Drebrin Regulates Collateral Axon Branching in Cortical Layer II/III Somatosensory Neurons

Visualizing individual cortical excitatory neuron morphology using in utero electroporation sparse labeling

Layer II/III excitatory pyramidal CPNs extend interstitial collateral axon branches mainly in Layers II/III and V, both ipsilaterally and contralaterally (Greig et al., 2013; Hand et al., 2015). To target the Layer II/III neurons that populate the somatosensory cortex, we performed in utero electroporation (IUE) at embryonic day 15.5 (E15.5). We employed a Cre recombinase-dependent mCherry construct that contains a STOP cassette flanked by LoxP sites [Lox-Stop-Lox (LSL)] to label cell bodies and neurites and assess branching phenotypes at postnatal day 14 (P14), when cortical neuron process lamination is well established and potential interstitial axon branching phenotypes can be assessed (Hand et al., 2015). This enables sparse labeling by titrating the concentration of a co-electroporated Cre expression plasmid. We chose axon guidance receptors, signaling molecules and cytoskeletal regulators known to play roles in axon branching elsewhere in the nervous system and designed dominant negative (DN), overexpression (OE) DNA constructs, shRNAs and guide RNAs (gRNAs) to perturb their function in and performed an in vivo IUE candidate screen.

In our initial experiments, using this “single recombinase approach” described above, constructs were active from the time of electroporation (E15.5) and produced cortical neuron migration defects. One example is the migration defects observed after OE of the cytoskeletal regulatory protein drebrin (Fig. 1D,E). Since the regulation of cytoskeletal dynamics underlies both cell migration and axon branching, we hypothesized that constitutive Cre-driven gene-perturbations could preclude an assessment of laminar-specific interstitial axon branching (Cooper, 2013; Kapitein and Hoogenraad, 2015). Therefore, we bypassed these migration phenotypes by using a tamoxifen-inducible Cre construct (pCAG-CreERT2). Since Layer II/III CPN radial migration is complete by P0 and axon collateral branch initiation begins at P2.5 (Franco et al., 2011; Hand et al., 2015; Yoshinaga et al., 2021), we administered tamoxifen at P1 and then again at P2 using CreERT2. Use of CreERT2 in this experimental paradigm overcame cortical neuron migration phenotypes resulting from the use of constitutively active Cre in combination with various DNA constructs. However, we were unable to obtain robust neuronal labeling and had difficulty efficiently controlling the levels of Cre-mediated recombination (data not shown). We hypothesized that when using CreERT2, the low levels of Cre-mediated recombination and subsequent GFP expression were due to the short half-life of tamoxifen (Reid et al., 2014; Valny et al., 2016).

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Figure 1.

The bipartite in utero electroporation approach enables sparse and robust labeling of Layer II/III cortical neurons in vivo. A, Schematic of the “bipartite screening approach.” Inducible Cre recombinase and plasmids encoding Frt-Stop-Frt cassettes are used. B–C'', WT Layer II/III neurons electroporated with pCAG-YPET (1 μg/μl), pCAG-FSF-mCherry (1 μg/μl), pEF1-Flex-FlpO (500 ng/μl), and pCAG-CreERT2 (500 ng/μl). B–B'', Confocal fluorescent maximum intensity projection images of WT CD1 Layer II/III neurons from P3 pups that did not receive tamoxifen. Scale bar represents 100 μm. B, Layer II/III neurons are labeled with pCAG-YPET. B', No mCherry signal is detected since Cre is not recombined without addition of tamoxifen. B'', Close up of cortical region outlined in B by dashed box. Scale box represents 25 μm. C–C'', Confocal fluorescent maximum intensity projection images of Layer II/III neurons from P3 pups that received tamoxifen at P1 and P2. C, Layer II/III neurons are labeled with YPET and mCherry. C', mCherry signal is present in Layer II/III neurons. C'', Close up of cortical section demarcated in C by dashed box. D–G, Example confocal maximum intensity images of somatosensory coronal section from P14 brains from the two screening approaches. D, E, Examples using the constitutive Cre recombinase. Layer II/III neurons were electroporated with pCAG-LSL-mCherry (1 μg/μl; cell fill), pCAG-LSL-GFP (2 μg/μl; D), pCAG-LSL-Dbn1-IRES-GFP (2 μg/μl; E), and pCAG-Cre (500 ng/μl) at E15.5. DAPI staining was used to distinguish cortical layer boundaries. D, Control neurons show typical laminar patterning. Scale bar represents 100 μm. E, Dbn1 overexpression (OE) neurons show radial migration deficits (arrows). F, G, Examples using the “bipartite” approach. Layer II/III neurons were electroporated with pCAG-FSF-mCherry (1 μg/μl), pCAG-FSF-GFP (2 μg/μl; E), pCAG-FSF-Dbn1-IRES-GFP (2 μg/μl; F), and pCAG-CreERT2 (500 ng/μl) at E15.5. Tamoxifen was administered at P1 and P2. F, Single control neuron showing typical Layer II/III pyramidal neuron branching morphology. Dashed lines represent cortical layer boundaries. Scale bar represents 100 μm. Inset scale bar represents 5 μm. G, Single Dbn1 OE neuron. No radial migration phenotype is observed. Boxed region highlights area of ectopic protrusions (asterisk). H, Volcano plot depicting the distribution and density of axon branches within Layer IV as assessed in P14 cortices. Each dot represents an individual branch from a Layer II/III neuron. Data were collected from 279 branches observed in 50 neurons across three independent electroporation experiments. Branch position percentage was calculated by measuring the entire length of the axon and determining where the branch fell with respect to soma position. Green areas denote branch positions in the superficial and deeper quarters of Layer IV. I, Graph showing the distribution of Layer IV primary branches versus soma distance from Layer II/III/IV boundary. Each black dot represents a single neuron. Data were captured from 50 neurons across three independent electroporation experiments. We then used a linear regression model to determine a soma distance cutoff from the Layer II/III boundary for the purposes of scoring Layer II/III neurons for branching parameters. We used the assumption that Layer II/III neurons do not branch in the middle of Layer IV. Therefore, for this linear regression, the Y-intercept, where Xi = 0 branches, is 50.817. The 95% confidence interval for the intercept is (39.00866–62.6247634). We used the cutoff of Layer II/III neurons with somas greater than 39 μm from the Layer II/III/Layer IV border for scoring P14 neurons. The same calculations were performed for P21 neurons. The Y intercept where Xi = 0 branches is approximately 79.9 μm with a 95% confidence interval range of [71–88]. We therefore analyzed neurons with somas greater than 71 μm from the Layer II/III/Layer IV border for scoring P21 neurons.

