Intracortical Projections

Intrahemispheric fiber tracts were strongly reduced in the absence of Neurod1/2/6.

Bidirectional axon tracing using the hydrophobic cyanine dye DiI, labeled only few dispersed long-range projections in fixed brains of newborn triple-deficient mice (fig 41a). FluorescentIHCfor the axonal cell adhesion moleculeL1cam, identified remaining axon bundles. The relative focus of L1cam immunofluorescence was shifted from the caudal to the rostral neocortex (arrows in fig41b).

The prominent upregulation ofRobo1 in theCPof Neurod2/6 double-deficient mice (sect2.4.4.5) was lost upon the additional inactivation of Neurod1 (asterisk in fig41c). In newborn triple-deficient animals, Robo1 was expressed at approx-imately normal levels. Robo1 IHC identified fasciculated axon bundles in the rostroventralICand in non-crossing aspects of theCC (double arrow in fig41c).

The above described axonal Cre-reporter allele (sect 2.4.4.6) was used to se-lectively visualize axons originating from Neurod6-lineage pyramidal neurons.

In control brains, all cortical projection tracts (CC, AC, HC, HF, IC, LOT) were stronglyGFP-positive, and theCPwas scattered with fluorescently labeled axons¹ (left panel in fig41d). In newborn Neurod1/2/6 triple-deficient brains, normal neocortical and archicortical projections were hardly identifiable. Instead, ab-normal fasciculated fiber bundles appeared to follow random trajectories within the neocortex (right panel in fig41d). The diffuse fluorescent labeling of the CP was not significantly reduced, suggesting the presence of short or unfasciculated axons originating from remaining neocortical Neurod1/2/6 triple-deficient pyra-midal neurons. The lateral olfactory tract (LOT), which originates from mitral and tufted cells of the paleocortical OB, was reduced but identifiable in triple-deficient mice (fig41d and data not shown). LacZhistochemistry confirmed that endogenousMapt promoter of the Cre-reporter construct was principally active in all areas and layers of the triple-deficient neocortex (fig41d).²

1The control animal in fig41d has the genotype Neurod1^Flox/Flox×Neurod2^Wt/Null×Neurod6^Cre/Cre. These mice showed delayed midline fusion, incomplete formation of theCCandPBs.

2Limitations apply as elaborated in sect 2.4.4.6 (footnote). It should be emphasized that the layering of theCP was severely disturbed and that many pyramidal neurons were weakly or notlacZ-positive in newborn Neurod1/2/6 triple-deficient mice.

Neurod1/2/6 Triple-Deficiency, Connectivity Results

Figure 41: Intracortical connectivity in Neurod1/2/6 triple-deficient mice

(a)DiI crystals (red asterisks) were placed in the centralCPand the paramedialCCof formalin-fixed brains from newborn mice. DiI molecules are very lipophilic and avoid intra- and intercellular fluids but readily diffuse along lipid membranes. The incubation of injected brains for several weeks in an aqueous solution resulted in bidirectional labeling of axons running in close proximity of DiI injection sites. Control tissue showed strong fluorescence in theCP,CRandCC. These structures were virtually devoid of specific signal in triple-deficient brains, suggesting that neocortical axon growth is severely affected in the absence of Neurod1/2/6.(b)IF for the axonal surface proteinL1cam (red) revealed the neocortical axonal compartment to be strongly reduced but not absent in parasagittal sections of newborn Neurod1/2/6 triple-deficient mice. In controls, the majority of L1cam-positive axons were located in the caudal neocortex, mostly contributing to the CC. In triple-deficient mice, the number of L1cam-positive axons was severely reduced in the caudal half but only mildly reduced in the rostral half of the neocortex. The relative focus of axonal connectivity had thus been shifted rostrally (arrows). Thedotted linerepresents the rostrocaudal center of the neocortex.(c)Chromogenic IHCforRobo1(red) in horizontal paraffin sections of newborn mice identified the rostralICin Neurod1/2/6 triple-deficient brains (double arrows). Robo1-upregulation in the CP (sect2.4.4.5a) was not observed.

(d)The previously described bicistronic Cre-reporter allele (sect2.4.4.6) was used to fluorescently label axons originating from Neurod6-Cre positive cortical pyramidal neurons. Newborn Neurod1/6 double-deficient mice carrying only one allele of Neurod2 showed dense axonal networks in the CC, IC, CR and lower CP.

