18Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. 19Mechanisms must be in place along the route to ensure they connect with their 20 targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, 21 but the importance of the diencephalon in TCA mapping is unknown. We report that 22 disruption of diencephalic development by Pax6 deletion results in a thalamocortical 23 projection containing mapping errors. We used conditional mutagenesis to test 24 whether these errors are due to the disruption of pioneer projections from 25 prethalamus to thalamus and found that, while this causes abnormal TCA 26 fasciculation, it does not induce topographical errors. To test whether the thalamus 27 contains important navigational cues for TCAs, we used slice culture transplants and 28 gene expression studies. We found the thalamic environment is instructive for TCA 29 navigation and that the molecular cues Netrin1 and Semaphorin3a are likely to be 30 involved. Our findings indicate that the correct topographic mapping of TCAs onto 31 the cortex requires the order to be established from the earliest stages of their 32 growth by molecular cues in the thalamus itself. 33 telencephalon requires interactions between the axons and signals released by cells 59 they encounter in this region (Bielle et al., 2011; Bonnin et al., 2007; Braisted et al., 60 1999; Dufour et al., 2003; Molnár et al., 2012; Powell et al., 2008). The importance of 61 earlier interactions within the diencephalon remains unclear. 62Here, we tested the effects of mutating the gene for the Pax6 transcription factor, 63 which is essential for normal diencephalic patterning (Caballero et al., 2014; Clegg et 64 al., 2015; Parish et al., 2016; Pratt et al., 2000; Stoykova et al., 1996; Warren and 65 Price, 1997), on the topographic mapping of TCAs onto the cortex. Pax6 starts to be 66 expressed in the anterior neural plate well before TCAs start to form (Walther and 67 Gruss, 1991). As the forebrain develops from the anterior neural plate, Pax6 68 expression becomes localized in (i) cortical progenitors that generate the target 69 neurons for TCAs, (ii) diencephalic (thalamic and prethalamic) progenitors and (iii) 70 prethalamic (but not thalamic) postmitotic neurons (Quintana-Urzainqui et al., 2018; 71 Stoykova et al., 1996; Warren and Price, 1997). We discovered that deletion of Pax6 72 from mouse embryos at the time when thalamic axons are starting to grow results in 73 the development of a thalamocortical projection containing mapping errors. Axons 74 from dorsolateral thalamus are misrouted medially and end up projecting abnormally 75 rostrally in the cortex. We went on to explore the reasons for this defect. 76We first used conditional mutagenesis to test whether misrouting is due to the loss of 77 Pax6 from prethalamic neurons, since previous work has shown that (i) Pax6 is not 78 required in the cortex for normal TCA topography (Piñon et al., 2008) and (ii) Pax6 is 79 neither expressed ...