Analysis of axon tract formation in Gli3 conditional mutant mice

The cerebral cortex is the largest subdivision of the human brain and is associated with higher cognitive functions. These functions are based on the interconnections between the neurons that form pre- and postnatally in the different telencephalic regions. The processes of neurons with similar functions and connectivity follow the same course and form axon tracts. There are three main axons tracts analysed in this thesis the corpus callosum, the corticothalamic/thalamocortical tracts and the lateral olfactory tract that transfers olfactory information to the telencephalon. In the mouse, these tracts are generated during embryogenesis as axons project to their target area. The mechanisms by which axons navigate still need to be elucidated. Studies of a number of mutant mice have shown that axon pathfinding is under the control of genes. Gli3 is a zinc finger transcription factor with known roles in axon pathfinding. Gli3 is widely expressed in progenitor cells of the dorsal and ventral telencephalon complicating the elucidation of the molecular mechanisms by which Gli3 controls axon tract formation. My aim here is to investigate the spatial and temporal requirements for Gli3 in axon pathfinding in the forebrain using Gli3 conditional mutants as a tool. Regarding the corpus callosum, my findings demonstrated a crucial role for Gli3 in the dorsal telencephalon, but not in the septum or medial ganglionic eminence, to control corpus callosum formation and indicated that defects in the formation of the corticoseptal boundary affect the positioning of callosal guidepost cells. Moreover, conditional inactivation of Gli3 in dorsal telencephalic progenitors led to few corticothalamic axons leaving the cortex in a restricted lateral neocortical domain. This restricted entry is at least partially caused by an expansion of the piriform cortex, which forms from an enlarged progenitor domain of the ventral pallium. Transplantation experiments showed that the expanded piriform cortex repels corticofugal axons. Moreover, expression of Sema5B, a chemorepellent for corticofugal axons produced by the piriform cortex, is similarly expanded. Hence, control of lateral cortical development by Gli3 at the progenitor level is crucial for corticothalamic pathfinding. Finally, by using Emx1Cre;Gli3fl/fl mutants I analysed the consequences of the expansion of the piriform cortex on the formation of the lateral olfactory tract (LOT). This analysis showed that LOT axons also appear to be medially shifted with LOT collaterals aberrantly colonising the expanded piriform cortex. Time course analysis confirmed an expansion of the paleocortical primordium from E13.5 onwards, coinciding with the arrival of the LOT axons. Hence, it is possible that the expanded piriform cortex contributed to the medial shift of the LOT. In conclusion, these findings support a strong link between Gli3 controlled early patterning defects and axon pathfinding defects and form the basis for future analysis of the molecular mechanisms by which Gli3 controls axon pathfinding in the forebrain. My findings also reveal how alterations in GLI3 function may contribute to connectivity defects in human patients with mutations in GLI3

Gli3 is required in Emx1+ progenitors for the development of the corpus callosum

AbstractThe corpus callosum (CC) is the largest commissure in the forebrain and mediates the transfer of sensory, motor and cognitive information between the cerebral hemispheres. During CC development, a number of strategically located glial and neuronal guidepost structures serve to guide callosal axons across the midline at the corticoseptal boundary (CSB). Correct positioning of these guideposts requires the Gli3 gene, mutations of which result in callosal defects in humans and mice. However, as Gli3 is widely expressed during critical stages of forebrain development, the precise temporal and spatial requirements for Gli3 function in callosal development remain unclear. Here, we used a conditional mouse mutant approach to inactivate Gli3 in specific regions of the developing telencephalon in order to delineate the domain(s) in which Gli3 is required for normal development of the corpus callosum. Inactivation of Gli3 in the septum or in the medial ganglionic eminence had no effect on CC formation, however Gli3 inactivation in the developing cerebral cortex led to the formation of a severely hypoplastic CC at E18.5 due to a severe disorganization of midline guideposts. Glial wedge cells translocate prematurely and Slit1/2 are ectopically expressed in the septum. These changes coincide with altered Fgf and Wnt/β-catenin signalling during CSB formation. Collectively, these data demonstrate a crucial role for Gli3 in cortical progenitors to control CC formation and indicate how defects in CSB formation affect the positioning of callosal guidepost cells

Cerebral cortex expression of Gli3 is required for normal development of the lateral olfactory tract

