Neurogenin2 regulates the initial axon guidance of cortical pyramidal neurons projecting medially to the corpus callosum
© Hand and Polleux; licensee BioMed Central Ltd. 2011
Received: 4 April 2011
Accepted: 24 August 2011
Published: 24 August 2011
The formation of the mammalian central nervous system requires the establishment of complex neural circuits between a diverse array of neuronal subtypes. Here we report that the proneural transcription factor Neurogenin2 (Ngn2) is crucial for the proper specification of cortical axon projections.
The genetic loss of Ngn2 in mice results in fewer callosal axons projecting towards the midline as well as abnormal midline crossing. shRNA-mediated knockdown of Ngn2 revealed its cell-autonomous requirement for the proper projection of axons from layer 2/3 pyramidal neurons to the midline in vivo. We found that the acute loss of Ngn2 in vivo induces the axon of superficial layer 2/3 neurons to project laterally towards aberrant cortical and subcortical targets.
These and previous results demonstrate that Ngn2 is required for the coordinated specification of cardinal features defining the phenotype of cortical pyramidal neurons, including their migration properties, dendritic morphology and axonal projection.
The mammalian nervous system consists of a tremendous diversity of neuronal subtypes forming complex functional circuits. In the cerebral cortex, long-distance-projecting glutamatergic pyramidal neurons arise from radial glial progenitors located in the dorsal telencephalon . During cortical neurogenesis in rodents, radial glial progenitors divide asymmetrically to generate another radial glial progenitor and an intermediate progenitor cell (IPC) that translocates to the subventricular zone (SVZ) . These IPCs display a transient multipolar morphology characterized by the dynamic extension and retraction of immature neurites, which might sense their micro-environment and respond to cues polarizing their leading process (future apical dendrite) dorsally towards the cortical plate and their trailing process (future axon) ventrally [2, 3]. During this polarization, the neuron adheres to a radial glial cell process and initiates radial migration through the cell-sparse but axon-rich intermediate zone (IZ) towards the pial surface. Upon reaching the top of the cortical plate, just below the pial surface, pyramidal neurons detach from the radial glial cell and undergo terminal translocation before elaborating both their dendritic and axonal processes. In mice, neurogenesis occurs between embryonic day 11 (E11) and E18 [4–6], giving rise to neurons accumulating in an 'inside-first outside-last' pattern where late-born neurons migrate to layers located more superficially than their predecessors . Ultimately, the location of progenitors, mode of migration, neurotransmitter expression, dendritic morphology, and axonal projections are used to define subtypes of cortical neurons .
One of the defining features of pyramidal neurons is the type of axonal projections they form within the brain and spinal cord. Pyramidal neurons in the deep layers of the cortex (layers 5/6) project laterally to exit the cortex towards subcortical regions [9, 10]. On the contrary, pyramidal neurons in superficial layers 2/3 mostly project to other cortical areas, including callosal projections through the midline that form the corpus callosum [9, 10]. This initial choice to project medially or laterally is one of the earliest and most critical axon guidance decisions made by pyramidal neurons since it defines these two large and distinct classes of pyramidal projection neurons. However, the molecular mechanisms underlying the specification of this key axon guidance choice is still poorly understood .
Neurogenin2 (Ngn2) is a proneural basic helix-loop-helix (bHLH) transcription factor was first identified for its ability to promote neuronal differentiation in the brain and spinal cord [11, 12]. Beyond its proneural function, Ngn2 also specifies the cardinal phenotypic features defining pyramidal neurons as a subpopulation, such as glutamatergic neurotransmitter expression, radial migration properties and their pyramidal dendritic morphology [13–17]. Here we show that Ngn2 also specifies the axon guidance choice made by superficial pyramidal neurons to project towards the midline in vivo.
Materials and methods
In this study we used several inbred strains of mice, including Balb/c and C57Bl/6 (for in utero electroporation). The Ngn2 green fluorescent protein (GFP) knockin mice were a generous gift from Dr Francois Guillemot and were maintained on a Balb/c background. Males and females were used indistinguishably for quantifications. All experiments were performed in strict accordance to IACUC protocols approved by UNC Chapel Hill.
