at the IZ-CP
boundary. Magnified images were depicted from Additional file 1: Video 1 (control) and 2 (iso2). c Tracing of control or the deficient neurons (iso2) in b. Migratory tracks of four representative cells were traced and shown as color lines. d Time-lapse imaging of control and the deficient neurons migrating in CP (Additional
file 3: Video 3 and Additional file 4: Video 4 for control and iso2, respectively). e Calculation of migration velocity of control and the deficient neurons in middle-upper
CP. Ten cells were analyzed in each experiment (n?=?3). Error bars indicate SD; **p??0.01 by Student’s t test. f pCAG-EGFP was electroporated with pCAG-PACKmKO1 together with pSuper control vector
or pSuper-mRbfox1-iso2 (iso2) into cerebral cortices at E14.5. Coronal sections were
prepared at E17.5 and immunostained with anti-GFP. Centrosome (red) and nuclei (blue) were also visualized. Representative images of migrating neurons in the lower CP
were shown. Bar, 5 ?m. g Quantification of the length of leading process of control and the deficient neurons
(iso2) in f. Numbers of cells used for calculation in g and h are 100 in each brain (n?=?3). Error bars indicate SD. h Distance between centrosome and the top of nucleus was measured for control and the
deficient (iso2) cells. Electroporation was done as in f. Error bars indicate SD; **p??0.01 by Student’s t test

It is notable that many mRbfox1-iso2-deficient cells still crossed IZ and moved into
CP. We thus monitored the migration of such cells in CP. While control cells showed
smooth locomotion in CP (Fig. 2d, control panels, Additional file 3: Video 3), the deficient cells exhibited characteristic migration defects (Fig. 2d, iso2 panels, e and Additional file 4: Video 4). While the deficient cells maintained apparently normal bipolar shape during
radial migration (Fig. 2f, g), the distance between the nucleus and the preceding centrosome (N-C distance) was
abnormally longer, suggestive of the impaired nucleokinesis (Fig. 2h). Nucleokinesis is the process of translocation of the nucleus into the proximal
leading process during radial migration and dependent on the microtubule dynamics
33]. Although dynamic changes of the microtubule function significantly affect the radial
migration profile and we showed the N-C distance abnormality (Fig. 2h), cell shapes in snapshot data do not satisfactorily reveal changes in the microtubule
dynamics and thus time-lapse imaging analyses are very useful. Collectively, mRbfox1-iso2
may be involved in the crossing over the IZ-CP border after multipolar-bipolar transition
in the upper IZ and subsequent radial migration in CP. We suppose that the migration
defects observed depend on the degree of knockdown; while migration defects might
take place at the IZ-CP border when the RNAi effect is strong, the aberrant migration
phenotype might be observed in CP after crossing the border when the RNAi effect is
relatively weak.

Involvement of mRbfox1-iso2 in the terminal translocation of cortical neurons

At the end of radial migration in CP, the migratory mode of cortical neurons changes
to the terminal translocation, a crucial step for the completion of neuronal migration,
just beneath the marginal zone (MZ) 34]. We asked if mRbfox1-iso2-knockdown has some effects on the terminal translocation.
As shown in Fig. 3, terminal translocation was not completed when mRbfox1-iso2 was silenced. The deficient
neurons could not enter the outermost region of the CP termed primitive cortical zone,
although the tip of the leading process reached MZ. These results indicate that mRbfox1-iso2
is involved in the terminal translocation as well as radial migration. Since neuronal
somas move quickly in a radial glia-independent manner during the terminal translocation,
nucleokinesis is most likely to be essential for the terminal translocation. Therefore,
migration defects observed in the radial migration and terminal translocation may
be caused by the same molecular mechanism (impaired nucleokinesis), although further
molecular analyses are crucial to address this issue.

Fig. 3. Role of mRbfox1-iso2 in the terminal translocation of cortical neurons. a mRbfox1-iso2-knockdown hampers the terminal translocation. Cerebral cortices were
electroporated with pCAG-EGFP together with pSuper vector (control) or pSuper-mRbfox1-iso2
(iso2) at E15.5. Coronal sections were prepared at P3 and immunostained with anti-GFP
(white) and DAPI (blue). Dotted lines represent the pial surface (upper) and top of CP (lower). MZ marginal zone, PCZ primitive cortical zone. b Statistical analyses of a. Distance between the top of CP and the cell soma was measured. Representative slices
form 5 (control) or 4 (iso2) brains were analyzed for each analysis. Numbers of cells
used for each calculation were 100. Error bars indicate SD; **p??0.01 by Student’s t test

