Pax6 guides a relay of pioneer longitudinal axons in the embryonic mouse forebrain

Abstract

We have characterized a system of early neurons that establish the first two major longitudinal tracts in the embryonic mouse forebrain. Axon tracers and antibody labels were used to map the axon projections in the thalamus from embryonic days 9.0-12, revealing several distinct neuron populations that contributed to the first tracts. Each of the early axon populations first grew independently, pioneering a short segment of new tract. However, each axon population soon merged with other axons to form one of only two shared longitudinal tracts, both descending: the tract of the postoptic commissure (TPOC), and, in parallel, the stria medullaris. Thus, the forebrain longitudinal tracts are pioneered by a relay of axons, with distinct axon populations pioneering successive segments of these pathways. The extensive merging of tracts suggests that axon-axon interactions are a major guidance mechanism for longitudinal axons. Several axon populations express tyrosine hydroxylase, identifying the TPOC as a major pathway for forebrain dopaminergic projections. To start a genetic analysis of pioneer axon guidance, we have identified the transcription factor Pax6 as critical for tract formation. In Pax6 mutants, both longitudinal tracts failed to form due to errors by every population of early longitudinal axons. Taken together, these results have identified potentially important interactions between series of pioneer axons and the Pax6 gene as a general regulator of longitudinal tract formation in the forebrain.

Fig. 1

Early longitudinal axons project through the Pax6⁺ thalamic region. Wild-type whole-mount embryos labeled with β-III tubulin (A–C) or Pax6 antibody (D). A: Two main clusters of neurons and fibers were labeled in anterior forebrain at embryonic day (E) 9.5. TPOC (white arrow) and A13 neurons (open arrow) appear very early in forebrain development. B: By E10.5, increased numbers of TPOC and A13 axons project through VT. C: Many more axons and neurons were labeled by E11.5, with the labeling becoming too crowded to resolve individual tracts. However, a new tract (SM) formed at the border between CV and VT (arrowhead), and axons project from the supraoptic region can be seen. D: Pax6 antibody labeling shows expression of Pax6 in forebrain at E10.5. Pax6 expression overlaps with TPOC in VT. Dashed lines, DT/VT boundary. For abbreviations, see list. Scale bar = 200 μm in D (applies to A–D).

Fig. 2

Time course of TPOC axon growth through the forebrain. A: Schematic representation of TPOC projection pattern. The gray line represents axons; the circle indicates cell bodies. B–D: TPOC neurons are located at the base of the optic stalk, and TPOC axons were labeled with DiI in embryos at different developmental stages. B: At 30 somites (som), the leading TPOC axons were in the VT. C: At 32 somites, the first TPOC axons entered DT. The majority of the axons were close to the VT/DT boundary but had not crossed yet. D: At 34 somites, more axons crossed the boundary, with the longest projecting into midbrain. Dashed lines, DT/VT boundary; asterisks, location of DiI crystals. For abbreviations, see list. Scale bar = 200 μm in D (applies to B–D).

Fig. 3

A13 axons precede TPOC axons across the interthalamic boundary. A: Cell bodies of A13 axons retrogradely labeled with DiI from DT. Inset shows the location of DiI crystal (red dot) and angle of section (red dashed line) for E and F. B,C: TH antibody labeling showed that TH⁺ cell bodies were distributed over VT (arrowhead in B), whereas the source of TPOC was TH⁻ (B); the longest TH+ projections reached DT by the 30-somite (som) stage (C). D: Anterograde labeling of TPOC with DiI (red) combined with retrograde labeling of A13 axons with DiO (green). A13 neurons were located in the TPOC pathway; thus, these two axon populations share the TPOC tract. E: Section. A13 neurons retrogradely labeled with DiI and photoconverted (gray), then labeled with Pax6 antibody. Arrows indicate Pax6 expression in A13 neurons. F: A subset of the A13 neurons were double labeled with Pax6 and TH (n = 2; arrows). Dashed lines, interthalamic boundary; asterisks, location of DiI and DiO crystals. For abbreviations, see list. Scale bars = 50 μm in C (applies to A,C), 100 μm in B, 150 μm in D, 10 μm in E,F.