The number of labeled cortical axons was strongly reduced in the Neurod1/2/6 triple-deficient littermate (the integrated fluorescent density was reduced by 80 %). Corticofugal projections (arrow heads) were hardly visible, but definitely present (the inset shows an intentionally overexposed image from the boxed region of a more caudal section).(e)LacZstaining showed that the endogenous Mapt-promoter driving the bicistronic reporter allele was transcriptionally active in pyramidal neurons of the triple-deficient neocortex.

2.5.6.2 Subcortical Projections

In newborn control mice, a large number of fluorescently labeled corticofugal fiber tracts had penetrated the striatum to form the IC and CST (fig 41d).

In Neurod1/2/6 triple-deficient animals, the striatum was nearly devoid of fluorescent signals. However, a thin band of GFP-positive axons formed a

Neurod1/2/6 Triple-Deficiency, Connectivity Results

Figure 42: Subcortical connectivity in Neurod1/2/6 triple-deficient mice

(a)The described Cre-reporter allele (sect2.4.4.6) was used to label axons (green) and cell bodies (blue) of the Neurod6-lineage in theSCof newborn mice. TheCST(arrow) could be identified in control and Neurod1/2/6 triple-deficient mice at various spinal levels. The reporter was active in neocortical pyramidal neurons (fig42e) and afferentDRGprojections neurons, but not in spinal Neurod6-lineage interneurons.

(b)IF for (red) in coronal brain sections identified a large number of axons in theIC,TCTandCPdof Neurod1/2/6 triple-deficient mice. ThePSPBwas only penetrated by very few L1cam-positive axons in these sections (arrow head). The presence of a ventrally oriented aberrant fiber bundle (double arrow) suggests that corticospinal axons had followed an abnormal trajectory to eventually reach theCPdandSC.

rudimentary CST that could only be identified in a few mid-coronal brain sections (arrow heads in fig41d).

In principle, Neurod1/2/6 triple-deficient motoneurons were able to form long range projections that could reach the spinal cord (SC). The dorsal CST of newborn triple-deficient mice was weakly GFP-positive and the CST cross section area was strongly reduced (arrow fig42a).¹ Because theMapt-promoter driven reporter allele was clearly active in neocortical motoneurons (fig41e), low GFP signal intensity in the spinal CST most probably results from a very low number of corticospinal axons.

Noteworthy, the Mapt-promoter of the Cre-reporter allele was not universally active. Expression was robust in in triple-deficient sensory neurons located in dorsal root ganglions (DRGs), but barely detectable in peptidergic neurons located in the dorsal horn of the spinal gray matter (fig42a).²

1The size and signal intensity of the Neurod1/2/6 triple-deficientCSTwas not quantified because usable tissue was limited. Membrane associatedGFPquickly diffused in fixed tissue sections and breeding was complicated due to Mapt and Neurod2 being located on the same chromo-some (footnote in sect 2.4.4.6). The fluorescently labeled axon tract, however, was clearly identifiable at different spinal levels and the shape of the surrounding dorsal funiculus was not significantly reduced in the absence of Neurod1/2/6 (Bröhl et al. 2008, fig 7).

2This is compatible with our previous finding that the identity of dynorphin- and galanin-expressing neurons in the dorsal horn depends on the expression of Neurod1/2/6 (Bröhl et al. 2008, fig 7–9) and suggests that these cells might fail to undergo terminal neuronal differentiate and do not acquire an alternative fate in the absence of Neurod1/2/6.

Neurod1/2/6 Triple-Deficiency, Connectivity Results

Fluorescent immunostaining for L1cam in coronal brain sections identified comparable numbers of afferent projections in the IC, corticothalamic tract (CTT)andcerebral peduncle (CPd)of newborn control and Neurod1/2/6 triple-deficient mice (fig42b). However, afferents that crossed thePSPBand invaded the Neurod1/2/6 triple-deficient neocortex were rare (arrow in fig42b). These afferent axons originate from subcortical neurons that are not part of the Neuod6 lineage (sect2.1). Their inability to normally invade the neocortex can thus not be explained by cell-intrinsic defects, but is most probably caused by the absence of some attractive signal originating in the neocortex.