Role of mRbfox1-iso2 in the proliferation of neuronal progenitor and stem cells

Since prolonged cell cycle causes delayed cortical neuron migration during corticogenesis
35], we analyzed the effects of mRbfox1-iso2-silencing on the cell cycle of progenitor
and stem cells in VZ/subventricular zone (SVZ). When S-phase cells were labeled with
EdU to detect DNA replication, mRbfox1-iso2-deficient cells entered S-phase to an
extent similar to the control cells (Fig. 4a, b). These results strongly suggest that mRbfox1-iso2 does not participate in the proliferation
of VZ/SVZ cells. Also, distribution of the EdU/EGFP-double-positive cells in VZ/SVZ
was not altered by the knockdown (Fig. 4a).

Fig. 4. Effects of mRbfox1-iso2-silencing on the cell cycle of VZ/SVZ progenitor and stem
cells. a Effects of mRbfox1-iso2-silencing on EdU incorporation were examined. Cortices were
electroporated in utero with pCAG-H2B-EGFP together with pSuper vector (control) or
pSuper-mRbfox1-iso2 (iso2). Coronal sections were visualized for GFP (green) and EdU (red). Arrowheads indicate EdU/GFP double positive cells. Dotted lines represent ventricular surface. Bar, 5 ?m. b Quantification of EdU/GFP double positive cells among GFP-positive ones (n?=?100) in a. Two sections were used per brain (control, n?=?5; iso2, n?=?4). Error bars indicate SD. c Effects of mRbfox1-iso2-silencing on cell cycle exit. Differentiated neurons are
EdU/GFP double positive while EdU/Ki67/GFP-triple-positive cells maintain progenitor
potency. Arrowheads indicate triple-positive cells. d Quantification of EdU/Ki67/GFP-triple-positive cells among GFP/EdU double positive
ones (n?=?100) in c. The ratio of the triple-positive cells over the total double positive ones in mRbfox1-iso2-silencing
experiments was similar to that in the control ones. Two sections were used per brain.
Error bars indicate SD (control, n?=?3; iso2, n?=?3)

We further looked into the effects of mRbfox1-iso2-silencing on the proliferation
of neuronal progenitor and stem cells by triple staining for EdU, GFP, and Ki67, a
marker for all active phases of the cell cycle except the quiescent G0 state. In this
analysis, cells still in proliferating after EdU incorporation could be identified
as EdU/Ki67-double-positive while neurons that differentiated after EdU incorporation
would be EdU-positive but Ki67-negative. Consequently, between control and mRbfox1-iso2-deficient
cells, no statistical differences were observed in the ratio of EdU/Ki67/GFP-triple-positive
cells to EdU/GFP-double-positive cells, indicating that mRbfox1-iso2-deficient progenitor
and stem cells differentiated to neuronal cells at a rate similar to the control cells
(Fig. 4c, d).

Taken together with the result that mRbfox1 was limitedly expressed in CP and upper
IZ but not VZ/SVZ during corticogenesis 20], we concluded that the positioning defects by mRbfox1-iso2-silencing resulted from
abnormal neuron migration.

mRbfox1-iso2 regulates axon elongation and dendrite development in vivo

Synaptic dysfunction and disrupted synaptic network are known to be involved in pathogenesis
of neurodevelopmental and psychiatric disorders. We thus looked into the effects of
mRbfox1-iso2-silencing on axon elongation into the contralateral hemisphere. Consequently,
axon density became lower after leaving the corpus callosum (Fig. 5a, b), and the phenotype was rescued by coexpression of mRbfox1-iso2R (Fig. 5b). Notably, axons from the hemisphere containing the deficient cells eventually ended
up elongating and reaching the contralateral white matter at P7 (data not shown) and
P16 (Fig. 5c). However, it should be noted that such axons did not extend efficiently into the
cortical layer structure even at P16 (Fig. 5c). These results suggest that mRbfox1-iso2 plays a role in the axon growth and extension
into the contralateral cortex.