For this study, we created the pSCV2 construct from the pSilencer2.1 (Ambion (Austin, TX- USA)) vector. To achieve this, we inserted a CAG-Venus-pA cassette into the backbone of the pSilencer2.1 vector. All short hairpin RNA (shRNAs) are inserted downstream of the U6 promoter using BamHI and HindIII restriction sites. The targeting sequence of control shRNA is ACTACCGTTGTTATAGGTG. The target sequence of the shRNA targeting Ngn2 is CCAACAACCGCGAGCGCAA. To create the rescue mutant of Ngn2, a noncoding point mutation was generated by mutating nucleotide 360 from cytosine to adenine using the QuickChangeII mutagenesis kit from Stratagene (Santa Clara, CA - USA).
Antibodies and immunostaining
All immunofluorescent staining was performed as previously described . Primary antibodies were: anti-L1 (1:2,500; Millipore(Millipore (Billerica, MA-USA)), anti-GFP (1:2,000; Aves (Aves (Tigard, OR- USA)), anti-NF165 (1:1,000; Developmental Studies Hybridoma Bank at the University of Iowa), anti-CTIP2 (1:2,000; Abcam (Cambridge, MA- USA)), anti-Tbr1 (1:1,000; Chemicon (Billerica, MA- USA)), and anti-Cux1 (1:500; Santa Cruz Biotechnology (Santa Cruz, CA- USA)). Secondary antibodies were: goat anti-chicken Alexa 488, goat anti-rat Alexa 546, goat anti-rat Alexa 637, goat anti-rabbit Alexa 546, goat anti-rabbit Alex 637, and goat anti-mouse Alexa 546. Streptavidin conjugated to Alexa 546 was used to detect the biotin-labeled dextrose amines.
Anterograde axonal tracings
Briefly, E18.5 brains dissected and injected with 0.1 mg/ml 10,000MW BDA Molecular Probes (Carlsbad, CA - USA) in phosphate-buffered saline (PBS) with 0.1% fast green for visualization. Then the brains were incubated in artificial cerebrospinal fluid at 37°C and oxygenated with 95/5% O2/CO2 for 8 hours. After 8 hours, brains were fixed overnight at 4°C in 4% paraformaldehyde in PBS pH7.4. The brains were then sectioned and immunostained.
Ex vivo electroporation and organotypic slice culture
All ex vivo electroporation and organotypic slice cultures were performed essentially as described previously .
In utero electroporation
Briefly, E15.5 mice were deeply anesthetized using 2.5% 2,2,2 tribromoethanol in PBS. A small 1- to 2-cm incision was made along the midline and the uterine horns were removed from the abdominal cavity and placed on sterile gauze. The lateral ventricles of the embryos were injected with 2 μg/μl of plasmid in 1 × PBS, and 0.1% fast green dye (for visualization). The embryos were subsequently electroporated as follows: 4 pulses of 30V for 50 ms with a 500-ms interval. After the embryos had been injected and electroporated, the uterine horns were placed back in the abdominal cavity and the incision was sutured. Mice were allowed to recover and embryos were harvested at postnatal day 14. All surgeries strictly adhered to IACUC approved protocols.
To visualize if late Ngn2 expression in the SVZ/IZ matches temporally with axon initiation of callosally projecting neurons that are generated between E15.5 and E18.5 , we used ex vivo electroporation coupled with organotypic slice culture. This technique effectively introduces cDNA into NPCs, and within 24 hours post-electroporation, neurons begin to differentiate from the NPCs . To ensure we labeled superficial layer neurons, we electroporated cortices at E15.5, a time point when only superficial neurons would be labeled [6, 22]. To visualize the emerging axons, we optimized electroporation conditions to allow for single cell resolution using a plasmid encoding the yellow fluorescent protein (YFP) variant Venus and imaged cells 36 hours post-electroporation. At this time point, we found many newly differentiated neurons containing a long single neurite (Figure 1I). Interestingly, these single long neurites grew medially towards the corpus callosum within the IZ, strongly suggesting that these are presumptive axons. In these neurons, the emergence of axons appears to precede the formation of a leading process and the initiation of radial migration. This refines previously published data [23, 24] and shows that the directed emergence of the axon occurs very soon after cell cycle exit, correlating well with Ngn2 expression. Importantly, these results strongly suggest that the decision for a neuron to project an axon medially and to become a callosally projecting neuron is taken extremely early during neuronal differentiation, well before the neurons reach their final position in the cortical plate.