To overcome these issues, we developed a “bipartite screening approach” (BP; Fig. 1A) that uses inducible Cre-dependent DNA expression constructs that are not reliant on continuous tamoxifen administration. We employed a combination of Cre recombinase-dependent and FlpO recombinase-dependent DNA expression constructs (Fig. 1A). We designed plasmids containing genes of interest embedded in a Frt-STOP-Frt (FSF) cassette instead of a Lox-STOP-Lox (LSL) cassette. We then delivered a combination of 3 or 4 plasmids (depending on the experimental paradigm) via IUE to Layer II/III CPNs at E15.5 that included: pCAG-FSF-mCherry (cell fill), pCAG-FSF-GENEX-IRES-GFP (gene of interest), pCAG-CreERT2, and pEF1-Flex-FlpO (Fig. 1A). We also engineered an IRES-GFP into our gene expression constructs in order to monitor construct expression in electroporated cells. In this experimental paradigm FlpO expression is CreERT2-dependent. We administered tamoxifen at P1 and then again at P2, activating pCAG-CreERT2 which in turn recombines pEF1-Flex-FlpO. FlpO is then constitutively “on” and can recombine the FlpO-dependent constructs to a much greater extent than we observed with a single tamoxifen-dependent Cre recombinase (Fig. 1A; see below). This approach allows for temporal control by employing a tamoxifen-dependent Cre recombinase, but also produces robust and sparse expression by titrating the expression of a Cre-dependent FlpO recombinase.

Robust and sparse labeling of Layer II/III neurons and visualization of collateral branching in vivo

Our bipartite screening approach (BP) enables robust and sparse labeling of Layer II/III neurons postmigration and satisfies the challenge of obtaining single cell resolution of cortical neuron axons and their branches to assess collateral branching phenotypes in vivo. To validate our method, we first performed bulk labeling of Layer II/III neurons using a pCAG-YPET DNA construct in addition to the other BP constructs, but without tamoxifen treatment. YPET is a yellow fluorescent protein (YFP) modified for FRET imaging, which we have found is robustly detected using antibodies directed against GFP in our electroporation paradigm and thus was used for these initial experiments (Nguyen and Daugherty, 2005). By P3, just after the onset of axon branch initiation, YPET-labeled Layer II/III neurons identified by immunohistochemistry (IHC) have cell bodies that populate the upper layers of the cortex (Layers II and III) and begin to extend axon branches in Layer V (Fig. 1B–B''). However, we could not visualize individual axon branches in detail using bulk labeling in conjunction with the BP approach without tamoxifen (Fig. 1B''). Importantly, IUE with this combination of plasmids in the absence of tamoxifen is not leaky, since IUE of FlpO-dependent mCherry in combination with the other constructs does not result in mCherry expression (Fig. 1B'). However, tamoxifen administration in conjunction with IUE of BP plasmids results in sparse and robust mCherry labeling of axons, allowing for detailed assessment of collateral branches across all cortical layers at the single cell level (Fig. 1C–C''). Together, these results demonstrate that the BP approach allows for: (1) temporal regulation of labeling and perturbation constructs; (2) segregation of neuronal migration from axon branching phenotypes; and (3) sparse Layer II/III neuron labeling with the ability to titrate the number of neurons labeled without sacrificing robust gene expression.

Using the BP approach, we analyzed the distribution of cortical Layer II/III neuron interstitial axon branches across all cortical layers in wild-type mice at P14. We found that Layer II/III CPNs do show a modest degree of collateral axon branching in Layer IV; however, these branches mainly occur at the boundaries between Layers II/III and IV (Fig. 1H,I). We segmented Layer IV in order to determine the distribution of branching within this layer, and we observed that very little axon branching occurs in the middle half of Layer IV (Fig. 1H). Interestingly, we observe a correlation between cell-body depth in Layer II/III with the number of axon collaterals formed in middle Layer IV; neurons closer to the Layer II/II–IV boundary exhibit collateral branches at a greater frequency within the middle of Layer IV than II/III neurons with cell bodies closer to Layer I (Fig. 1I). These observations are in line with previous findings showing that Layer II/III CPNs with cell bodies located more superficially lack axon arbors in Layer IV, while CPNs whose cell bodies are closer to the Layer IV border exhibit substantial axon branching within Layer IV in the developing mouse brain (Larsen and Callaway, 2006). Together, these results highlight the existence of distinct classes of Layer II/III neurons and support the idea that soma position of these neurons predicts axonal branching patterns.

Using these data, we identified a cutoff soma position to exclude Layer II/III neurons that reside close to the border of Layer IV and so are more prone to forming collateral branches in Layer IV (Fig 1I; see Materials and Methods). We chose to focus here only on Layer II/III neurons that normally do not branch in Layer IV in our search for determinants of laminar specific interstitial axon branching patterns.

Dbn1 overexpression leads to an increase in axon protrusions in Layer II/III CPNs

We initially screened candidate genes encoding cytoskeletal regulators using our constitutive Cre-dependent Single Recombinase Approach. Among these candidates were Dbn1, the Ras-family GTPase RalA, and the serine/threonine kinase GSK3β (Lalli and Hall, 2005; Kim et al., 2011; Ketschek et al., 2016). Overexpression of WT Dbn1 using the Single Recombinase Approach resulted in significant Layer II/III radial neuronal migration deficits as assessed at P14 (Fig. 1E). Because overexpression of several candidate genes, including Dbn1 and also a constitutively active-RalA (CA-RalA: RALAG23V; Lopez et al., 2008), resulted in severe layer 2/3 cortical neuron migration defects, we re-screened these candidates using the BP approach (Figs. 1F,G, 2A–D').

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Figure 2.