Fig. 5. Role of mRbfox1-iso2 in the axon growth of cortical neurons in vivo. a pCAG-RFP was electroporated with control pSuper vector (control) or pSuper-mRbfox1-iso2
(iso2) into cerebral cortices at E14.5. Coronal sections were prepared at P3. Hematoxylin
staining of a slice was also shown. Bar, 1 mm. b Quantitative analyses of the ratio of the intensity of RFP-positive axons in the
area (white) of contralateral cortex to that in the area (green) of ipsilateral one in a. Rescue experiments were also done by cotransfection with pCAG-Myc-mRbfox1-iso2R
(iso2R). Error bars indicate SD; control (n?=?5), iso2 (n?=?5), iso2?+?iso2R (n?=?4); **p??0.01 by Tukey-Kramer LSD. c Representative images of the terminal arbors of axons expressing GFP with pSuper
vector (control) or pSuper-mRbfox1-iso2 at P16. Note that axons of the deficient neurons
reached contralateral hemisphere at the time point, whereas they did not extend efficiently
into the cortical layer structure. Densitometric analyses of GFP fluorescence intensity
were also carried out. Blue (control) and red (iso2) lines, average; shadow, SD (control, n?=?6; iso2, n?=?6). Bar, 200 ?m

We next examined the role of mRbfox1-iso2 in dendritic arbor formation in vivo. Introduction
of pSuper-mRbfox1-iso2 at E14.5 into VZ cells resulted in highly abrogated dendritic
arborization at P30 (Fig. 6a). As shown in Fig. 6b, c, the total length of dendrites per cell and branching point number at each successive
10-?m Sholl radius were both significantly decreased in mRbfox1-iso2-deficient neurons,
strongly suggesting that mRbfox1-iso2 is essential for dendritic arbor formation and
maintenance. Taken together, functional loss of mRbfox1-iso2 is most likely to impair
neuronal connectivity through defective axon and dendrite development.

Fig 6. Role of mRbfox1-iso2 in the dendrite growth in cortical neurons in vivo. a pCAG-loxP-GFP was electroporated for sparse expression with pCAG-M-Cre together with
pSuper vector (Control) or pSuper-mRbfox1-iso2 (iso2) at E14.5. Analyses were carried
out in cortical slices at P30. Representative average Z-stack projection images of
GFP fluorescence of upper layer cortical neurons were shown. b, c One section from each brain was analyzed for control (n?=?4) and iso2 (n?=?6) experiments. Representative data were shown. b Total dendritic length was calculated for neurons observed at P30. Error bars indicate SD; **p??0.01 by Student’s t test. c Branch points of dendrites were analyzed by Sholl test. Error bars indicate SD (n?=?19 neurons for control, n?=?20 for iso2); **p??0.01 by Tukey-Kramer LSD.

Involvement of mRbfox1-iso2 in spine morphology in vitro

To further test the possibility of the involvement of mRbfox1-iso2 in the spine morphology,
we carried out in vitro experiments. To this end, primary cultured mouse hippocampal
neurons were transfected with p?Act-EGFP together with the control vector or pSuper-mRbfox1-iso2
immediately after isolation, fixed at 21 div (days in vitro) and stained for GFP.
Under the conditions, spine density was decreased in neurons transfected with pSuper-mRbfox1-iso2
(Fig. 7a, b). We then looked into the spine morphogenesis in the deficient neurons by counting
four established spine morphology groups (i.e., mushroom, stubby, thin filopodia-like,
and branched spines) (Fig. 7c). When compared to the control neurons, relative percentage of mushroom (mature)
spine decreased and that of stubby spine increased concomitantly in the deficient
neurons (Fig. 7d). It is notable that the ratio of the branched spine was less than 2 % in these assay
conditions.

Fig. 7. Role of mRbfox1-iso2 in the dendritic spine morphology of primary cultured hippocampal
neurons. a Neurons were transfected with p?Act-EGFP together with pSuper vector (control) or
pSuper-mRbfox1-iso2 (iso2) when isolated, fixed at 21 div and stained for GFP. Bar, 20 ?m. Magnified images of dendrites are shown. Bars, 10 ?m (upper panels) and 5 ?m (lower panels). b Quantitative analyses of density of dendritic spines for each condition in a. Error bars show SD of the results from control (n?=?17) and iso2-transfected neurons (n?=?23). Experiments were repeated three times with similar results and representative
data were shown. **p??0.01 by Student’s t test. c Typical examples of mature (mushroom) and immature (stubby, thin filopodia, and branch)
spines. d Relative abundance of the four different spine types in neurons transfected with
pSuper vector (control) or pSuper-mRbfox1-iso2 (iso2) was analyzed. Relative percentages
of spine types were indicated in graphs. Error bars show SD of the results from 150 spines (control, 17 cells; iso2, 23 cells). Experiments
were repeated three times with similar results and representative data were shown.
**p??0.01 by Student’s t test. Note that branched spine was hardly detected under the conditions used (less
than 2 % of total spine number)