Ngn2 is required for callosal axon projection and midline crossing
Ngn2 regulates the initial guidance of callosal axons
Ex utero cortical electroporation coupled with organotypic slice culture is ideal for rapidly assessing the many aspects of pyramidal neuron differentiation and migration [15, 23], so we began by performing a developmental time course to assess if the axonal projections of cortical neurons are maintained in slice cultures in vitro. Embryonic cortices were electroporated at times ranging from E13.5 to E16.5 and organotypic slice cultures were prepared and cultured for 5 days in vitro (DIV), allowing for generation, migration and axon projection of electroporated pyramidal neurons (Figure 3E-H). We found that the axonal projections of pyramidal neurons cultured in vitro mimicked those found in vivo. At the earliest time points (E13.5 and E14.5), we found a significant proportion of axons were projecting laterally as we would expect since deep layer neurons are generated at these developmental time points (Figure 3E, F). At later time points (E15.5 to 16.5), the axons of electroporated neurons almost exclusively projected medially (Figure 3G, H) as only superficial neurons would be generated at this later time point .
Loss of Ngn2 does not alter the laminar fate of cortical neurons
Superficial pyramidal neurons lacking Ngn2 project axons to many areas postnatally
Here we demonstrate a novel role for Ngn2 during cortical development. We found that the genetic loss of Ngn2 results in a reduction of callosal axons and a malformation of the corpus callosum in vivo. When Ngn2 expression is knocked down acutely using shRNA in progenitors of superficial pyramidal neurons, many layer 2/3 neurons that normally project axons medially, now aberrantly project them laterally toward both cortical and subcortical brain regions. The change in axonal projection resulting from the loss of Ngn2 does not induce any dramatic change in laminar fate, at least with regard to the expression of transcriptional regulators such as CTIP2 or Cux1, although a small proportion of neurons permanently fail to reach their final position in layer 2/3 (Figures 5 and 8). Taken together our results show that Ngn2 coordinates the acquisition of many of the cardinal features of pyramidal neurons in the developing cortex, including neurotransmitter expression , migration properties and dendritic morphology , and axonal projections (the present study and ). Previous results  showed that expression of layer-specific markers of layers 5/6, such as Tbr1 and ER81, were upregulated in the Ngn2-/- compared to wild-type mice but that markers of layer 2/3, such as Cux1, were unchanged. Our results confirm that Ngn2 does not seem to have a major role in determining the transcriptional identity of superficial layer 2/3 neurons (Figures 4 and 5) but rather have a significant effect on the specification of their axon projections (see below).
The first interpretation of our results is that during the second half of neurogenesis (that is, after E14), when neurogenesis switches from producing subcortically projecting layer 5/6 neurons to producing mostly cortico-cortical and contralateral/midline projecting neurons (layer 2-4), Ngn2 plays an instructive function in intermediate progenitors by directly or indirectly specifying midline projection 'fate' (Figure 8G). The second alternative interpretation of our results is that the Ngn2 function during the second half of cortical neurogenesis is to repress the lateral projection fate during the production of superficial layer 2/3 neurons, thereby allowing neurons to acquire a 'cortical projection fate'  (Figure 8G). Future experiments will test these two mechanisms by identifying the transcriptional mechanisms (transactivation or repression) underlying their function in the cortex.
Since Ngn2 is expressed throughout neurogenesis (present results, but also see [13–15, 19], this suggests that the transcriptional function of Ngn2 changes over time. During early stages of cortical neurogenesis (such as E12.5), Ngn2 is primarily playing a proneural function due to high expression in dividing progenitors [18, 19], but at later time points Ngn2 has additional roles in the acquisition of the phenotypic traits associated with pyramidal neurons, including neurotransmitter expression [13, 14], migration [15, 16] and axon guidance (present study and ). This could be explained by the subtle change in expression pattern over time and/or by post-translational modifications affecting Ngn2's ability to partner with various transcriptional regulators [15, 17, 31]. Our data support this as we found Ngn2 expression in the upper SVZ and the IZ, and most of these cells also expressed Tbr2 (Figure 1E-H), a marker for IPCs at the later time point of E16.5, when superficial layer neurons are differentiating. Since the Ngn2 expression pattern changes as the cortex develops, we hypothesize that Ngn2 is capable of inducing differential gene expression as the cortex develops due to the differential expression of transcriptional co-activators and differences in accessibility of transcriptional targets due to epigenetic regulation by chromatin-modifying proteins. Future studies will be needed to identify which of the known transcriptional targets of Ngn2 [32–34] or novel transcriptional targets of Ngn2 regulate the switch in axonal projections over time, and to test how Ngn2 differentially regulates these gene(s) during cortical development.