Dbn1 overexpression in Layer II/III neurons in vivo causes ectopic and stable protrusions along primary axons. A–D'', Confocal immunofluorescent maximum intensity projection images of Layer II/III P14 neurons from CD1 WT mice electroporated with overexpression candidates from the single recombinase/constitutive Cre approach using the bipartite labeling approach. All neurons were electroporated with: pCAG-FSF-GENEX-IRES-GFP (2 μg/μl), pCAG-FSF-mCherry (1 μg/μl), pEF1-Flex-FlpO, and pCAG-CreERT2. Pups were administered tamoxifen at P1 and P2 (1 mg/ml). Brains were harvested and sectioned at P14 and immunostained with antibodies directed against GFP, against dsRED to detect mCherry, and DAPI to assess potential branching patterns that may have been masked by migration deficits in the original screening approach (these same staining conditions are used throughout all following figures except where specified). A, Control neurons were electroporated with pCAG-FSF-GFP (2 μg/μl) in addition to other three bipartite constructs. Scale bar represents 100 μm. Littermate controls were used for statistical calculations within each experiment. A', mCherry labeling of electroporated neurons (cell fill). A'', Zoomed in view of middle Layer IV axon region. Scale bar represents 25 μm. B, Dbn1 OE. Neurons were electroporated with pCAG-FSF-Dbn1-IRES-GFP in addition to other three bipartite constructs. B', mCherry labeling of electroporated neurons. B'', Zoomed in view of Layer IV region of Layer II/III axon. C–C'', CA-RALA. C, Neurons were electroporated with pCAG-FSF-CA-RALA-IRES-GFP in addition to other three bipartite constructs. C', mCherry labeling of Layer II/III neuron. C'', Zoomed in view of Layer IV region along Layer II/III neuron axon. D–D'', CA-GSK3β. D, Layer II/III neurons were electroporated with pCAG-FSF-CA-GSK3β-IRES-GFP in addition to other three bipartite constructs. D', mCherry labeling of Layer II/III neurons. D'', Zoomed in view of Layer IV region along Layer II/III axon. E–H, Quantification of protrusion and branching phenotypes. E, Total number of branches within Layer IV for Dbn1 OE neurons. No significant difference was found in the number of branches between control and OE neurons. F, Total number of protrusions for Dbn1 OE Layer II/III neurons across all layers. Dbn1 OE causes an increase in the total number of axon protrusions (branches <10 μm). 3 IUEs with 8 control mice and 3 Dbn1 OE mice were analyzed. N = 24 control neurons and 35 Dbn1 OE neurons. G, Total number of Layer IV branches (>10 μm). Overexpression of CA-RalA causes an increase in the number of branches in Layer IV. Two IUEs with two to three pups per conditions were analyzed. N = 38 control neurons and 83 CA-RALA neurons. H, Overexpression of CA-GSK3β causes an increase in axon branches in Layer IV. Three IUEs with two to three pups per condition per IUE were analyzed. N = 64 control neurons and N = 66 CA-GSK3β neurons. Statistics: unpaired t test. Lines represent mean and SEM. Symbols represent individual neurons obtained from independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001.

Using the BP approach, we found that both CA-RalA and CA-GSK3β showed the presence of ectopic axon branches with aberrant trajectories in Layer IV, demonstrating that bypassing CPN migration defects and manipulating genes of interest just prior to the onset of collateral branching enables identification of branching defects (Fig. 2C–C'',D–D'',G,H; RalA: unpaired t test, CA-RalA: t₍₁₁₉₎ = 2.537, p < 0.0125; GSK3β: t₍₁₁₂₎ = 5.540, p < 0.0001). Dbn1 OE, however, did not result in an increase in total axon collateral branches within Layer IV (p = 0.1141), but instead led to the formation of ectopic protrusions along the entire length of the axon (Fig. 2B–B'',F; unpaired t test, t₍₅₇₎ = 2.562, p = 0.0131). We define neurites or axon collaterals less than 10 micrometers (μm) in length extending from the main CPN axon shaft as protrusions (Ketschek et al., 2016). These protrusions resemble our previous observations showing in WT Layer II/III CPNs at P3 that axon collateral initiation events are uniformly distributed across all cortical layers; however, later in the development of Layer II/III CPNs, a subset of protrusions in Layers II/III and V extend to form long and stable collaterals, while those in Layer IV are retracted (Hand et al., 2015). Additional studies also show that filopodial axon protrusions serve as the precursors for axon branches (Gibson and Ma, 2011; Hand et al., 2015; Ketschek et al., 2016; Armijo-Weingart and Gallo, 2017). Therefore, in Dbn1 OE CPNs, persistent axon protrusions at P14 provide evidence of collateral branch initiation events that fail to undergo later elongation or retraction.

Septin7, like Dbn1, plays a role in regulating the axon cytoskeleton during neurite outgrowth (Hu et al., 2012; Kaplan et al., 2017; Radler et al., 2023). Septin7 binds to MTs and promotes the entry of axonal MTs into filipodia during the initial steps of collateral branching, similar to what is thought to be the mechanism underlying Dbn1 function (Hu et al., 2012; Ketschek et al., 2016). We assessed Septin7 overexpression using the BP approach, but we did not observe any changes in axon branching or protrusions in Layer II/III CPNs (data not shown; unpaired t test, Branches, t₍₂₆₎ = 0.03849, p = 0.9696; Protrusions, t₍₂₆₎ = 1.077, p = 0.2912). This suggests that Dbn1 overexpression phenotypes are specific to drebrin and not a general property of this class of cytoskeletal binding proteins.

Taken together, these data show that Dbn1 overexpression causes an increase in stable axon protrusions in Layer II/III CPNs throughout cortical development. Dbn1 overexpression did not result in a change in the number of collateral axon branches extending from the primary axon, suggesting that these aberrant protrusions do not develop into stabilized, mature, collateral branches.

Dbn1 loss-of-function in Layer II/III neurons results in ectopic collateral axon branching within Layer IV

We next analyzed how LOF in genes encoding cytoskeletal regulatory proteins of interest in this study affects the formation of stable axon collateral branches. For RalA LOF, we overexpressed a constitutively inactive GDP-bound RalA (G26A; Vitale et al., 2005) in Layer II/III neurons using the BP approach and did not see any abnormal branching phenotypes (data not shown). We also used the BP approach to overexpress a dominant negative GSK3β (K85A; Dominguez et al., 1995) and did not observe any branching phenotypes (data not shown). However, based on the strong collateral axon branching phenotypes we observed following Dbn1 LOF in Layer II/III CPNs (see below), we focused on drebrin.