<div><p>Formation of the lateral olfactory tract (LOT) and innervation of the piriform cortex represent fundamental steps to allow the transmission of olfactory information to the cerebral cortex. Several transcription factors, including the zinc finger transcription factor Gli3, influence LOT formation by controlling the development of mitral cells from which LOT axons emanate and/or by specifying the environment through which these axons navigate. <i>Gli3</i> null and hypomorphic mutants display severe defects throughout the territory covered by the developing lateral olfactory tract, making it difficult to identify specific roles for <i>Gli3</i> in its development. Here, we used <i>Emx1Cre</i>;<i>Gli3</i>^<i>fl/fl</i> conditional mutants to investigate LOT formation and colonization of the olfactory cortex in embryos in which loss of <i>Gli3</i> function is restricted to the dorsal telencephalon. These mutants form an olfactory bulb like structure which does not protrude from the telencephalic surface. Nevertheless, mitral cells are formed and their axons enter the piriform cortex though the LOT is shifted medially. Mitral axons also innervate a larger target area consistent with an enlargement of the piriform cortex and form aberrant projections into the deeper layers of the piriform cortex. No obvious differences were found in the expression patterns of key guidance cues. However, we found that an expansion of the piriform cortex temporally coincides with the arrival of LOT axons, suggesting that <i>Gli3</i> affects LOT positioning and target area innervation through controlling the development of the piriform cortex.</p></div

The expanded piriform cortex showed no obvious lamination defects in E18.5 <i>Gli3</i>^<i>cKO</i> mutants.

<p><b>(A)</b> Ctip2 labels neocortical neurons in neocortical layer V and layer II neurons in the piriform cortex in control brains. CR labels mitral cell axons comprising the LOT. <b>(B)</b> Higher magnification revealing the position of Ctip2+ layer II neurons with respect to the CR+ axons of the LOT. <b>(C-D)</b> In <i>Gli3</i>^<i>cKO</i> mutants, Ctip2+ cells occupy layer II in the piriform cortex below the CR+ axons. <b>(E, F)</b> Organization of the piriform cortex visualized by immunofluorescence analysis for CR, Map2 and Dapi. CR labels the LOT axons; Map2 identifies dendrites in layer I and the dense population of cells in layer II is stained with Dapi. <b>(G, H)</b> In <i>Gli3</i>^<i>cKO</i> mutants, no obvious defects are observed in piriform cortex organization. Abbreviations: ctx, neocortex; LOT, lateral olfactory tract; pc, piriform cortex. Arrows in A, D, E and H indicate the rhinal fissure and the transition from neocortex to piriform cortex. Scale bars: A-H:250μm.</p

Mitral and granule cell layers are formed in the early OB-like primordium of E14.5 <i>Gli3</i>^<i>cKO</i> embryos.

<p><b>(A, B, E, F, I, J)</b> In the olfactory bulb primordium (OBp), Tbr1, <i>Id2</i> and <i>Ap2e</i> are specifically expressed in mitral cells at the rostral tip of the OB of control embryos. <b>(C, D, G, H, K, L)</b> In <i>Gli3</i>^<i>cKO</i> mutants, Tbr1, <i>Id2</i> and <i>Ap2e</i> expressing cells form a thick band at the rostral tip of the OB-like primordium. <b>(M, N)</b> Control embryos show <i>ER81</i> expression in interneuron progenitors in the granule cell layer of the OB primordium. <b>(O-P)</b> In <i>Gli3</i>^<i>cKO</i> mutants, <i>ER81</i> transcripts are present in a distinct cell layer of the OB-like primordium but the <i>ER81</i>+ inner cell layer is extended into the outer mitral cell layer (arrowheads in O). Scale bars: A-P:250μm.</p

Mitral cells are present in an OB-like structure in E18.5 <i>Gli3</i>^<i>cKO</i> mutant brains.

<p><b>(A, D, E)</b> In control brains, Tbr2+ cells are present in the cortical ventricular zone and in the mitral and granule cell layers (arrowhead) of the OB which protrudes from the anterior end of the telencephalon. <b>(B, C, F-I)</b> In <i>Gli3</i>^<i>cKO</i> brains, Tbr2+ cells form a discernible mitral cell layer (B, F, G) or a cluster in an OB-like structure which does not form a protrusion (C, H, I). <b>(J, K)</b><i>Tbx2</i>.<i>1</i> expression is restricted to mitral cells in control brains. <b>(L-O)</b> In <i>Gli3</i>^<i>cKO</i> brains, <i>Tbx2</i>.<i>1</i> expressing mitral cells are present in the OB-like structure and either form a layer (L, M) or a cluster (N, O). Abbreviations: MCL, mitral cell layer; OB, olfactory bulb; OBLS, olfactory bulb like structure. Scale bars:A-D, F and H:250μm; E, G and I:50μm; J, L and N:100μm; K, M and O:50μm.</p

The piriform cortex is expanded at the arrival of LOT axons in <i>Gli3</i>^<i>cKO</i> mutants.