Our data suggest that the initiation of the axon is a directed process leading to the guidance of the axon medially within the intermediate zone. We found this initial projection often occurred in immature neurons prior to forming a leading process and before initiating migration (Figure 1I). This raises an interesting question of whether direct transcriptional targets of Ngn2 regulate the initial projection of the superficial pyramidal neuron or if downstream transcription factors are responsible for the observed phenotype. Several transcription factors are directly and indirectly downstream of Ngn2, including transcription factors expressed in the SVZ (Tbr2 and NeuroD4) [32, 35], in the IZ (NeuroD1) [14, 15], and in the cortical plate (NeuroD2 and MEF2C) [14, 33]. Recently, a downstream target of Ngn2, the small GTPase Rnd2, was found to play an important role in the control of radial migration of pyramidal neurons . Furthermore, Rnd2 was sufficient to rescue the inhibition of migration in Ngn2-/- embryos. Interestingly, Rnd2 is a direct target of both Ngn2 and NeuroD1 , suggesting that Ngn2 is capable of directly and indirectly regulating the transcription of a gene necessary for migration. Identifying the gene(s) that are directly responsible for the directed axon guidance of sub-cortical versus medially projecting neurons will be of great interest. Several studies have investigated genes downstream of Ngn2. Not surprisingly, receptors for several axon guidance ligands, including Netrins, Slits, Semaphorins, and Ephrins, are down-regulated in Ngn2-/- embryos [14, 33, 34], and some of these receptors were found to be direct targets of Ngn2, while others are presumably indirect transcriptional targets. Therefore, we believe that Ngn2 is likely regulating, both directly and indirectly, the gene expression underlying the initial projection of callosal axons medially. Future experiments will need to test which Ngn2 downstream targets participate in the guidance of layer 2/3 axons towards the midline.
We, along with others, have identified that Ngn2 is crucial for the proper formation of cortical circuitry. While Ngn2 was first identified as a proneural transcription factor, further studies have demonstrated that Ngn2 regulates many of the defining features of pyramidal neurons. Elegant genetic studies previously showed that Ngn2, along with Ngn1, a close homolog presenting an overlapping expression pattern, specify the neurotransmitter fate of pyramidal neurons . In addition, Ngn1 is largely sufficient to compensate for the proneural deficit associated with the loss of Ngn2, and likely to compensate for some other phenotypes associated with Ngn2. This may explain the partial penetrance of our phenotypes. In addition to the expression of glutamate as a neurotransmitter and the repression of ventral telencephalic fate through repression of Mash1 expression, Ngn2 is required for the proper location of pyramidal neurons and acquisition of pyramidal dendritic morphology. Neuronal morphology and laminar position are both crucial to the formation of proper neural circuits. The present study demonstrates that Ngn2 regulates how pyramidal neurons innervate target areas by regulating the first axon guidance decision made by layer 2/3 pyramidal neurons to project laterally towards the midline, which underlies the formation of cortical circuits. Further studies of Ngn2 transcriptional targets will lead to a better understanding of the molecular mechanisms underlying its function during cortical circuit formation.
biotinylated dextran amine
days in vitro
green fluorescent protein
intermediate progenitor cell
neural progenitor cell
short hairpin RNA
yellow fluorescent protein.
We would like to thank all the members of the Polleux lab for constructive discussions and Marie Rougié for excellent technical assistance. We also would like to thank Francois Guillemot for providing the Ngn2-GFP knockin line. This work was supported by NRSA Award (1 F31 MH078665-01; to RAH) and a RO1 grant from NINDS (R01NS047701-05; to FP).
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