To assess Dbn1 LOF in Layer II/III CPNs, we used the BP approach in the context of a Dbn1^flox conditional allele (Zhang et al., 2018), leading to Dbn1 LOF at ∼P3, the normal onset of collateral axon branch initiation. Since Dbn1 OE results in an increase in collateral branch initiation (manifested as protrusions) but not collateral axon branch stabilization (mature axon branches), we hypothesized that Dbn1 LOF would result in a decrease in protrusions and an overall reduction in Layer II/III neuron axon branches. In heterozygous Dbn1^fl/+ littermate control mice subjected to BP IUE there were very few to no collateral axon branches in Layer IV, including within the middle region of Layer IV (Fig. 3A–A''). However, Dbn1 LOF, assessed using the BP approach in Dbn1^fl/fl mice, resulted in an increase in collateral axon branching in Layer II/III neurons within Layer IV compared to heterozygous controls observed at P14 (Fig. 3B–F; unpaired t test, t₍₇₁₎ = 3.121, p = 0.0026). This increase was seen within the middle region of Layer IV, where control neurons rarely branch, and therefore the presence of increased numbers of collateral axon branches in this region, even just one or two, is statistically significant (unpaired t test, t₍₇₀₎ = 3.198, p = 0.0021). There was no difference in collateral axon branching along the primary axon within Layer II/III or Layer V following Dbn1 LOF in Layer II/III CPNs (Fig. 3H,I; unpaired t test, t₍₇₀₎ = 1.686, p = 0.0963, t₍₄₃₎ = 0.2683, p = 0.7898). In contrast to Dbn1 OE in Layer II/III neurons, Dbn1 LOF did not produce differences in the number of primary axon protrusions within Layer IV (Fig. 3G; t₍₇₀₎ = 0.5682, p = 0.5717). Therefore, Dbn1 OE and LOF in Layer II/III neurons lead to distinct phenotypes; overexpressing Dbn1 causes an increase in axon protrusions but not stabilized axon collateral branches, whereas Dbn1 LOF in Layer II/III neurons results in ectopic axon collateral branches in Layer IV.

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Figure 3.

Dbn1 LOF in Layer II/III neurons in vivo results in ectopic branching in the middle of Layer IV. A–D', Confocal immunofluorescent maximum intensity projection images of Layer II/III P14 neurons from Dbn1 floxed and heterozygous littermate controls on a mixed background labeled via electroporation with pCAG-FSF-mCherry (1 μg/μl), pEF1-Flex-FlpO (500 ng/μl), and pCAG-CreERT2 (500 ng/μl) using the bipartite approach. Tamoxifen was administered at P1 and P2. A–A'', Example of Dbn1 heterozygous Layer II/III LOF neurons. A, Merged image with scale bar that represents 100 μm. A', mCherry labeling. A'', Zoomed in example of Layer IV region along Layer II/III neuron axon. Scale bar represents 50 μm. B–B'', Example of Dbn1 mutant Layer II/III neuron. B', mCherry labeling of mutant neuron. B'', Zoomed in the middle Layer IV region along Layer II/III axon. Arrows point to the presence of ectopic branches in this region. C–D', Examples of neurons that show dramatic ectopic branching in Layer IV due to loss of Dbn1. C', D', mCherry staining of neurons. Arrows point to ectopic branches in the middle Layer IV region. Dashed lines demarcate layer borders. E–I, Quantification of Layer II/III neuron axon branches and protrusions. Symbols represent individual neurons obtained from: Fl/+: 34 individual neurons from five mice obtained from two IUEs, Fl/Fl: 38 individual neurons from four mice obtained from two IUEs. For Layer V quantification, 24 Fl/+ and 21 Fl/Fl neurons were analyzed. Statistics: unpaired t test. Lines represent mean and SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To verify the requirement for Dbn1 in suppressing ectopic axon collateral branching, we performed rescue experiments using the Dbn1^flox allele to provide cell-autonomous loss of Dbn1 in Layer II/III CPNs. We introduced plasmids to express Dbn1 in Dbn1^fl/+ and Dbn1^fl/fl Layer II/III CPNs using a slightly modified BP approach (see Materials and Methods) and assessed axon branching phenotypes at P14. We found, again, that Dbn1 LOF Layer II/III neurons resulted in ectopic branches in the middle region of Layer IV with no presence of ectopic protrusions in this region (Fig. 4A–B''; two-way ANOVA (interaction effect): Branches, F_(3,284) = 5.116, p = 0.0018, Tukey's multiple comparisons test, p = 0.0006; Protrusions, F_(3,284) = 5.977, p = 0.0006, Tukey's multiple comparisons test, p > 0.9999). Dbn1 expression in Dbn1^fl/fl Layer II/III neurons rescued the Dbn1 ectopic branching LOF phenotype in the middle region of Layer IV (Fig. 4B–B'',G; two-way ANOVA, Tukey's multiple comparisons test, p = 0.0012). Dbn1 expression in Dbn1^fl/+ but not in Dbn1^fl/fl Layer II/III CPNs (which lack Dbn1), resulted in an increase in axon protrusions within Layer IV (Fig. 4B–B'',F; two-way ANOVA, Tukey's multiple comparisons test: p = 0.0004 (Dbn1^fl/+), p > 0.9999 (Dbn1^fl/fl)), further indicating that high levels of Dbn1 levels result in protrusions. Using a construct expressing YFP-tagged Dbn1, we also observed localization of Dbn1 within CPN axons in Layer IV (Fig. 4B''). These data support the conclusion that Dbn1 suppresses the formation of collateral axon branches in the middle region of Layer IV.

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Figure 4.

Phosphorylation of Dbn1 at S142 is not required for Dbn1 suppression of ectopic collateral branching in Layer IV. A–D'', Confocal immunofluorescent images of Layer II/III neurons at P14 obtained from Dbn1^flox mice. Mice were electroporated using a modified version of the bipartite approach with the following constructs: pCAG-CreERT2 (0.3–0.5 μg/μl), pAAV-TRE-DIO-FlpO (0.01–0.015 μg/μl), and pCAG-FSF-turboRFP-IRES-tTA (0.75–1 μg/μl). All (') letters represent zoomed in images of dashed box region in respective letter image with tagRFP staining. All ('') represent zoomed in images of dashed box region in respective letter image with GFP staining (for Dbn1 localization). A, Layer II/III neurons were additionally electroporated with pCAG-FSF-IRES-GFP (1.5 μg/μl). Scale bar represents 100 μm. A', Scale bar represents 10 μm. B–D'', Layer II/III neurons were electroporated with an additional Dbn1 expression construct: (B–B'') pCAG-FSF-Dbn1-YFP (1.5 μg/μl), (C–C'') pCAG-FSF-Dbn1^S142D-YFP (1.5 μg/μl), (D–D'') pCAG-FSF-Dbn1^S142A-YFP (1.5 μg/μl). E, F, Quantification of axon branches and protrusions for experimental perturbations. The following number of neurons were analyzed per condition: Fl/+: IRES-GFP (control): 28 neurons across six mice from four independent IUEs, Dbn1 WT: 33 neurons across three mice from two IUEs, Dbn1^S142D: 56 neurons across four mice from two IUEs, Dbn1^S142A: 42 neurons from three mice, obtained from three IUEs. Fl/Fl: IRES-GFP: 50 neurons from six mice obtained from three IUEs, Dbn1 WT: 27 neurons from four mice obtained from two IUEs, Dbn1^S142D: 21 neurons from four mice obtained from two IUEs, Dbn1^S142A: 35 neurons from three mice obtained from two IUEs. Statistics: two-way ANOVA using Tukey's multiple comparisons test. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001.