Fig. 4

A13 and TPOC axons make errors in Pax6 mutants. A,B: Whole-mount antibody labeling against TH revealed projection pattern of A13 axons and SN in wild-type (+/+) and Pax6 mutant (−/−) embryos. A: A13 axons projected through DT in wild-type (embryonic day [E] 10.5). SN-VTA complex, located in midbrain floor plate, extended their axons rostrally through VT (A). B: In Pax6 mutants, A13 axons did not project into DT but instead turned dorsally at the interthalamic boundary. SN-VTA axons failed to project into DT. C: DiI labeling from dorsal region of VT, anterior to interthalamic boundary. Inset shows location of the label site. Cell bodies of A13 axons retrogradely labeled from this dorsal location confirmed that A13 axons did not project into DT but rather turned dorsally at the boundary. D: Errors in guidance of TPOC axons are shown by DiI labeling. Similar to A13 axons, TPOC axons failed to cross the inter-thalamic boundary and instead turned dorsally along it. Some of the TPOC axons projected dorsally into CV. Dashed lines, DT/VT boundary. Scale bar = 400 μm in D (applies to A–D).

Fig. 5

Supraoptic tract (SOT) axons project along TPOC pathway and make errors in Pax6 mutants. A: Location of crystals and schematic representation of SOT. The blue line indicates the angle of section in F. B,C: SOT axons started to project at embryonic day (E) 11.5, coursed ventrally and merged with the TPOC. DiI labeling of SOT at E11.5 (B) and E12.5 (C) shows the projection pattern of SOT. SOT axons formed two branches. The ventral branch joined TPOC and the dorsal branch projected close to the cerebral vesicle–thalamus boundary, merging with SM. D: Labeling of SOT with DiO (green) and TPOC with DiI (red). SOT axons and TPOC axons had an overlapping projection pattern. E: Double labeling of SOT cell bodies with DiI (brown) and Pax6 (green; n = 2). A subpopulation of SOT neurons expressed Pax6 (white and green arrowheads), whereas some photo-converted SOT neurons did not express Pax6 (white arrowheads) and some Pax6⁺ cells were not back-labeled with DiI (green arrowhead). F: DiI label of SOT axons in Pax6 mutant embryo. The majority of SOT axons turned dorsally at the boundary, although a few axons projected into DT and formed loops. Dorsal tracts, including the stria medullaris (SM) and tract of the zona limitans (TZL) are disrupted in Pax6 mutants. G: Schematic representation of projection pattern TZL (blue) and SM (pink). H: Anterograde labeling of TZL and retrograde labeling of SM with DiI from dorsal aspect of VT anterior to interthalamic boundary. TZL projected along interthalamic boundary and turned caudally into DT, while SM projected through DT parallel to thalamus and CV boundary. I: Labeling of TPOC with DiI (red) and SM and TZL with DiO (green). J: Higher magnification of boxed area in B. TZL axons merged with TPOC in DT. K: DiI labeling from dorsal region of VT in Pax6 mutants. Both TZL and SM failed to be labeled, but A13 neurons were back-labeled. Dashed lines, interthalamic boundary; asterisks, location of DiI and DiO crystals. For abbreviations, see list. Scale bars = 500 μm in B (applies to B–D,F), 10 μm in E, 500 μm in J, 500 μm in H (applies to H,I,K).

Fig. 6

Pax6 is a common regulator of several distinct axon populations in the forebrain. A,B: Schematic map of tracts and their sources in the forebrain of wild-type (A) and Pax6 mutant (B) embryos. The gray area represent tracts, and colored lines indicate different populations of axons projecting along those tracts. For abbreviations, see list.