<p><b>(A-D)</b> At E12.5, <i>Nrp2</i> is expressed in the presumptive paleocortex (green dotted region in B and D) and in lot cells of both control (A, B) and <i>Gli3</i>^<i>cKO</i> brains (C, D). <b>(E, H)</b> Calretinin labels mitral cell axons and indicates the position of the LOT at the ventro-lateral margin of the telencephalon in E13.5 control (E) and <i>Gli3</i>^<i>cKO</i> mutant embryos (H). <b>(F, G)</b> In control embryos, <i>Nrp2</i> expression is confined to neurons of the piriform cortex and to lot cells while the area dorsal to the LOT (*) is <i>Nrp2</i> negative. <b>(I, J)</b><i>Nrp2</i> expression expands dorsally beyond the LOT site in E13.5 <i>Gli3</i>^<i>cKO</i> mutants. The arrows in (G and J) indicate the dorsal boundary of the <i>Nrp2</i> expression domain. Scale bars: A-J:100μm.</p

Innervation of the piriform cortex is disorganized in P7 <i>Gli3</i>^<i>cKO</i> brains.

<p><b>(A)</b> In P7 control brains, Satb2 is expressed in layer II/III neocortical neurons and Tbr1 in layer VI neocortical neurons and layer II neurons of the piriform cortex. <b>(B)</b> Magnification of the transition area from neocortex to piriform cortex. <b>(C, D)</b> In <i>Gli3</i>^<i>cKO</i> brains, the transition between neocortex and piriform cortex is shifted medially. <b>(E, F)</b> Ctip2+ neurons are positioned in layer II of the control piriform cortex. <b>(G, H)</b> In <i>Gli3</i>^<i>cKO</i> brains, Ctip2+ neurons occupy a similar layer position but layer II is expanded medially. <b>(I, J)</b> Cellular organization of the piriform cortex visualized by immunofluorescence analysis of CR, Map2 and Dapi. In control brains, CR+ LOT axons extend to layer Ia; Map2 dendrites are present in layer Ia and Ib and Dapi+ cells occupy layer II. <b>(K, L)</b> In <i>Gli3</i>^<i>cKO</i> brains, CR+ LOT axons extend in layer Ia but some CR+ axons aberrantly project in layer Ib and II. Map2+ dendrites are disorganized in layers Ia and Ib. (<b>M</b>, <b>N</b>) <i>Gad67</i> in situ hybridization and Calretinin immunolabeling revealed interneurons and LOT axons in the piriform cortex of control brains, respectively. (<b>O</b>, <b>P</b>) In <i>Gli3</i>^<i>cKO</i> brains, there is no overlap in Gad67 and Calretinin staining in layer II of the piriform cortex. Arrows in A, D, E, H, I and L indicate the position of the rhinal fissure, arrows in N and O indicate <i>Gad67</i>+CR+ interneurons. Scale bars: A-P:250μm.</p

Afferent input from the olfactory bulb expands into more medial positions in the <i>Gli3</i>^<i>cKO</i> cortex.

<p><b>(B, D, H, J)</b> DiI crystal placement in the olfactory bulb (OB) and OB-like structure of control (B, D) and <i>Gli3</i>^<i>cKO</i> brains (H, J), respectively (arrow). (J) In P7 <i>Gli3</i>^<i>cKO</i> brains, the OB protrusion is more prominent compared to E18.5 but not as much as in wild-type brains. <b>(A, C, G, I)</b> Anterograde labeling of LOT axons and their collateral branches in E18.5 (A, C) and P7 (G, I) control brains. In P7 brains (G, I), the LOT occupies the outer piriform cortex layers and DiI labelling extends into layer III. Note the distinct gap between DiI labelling in the LOT and in layer III (I). <b>(E, F)</b> In E18.5 <i>Gli3</i>^<i>cKO</i> brains, the LOT position is shifted medially and the LOT formation appears lense densely packed (arrowhead). <b>(K)</b> A dense population of DiI labelled branches is present in layer III with a barely discernible gap between the LOT and layer III (K, arrowheads). <b>(L)</b> Mitral cell axons occupy a medially expanded region in P7 <i>Gli3</i>^<i>cKO</i> brains. Note the aberrant formation of an axon bundle that projects into the ventral telencephalon (L, <b>arrow</b>). Scale bars: A, G, F and L:50μm; B, D, H and J:0.5μm;C-E, I-K:250μm.</p