Ectopic collateral axon branching following Dbn1 loss-of-function persists at later developmental stages

Since synaptic pruning and axon elimination can occur both early and later in postnatal development (L. Luo and O'Leary, 2005; Riccomagno and Kolodkin, 2015), we asked whether Dbn1 LOF phenotypes are observed at more advanced postnatal developmental stages. We assessed axon branching in Dbn1 LOF Layer II/III CPNs at P21 in vivo. P21 Dbn1^fl/fl Layer II/III neurons do show ectopic branches in the middle region of Layer IV that are typically absent in Dbn1^fl/+ Layer II/III neurons (Fig. 5A–E; unpaired t test: Primary interstitial branches, t₍₂₀₃₎ = 3.251, p = 0.0013; Total branches, t₍₂₀₃₎ = 4.540, p < 0.0001). Further, Dbn1 LOF CPNs do not show the presence of excess protrusions in Layer IV (Fig. 5F; unpaired t test, t₍₂₀₃₎ = 0.5688), p = 0.5701). These data suggest that ectopic axon branches observed at P14 due to Dbn1 LOF persist into late postnatal development. The absence of primary ectopic protrusions within the middle region of Layer IV also suggests that no new ectopic branches are formed between P14 and P21. Similar to P14, Dbn1 LOF CPNs exhibit no difference in the number of branches within Layer II/III (unpaired t test, t₍₂₀₃₎ = 0.3527, p = 0.7246) or V (unpaired t test, t₍₁₃₃₎ = 0.3135, p = 0.7544) compared to Dbn1^fl/+ CPNs (Fig 5G,H). These data suggest Dbn1 restricts the growth of ectopic axon branches in the middle region of Layer IV, where branching is normally absent, and that these effects persist and are not corrected.

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Figure 5.

Dbn1 LOF ectopic branching phenotypes are persistent throughout later postnatal developmental time points. A–B'', Confocal immunofluorescent maximum intensity projection images of Layer II/III P21 neurons from Dbn1 floxed and heterozygous littermate controls labeled using the “bipartite” approach with the following constructs: pCAG-FSF-mCherry (1 μg/μl), pEF1-Flex-FlpO (500 ng/μl), and pCAG-CreERT2 (500 ng/μl). Tamoxifen was administered at P1 and P2. A–A'', Example heterozygous neuron. A, Merged image of mCherry and DAPI staining. Dashed lines demarcate cortical layer boundaries. A', mCherry staining. Dashed box represent region along the axon within the middle of Layer IV. A'', Magnified image of boxed region in A'. Scale bar represents 25 μm. B–C'', Example of Dbn1 null neuron. B, C, Merged image of mCherry and DAPI staining. Dashed lines demarcate cortical layer boundaries. B', C', mCherry staining. Dashed box represents region along the axon within the middle of Layer IV. B'', C'' Magnified image of boxed region in B'. Arrows point to ectopic branches in Layer IV. D–F, Quantification of Layer IV branches and protrusions. G, Quantification of Layer II/III branches. H, Quantification of Layer V branches. Symbols represent individual neurons obtained from: Fl/+: 95 individual neurons from four mice obtained from three IUEs, Fl/Fl: 110 individual neurons from seven mice obtained from three IUEs. For Layer V analyses: 50 individual Fl/+ neurons and 85 individual Fl/Fl neurons obtained from the same experiments were used due to additional LV cutoff (see Materials and Methods). Statistics: unpaired t test. Lines represent mean and SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The requirement for Dbn1 to regulate axon collateral branching is confined to an early postnatal developmental window

To determine whether ectopic collateral axon branching observed in Dbn1-deficient Layer II/III CPNs is confined to the narrow developmental window around the time of collateral axon branch initiation (∼P2–P3) after cell migration is complete (P0), we performed LOF experiments in Dbn1^flox mice, as described above, but administered tamoxifen at later postnatal stages (P4 and P7; Fig. 6A–D; data not shown). Dbn1^fl/fl mice that received tamoxifen at later times, at either P4 or P7, and were subsequently sacrificed at P14 resembled Dbn1^fl/+ littermates, with Layer II/III neurons exhibiting no ectopic collateral axon branching or protrusion phenotypes in the middle region of Layer IV and Layer IV, respectively (Fig. 6C,D; data not shown; Branches, unpaired t test: P4, t₍₅₂₎ = 0.9932, p = 0.3252; Protrusions, unpaired t test: P4, t₍₅₂₎ = 0.9579, p = 0.3426). Therefore, Dbn1 LOF CPN interstitial branching phenotypes are developmentally constrained such that only removal of Dbn1 just prior to the onset of collateral axon branching results in axon collateral branching phenotypes.

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Figure 6.

Dbn1 LOF ectopic branching phenotypes are developmentally fixed. A–D', Neurons were electroporated with pCAG-LSL-GFP (1 μg/μl), pCAG-FSF-mCherry (1 μg/μl), pEF1-Flex-FlpO (500 ng/μl), and pCAG-CreERT2 (500 ng/μl). A–B', Dbn1^fl/+ (A–A') pups and Dbn1^fl/fl (B–B') pups were administered tamoxifen at P4 after branching has already initiated. Confocal fluorescent maximum intensity projection image of P14 cortical section stained with mCherry, GFP, and DAPI. No branching defects were observed. Scale bar represents 50 μm. Dotted lines demarcate layer boundaries. C, D, Quantification of Layer II/III neuron axon branches and protrusions within the middle region of Layer IV and Layer IV, respectively. For P4 tamoxifen treatment symbols represent individual neurons obtained from: 25 Fl/+ neurons from three mice and 29 Fl/Fl neurons from three mice obtained from two IUEs. Statistics: unpaired t test. Lines represent mean and SEM.

Dbn1 overexpression and loss-of-function axon protrusion and branching phenotypes are recapitulated in vitro

Dbn1 overexpression in DRGs and in embryonic cortical neurons results in an increase in axon branches, filopodial protrusions and neurites in vivo and in vitro (Geraldo et al., 2008; Ketschek et al., 2016; Poobalasingam et al., 2022). Additionally, knock-down of Dbn1 using RNAi leads to a reduction in filopodia, lamellipodia and axon branching in vitro in several neuronal cell types, including cortical neurons and sensory neurons (Geraldo et al., 2008; Ketschek et al., 2016; Miyata and Hayashi, 2022). To complement these findings and our in vivo results on interstitial axon branching regulators we manipulated Layer II/III CNPs in vitro. We performed ex vivo electroporation on Dbn1^fl/fl mouse embryos at E15.5 using plasmids expressing Cre recombinase (for Dbn1 LOF) or only GFP (control) to label Layer II/III neurons. Then, we immediately dissociated electroporated cortices, cultured isolated neurons, and subsequently assessed cortical neuron axon morphology after 6 d in vitro (DIV), a time point that corresponds to the postnatal stage at which we normally observe the onset of Layer II/III collateral axon branching in vivo (∼P2; Hand et al., 2015). Dbn1^fl/fl neurons electroporated with Cre exhibit a loss of Dbn1 protein compared to control neurons electroporated with GFP, demonstrating efficient inactivation of Dbn1 (Fig. 7A–B').

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Figure 7.

Dbn1 LOF in Layer II/III neurons in vitro results in longer and more axon branches. A–D, Confocal immunofluorescent images of Layer II/III neurons at DIV6 obtained from Dbn1^fl/fl mice using ex vivo electroporation at E15.5. Cells were stained with antibodies directed against GFP, against dsRED, against Dbn1, and DAPI for analyses. A, Dbn1^fl/fl embryos were electroporated at E15.5 with pCAG-GFP (2 μg/μl; control neurons). Example of control neuron. Arrow points to GFP-positive soma. A'', Dbn1 staining showing the presence of Dbn1 in the GFP labeled neurons. Arrow points to GFP-positive cell body. Boxed region highlights example of average branch size found in control neurons. B–B', Neurons from Dbn1^fl/fl were electroporated with pCAG-LSL-GFP (2 μg/μl) and pCAG-Cre (1 μg/μl; LOF neurons). B, A GFP labeled Dbn1 LOF Layer II/III neuron soma. B', Dbn1 staining shows the absence of Dbn1 protein in this GFP-positive neuron. Arrowhead indicates absence of Dbn1 in the GFP-positive cell body. Boxed region shows zoomed in image of average size of an axon branch found in LOF neurons. C, D, Example of Control (Dbn1^fl/+) versus Dbn1 LOF (Dbn1^fl/fl) neuron. Arrows point to axon branches. Scale bars represent 50 μm. E–I, Quantification of axon branches. Control (-Cre): 95 neurons obtained from two to three pups across two individual experiments, LOF: (+Cre): 92 neurons obtained from two to three pups across two individual experiments. Statistics: unpaired t test. Lines represent mean and SEM. *p < 0.05, **p < 0.01. J–K', Confocal immunofluorescent images of Layer II/III neurons at DIV6 obtained from WT CD1 mice using ex vivo electroporation at E15.5. Neurons were electroporated with pCAG-FSF-mCherry (1 μg/μl), pCAG-FSF-GFP (J), pCAG-FSF-Dbn1-IRES-GFP (2 μg/μl; K), and pCAG-FlpO (1 μg/μl). J, Example of WT control neuron. Dashed box represents region expanded in J'. Scale bar represents 100 μm. J', Expanded view of boxed region with dsRed staining. Scale bar represents 50 μm. K, Confocal immunofluorescent images of Dbn1 OE Layer II/III neurons at DIV6 obtained from WT CD1 mice using ex vivo electroporation at E15.5. Dashed box represents region expanded in K'. K', Zoomed in image of axon protrusions; asterisks indicate protrusions. L–N, Quantification of axon branches and protrusions. Forty-six control neurons and 41 Dbn1 overexpressing neurons were analyzed from two to three brains obtrained from 2 separate cultures. Statistics: unpaired t test. Lines represent mean and SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Dbn1 LOF in cultured Layer II/III neurons resulted in an increase in axon branching (Fig. 7C,D,F,H,I; unpaired t test, t₍₁₈₅₎ = 2.236, p = 0.0265). However, we observed no difference in overall primary axon length measurements (Fig. 7E; unpaired t test, t₍₁₈₅₎ = 1.641, p = 0.1024). Interestingly, there was also an increase in long collateral axon branches (unpaired t test, t₍₁₈₅₎ = 2.222, p = 0.0275), including those longer 50 μm, as compared to control neurons (Fig. 7C,D,I; unpaired t test, t₍₁₈₅₎ = 2.427, p = 0.0162). Additionally, Dbn1-deficient neurons did not show an increase in axon protrusions, as seen following Dbn1 OE in vivo (Figs. 1G, 2B''; unpaired t test, t₍₁₈₅₎ = 1.217, p = 0.225). Therefore, because Dbn1-deficient neurons form more axon branches but not more axon protrusions, Dbn1 LOF regulates axon branch elongation in vitro in a manner similar to what we observe following LOF in vivo (Fig. 3–5).

To characterize Dbn1 overexpression effects on cortical neuron morphology in vitro, we used a Dbn1 overexpression construct tagged with YFP to enable localization of Dbn1 in electroporated Layer II/III neurons employing ex vivo electroporation (Geraldo et al., 2008). Dbn1 OE in Layer II/III CPNs cultured in vitro resulted in an increase in both axon branches (unpaired t test, t₍₆₈₎ = 3.052, p = 0.032) and axon protrusions (unpaired t test, t₍₆₈₎ = 4.694, p < 0.0001), compared to control neurons electroporated with GFP (Fig. 7J–K',M). Further, there was no difference in primary axon length and average branch length in Dbn1 OE neurons compared to control neurons (data not shown). Interestingly, in contrast to Dbn1 LOF CPNs in vitro, there was no difference in the number of long axon branches (those greater that 50 μm) compared to control neurons (Fig. 7N; unpaired t test, t₍₆₈₎ = 0.2146), p = 0.8307). These data suggest that Dbn1 OE enables the formation of ectopic protrusions and short branches, but these branches fail to become long, stable, collateral branches, as seen in Dbn1 LOF CPNs. The observation that Dbn1 OE in vitro results in excess shorter branches could be due to the lack of extrinsic signals that are present in these in vivo conditions. Further, our results are similar to previous work showing that both activation and inhibition of VEGF/VEGFR signaling lead to an increase in axon branching in hippocampal neurons (Luck et al., 2019). Interestingly, only the branches resulting from VEGF stimulation were mature and functional, whereas the LOF branches were not (Luck et al., 2019). Similarly, the aberrant short branches we observe in Dbn1 OE neurons are likely to be immature and unstable, since they fail to elongate. These results are in line with our in vivo data showing that Dbn1 OE results in an increase in axon branch initiation as assessed by protrusion number, but not branch elongation. Further, they highlight distinct phenotypes for Dbn1 OE and LOF phenotypes, which can be assessed in vitro as well as in vivo.

Dbn1 actin-binding and MT-coupling domains are necessary for Dbn1 overexpression-mediated protrusions

There are two major mammalian drebrin isoforms, E and A, and they are produced by alternative splicing from a single Dbn1 transcript. Dbn1-E is encoded by the embryonic splice variant Dbn1-E and is expressed throughout the body. Within the brain, Dbn1-E is expressed prominently in the hippocampus and is also distributed across cortical layers within the developing somatosensory cortex (Ao et al., 2014). Dbn1-A, on the other hand, exhibits restricted expression in the brain, is enriched in dendritic spines and contains an additional amino acid sequence called “Insert 2” (Ins; Koganezawa et al., 2017; Kojima, 2017; Shirao and Sekino, 2017). These Dbn1 isoforms contain several shared protein domains that include locations where Dbn1 binding partners associate and thereby facilitate cytoskeletal regulatory functions (Dun et al., 2012; Worth et al., 2013; Kojima, 2017; Shirao and Sekino, 2017). Dbn1 contains several functional domains, including: in its N-terminal region (eliminated below in the Dbn1ΔN deletion construct) an actin-depolymerizing factor homology domain (ADF-H), two actin binding domains (AB1/2) that include a coiled-coiled domain (CC) and a helical domain (Hel); and in its C-terminal domain (eliminated below in the Dbn1ΔC deletion construct) a proline rich domain (PP), and a region implicated in MT plus-end coupling through end binding protein 1 (EB1; Fig. 8A; Shan et al., 2021). These domains associate with unique Dbn1 binding partners and serve specific cytoskeletal regulatory functions (Dun et al., 2012; Worth et al., 2013; Kojima, 2017; Shirao and Sekino, 2017).

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Figure 8.

Actin-binding and MT-coupling domains of Dbn1 are necessary for protrusion formation upon overexpression. A, Diagram of Drebrin protein domain organization in Dbn1-E. ADF-H: actin-depolymerizing factor homology domain; S142: Cdk5 phosphorylation site; AB1/2: actin binding 1 and 2 domains; PP: proline-rich domain. B–G, Confocal immunofluorescent images of an electroporated Layer II/III neuron within somatosensory cortical sections from CD1 mice that underwent electroporation at E15.5 via the bipartite approach with pCAG-FSF-mCherry (1 μg/μl), pEF1-Flex-FlpO (500 ng/μl), and pCAG-CreERT2 (500 ng/μl) and a control or specific deletion construct for each experiment detailed below. Tamoxifen was administered to electroporated pups at P1 and P2. Images of Layer II/III neurons are stained with antibodies against dsRED and DAPI. B, pCAG-FSF-GFP (2 μg/μl). Scale bars represent 100 μm. C, pCAG-FSF-Dbn1ΔN-terminal-IRES-GFP (2 μg/μl). D, pCAG-FSF-Dbn1ΔAB1/2-IRES-GFP (2 μg/μl). E, pCAG-FSF-Dbn1ΔPP-IRES-GFP (2 μg/μl). F, Dbn1ΔC-terminal-IRES-GFP (2 μg/μl). G, pCAG-FSF-Dbn1ΔADF-H-IRES-GFP (2 μg/μl). B', C', D', E', F', G', mCherry labeling of zoomed in region of Layer II/III axon within Layer IV. Scale bar represents 25 μm (B'). H–Q, For all experimental manipulations, only the Layer IV region was traced for axon branches and protrusions since this was the region of interest for us. Thirty-eight Dbn1ΔN-terminal OE and 32 littermate control Layer II/III neurons across two IUEs with 4 pups per group were analyzed; 24 Dbn1ΔAB1/2 OE and 33 littermate control Layer II/III neurons across two IUEs with two to three pups per group analyzed; 35 Dbn1ΔPP OE and 28 littermate control Layer II/III neurons across two IUEs with four mice per group analyzed; 22 Dbn1ΔC-terminal OE and 23 littermate control Layer II/III neurons across four IUEs with six mice per group analyzed. Twenty-four Dbn1ΔADF-H OE and 32 littermate control Layer II/III neurons across two IUEs with two to three pups per experiment were analyzed. Statistics: unpaired t test. Lines represent mean and SEM. **p < 0.01.

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Figure 9.

Phosphorylation of Drebrin at S142 regulates Dbn1 overexpression axon protrusion initiation phenotype. A–C'', Confocal immunofluorescent images of P14 Layer II/III neurons from CD1 following IUE at E15.5 using the bipartite approach. Sections were stained with antibodies against dsRED, against GFP to detect YFP, and DAPI. Neurons were electroporated with the following constructs: pCAG-FSF-mCherry (1 μg/μl), pEF1-Flex-FlpO (500 ng/μl), pCAG-CreERT2 (500 μg/μl), and individual plasmids for each experimental manipulation. Tamoxifen was administered at P1 and P2. A, Merged image of an example control Layer II/III neuron electroporated with pCAG-FSF-GFP (2 μg/μl) in addition to bipartite plasmids. Cortical layer boundaries are demarcated using DAPI staining. Scale bar represents 100 μm. A', mCherry labeling of control neuron. Boxed region is magnified in A''. A'', Zoomed in image of region of Layer IV along Layer II/III axon. No atypical branching or protrusion phenotypes are observed. Scale bar represents 50 μm. B, Example of a Layer II/III neuron overexpressing the phospho-mutant Dbn1 mutant construct pCAG-FSF-Dbn1^S142A-YFP (2 μg/μl). B', mCherry labeling of Layer II/III neuron. Boxed region is expanded in B''. B'', Zoomed in image of a region of axon within Layer IV. No atypical morphologies are observed. C, Example of a Layer II/III neuron overexpressing the phosphomimetic Dbn1 mutant construct pCAG-FSF-Dbn1^S142D-YFP (2 μg/μl) in addition to other listed bipartite constructs. C', mCherry labeling of Layer II/III neuron. Box region is amplified in C''. C'', Zoomed in image of Layer IV region along Layer II/III neuron axon. The presence of axon protrusions in this region is observed. D, E, Quantification of Layer II/III neurons overexpressing Dbn1^S142D. Thirty-three control neurons and 37 Dbn1^S142D OE neurons were analyzed from two IUEs across two mice per experiment. Statistics: unpaired t test. Lines represent mean and SEM. *p < 0.05. F, G, Quantification of Layer II/III neurons overexpressing Dbn1^S142A. Twenty-nine control neurons and 40 Dbn1^S142A OE neurons across two IUEs from two to three mice per experiment.

To investigate molecular mechanisms by which Dbn1 regulates collateral branch initiation and elongation, we generated Dbn1E overexpression constructs lacking select protein domains and assessed collateral axon branching and protrusion phenotypes within Layer IV in CD1 WT mice in vivo using the BP approach. We focused on Dbn1E for this analysis since it is highly expressed during stages of cortical developmental when collateral branching events are initiated, whereas Dbn1A is expressed in later postnatal cortical development and spine maturation (Aoki et al., 2005). We observed that OE of Dbn1ΔN (unpaired t test: Branches, t₍₆₈₎ = 0.3894, p = 0.6982; Protrusions, t₍₆₈₎ = 1.009, p = 0.3165) in Layer II/III CPNs did not cause ectopic branching or protrusion phenotypes (Fig. 8B–C',H,M). Similarly, OE of Dbn1ΔAB1/2 (unpaired t test: Branches, t₍₅₅₎ = 1.197, p = 0.2364; Protrusions, t₍₅₅₎ = 0.3437, p = 0.7324) in Layer II/III CPNs did not cause any ectopic branching or protrusion phenotypes (Fig. 8D,D',I,N), and neither did OE using Dbn1ΔC (unpaired t test: Branches, t₍₄₃₎ = 0.4178, p = 0.6782; Protrusions, t₍₄₃₎ = 1.633, p = 1.098) or Dbn 1ΔPP (unpaired t test: Branches, t₍₆₁₎ = 0.9488, p = 0.3465; Protrusions, t₍₆₁₎ = 0.6328, p = 0.5292); this suggests that these domains are all necessary for Dbn1 OE axon protrusion phenotypes (Fig. 8E–F',J,K,O,P). Interestingly, Dbn1ΔADF-H OE phenocopies full-length Dbn1-E OE, leading to axon protrusions within Layer IV (Fig. 8G–G',L,Q; unpaired t test: Branches, t₍₅₄₎ = 0.7207, p = 0.4742; Protrusions, t₍₅₄₎ = 2.861, p = 0.0060). Therefore, the ADF-H domain is dispensable for axon branch initiation as evidenced by the presence of protrusions in its absence (Fig. 8L). We also assessed Dbn1A OE and observed no ectopic protrusion phenotypes, suggesting that Dbn1A and E play distinct roles in cortical development (data not shown).

Taken together, these results suggest diverse roles for Dbn1 domains in cortical Layer II/III neuronal development. Each of the Dbn1 domains known to mediate F-actin bundling and MT-coupling is required for the formation of primary axon protrusions, which likely serve as the precursors for collateral axon branches (Hand et al., 2015; Ketschek et al., 2016; Shan et al., 2021).

Phosphorylation of Dbn1 at S142 regulates interstitial axon protrusions

Dbn1 is phosphorylated by the Cdk5 serine/threonine kinase on serine 142 (S142), which lies within the Dbn1 N-terminal domain, and this posttranslational modification has been implicated in neuritogenesis, cell migration, growth cone turning, and synapse formation (Worth et al., 2013; Tanabe et al., 2014; Gordon-Weeks, 2017; Chen et al., 2022). Previous work identifies a role for Dbn1 phosphorylation by CDK5 at S142 in the regulation of F-actin bunding, which serves as a precursor for axon protrusions that in turn precede axon branch initiation (Worth et al., 2013; Kalil and Dent, 2014; Hand et al., 2015). To investigate further the molecular mechanisms underlying Dbn1 regulation of Layer II/III neuron axon branching, we used DNA constructs encoding phosopho-mimetic (S142D) or phospho-dead (S142A) Dbn1 fused to a C-terminal YFP-tag in our BP electroporation screening paradigm and performed IUE at E15.5. Dbn1^S142A OE did not cause any changes in overall Layer II/III axon branching, including in the middle region of Layer IV, or in protrusions across cortical layers compared to control neurons (Fig. 9A–B'',F,G; t test: Branching, t₍₆₆₎ = 0.2810, p = 0.7796; Protrusions, t₍₆₆₎ = 0.3605, p = 0.7197). Though we did not observe an overall increase in the total number of axon branches within the middle region of Layer IV following Dbn1^S142D overexpression, there was an increase in the number of total primary protrusions, similar to what we observed with WT Dbn1 OE (Fig. 9C–E; see Fig. 2B–B'', E,F''; unpaired t test: Branches, t₍₆₈₎ = 1.539, p = 0.1285; Protrusions, t₍₆₈₎ = 2.046, p = 0.0446). These in vivo data suggest that phosphorylation of Dbn1 at S142 positively regulates axon protrusion formation since we do not observe ectopic protrusions when Dbn1 cannot be phosphorylated (Dbn1^S142A OE) but do observe these phenotypes in the presence of constitutive Dbn1 phosphorylation (Dbn1^S142D OE). Interestingly, in vitro analysis of cultured Layer II/III cortical neurons shows that OE of both Dbn1 phospho-mimetic and phospho-dead mutants does not cause an increase in long axon branches (those greater than 50 μm), as we observed in Dbn1-deficient neurons in vivo, but did cause an increase in axon protrusions, showing that both Dbn1 phospho-mutants mimic Dbn1 overexpression phenotypes rather than Dbn1 LOF (data not shown). Taken together, these data show that Dbn1 OE regulates axon protrusion initiation in a manner dependent on the Dbn1 phosphorylation at S142.

Phosphorylation of Dbn1 at S142 is not required for suppression of collateral branching in Layer IV

To determine whether phosphorylation of Dbn1 at S142 is necessary for the normal suppression of collateral axon branching by Dbn1, we performed rescue experiments using the conditional Dbn1^flox allele and phosopho-mimetic or phospho-mutant constructs in a slightly modified labeling version of our BP approach (see Materials and Methods). As we saw previously, Dbn1^S142D overexpression resulted in an increase of axon protrusions in Dbn1^fl/+ Layer II/III neurons, but not in Dbn1^fl/fl Layer II/III neurons (Fig. 4C–C'',F''; two-way ANOVA, Tukey's multiple comparisons test: Dbn1^fl/+, p = 0.0023, Dbn1^fl/fl, p > 0.9999). Further, Dbn1^S142D expression in the context of Dbn1^fl/fl LOF eliminated the ectopic axon branching phenotype observed in Dbn1 LOF neurons (Fig. 4C–C'',G; p = 0.0016). On the other hand, as expected, Dbn1^S142A expression in Dbn1^fl/+ Layer II/III CPNs did not result in an increase in axon protrusions within Layer IV (Fig. 4D–D'',E; p > 0.9999). Interestingly, Dbn1^S142A expression in Layer II/III neurons suppressed the Dbn1 LOF Layer IV ectopic branching phenotype (Fig. 4D–D'',F; p < 0.0001). These results suggest that Dbn1 normally suppresses ectopic collateral branching within Layer IV and that phosphorylation at S142 is not required for this aspect of branch regulation.