and Slit2 are most likely not direct targets of Pax6. Additional experiments suggest that Slit1 and Slit2 may also act as repellent to push mms neurons to the marginal migration route during the initial phase of migration (data not shown). Pou4f2 controls ipsilateral versus contralateral settling of LRN neurons and causes a migrational arrest at the midline Two POU transcription factors were among the genes whose expression pattern was altered in the mms of Pax6Sey/Sey embryos. Pou4f2 (also: Brn3b) was strongly expressed in about 18.6% Figure 4. The Slit-Robo pathway controls the settlement of LRN neurons. (A,A9,B,B9) In situ hybridization of E13.5 coronal wt (A, B) and Pax6Sey/Sey (A9,B9) vibratome sections. The Slit receptor Robo3 is expressed in neurons at the basal and alar plate and in the mms (A). In Pax6Sey/Sey embryos basal and alar plate expression of Robo3 is identical to the wt expression (A9). However, Robo3 expression in the Pax6Sey/Sey mms again demonstrates that mutant cells are delayed in midline crossing (white arrow in A9). In wt embryos Slit2 is expressed in a small group of cells at the midline, in the hypoglossal nuclei (arrowheads in B), in two small groups of cells at the site of the future LRN (black arrow in B), and at the ventricular zone of the rhombic lip. In Pax6Sey/Sey embryos the hypoglossal nuclei and therefore also the hypoglossal Slit2 expression are lost, whereas, other sites of Slit2 expression are unchanged (B9). shRNA driven knockdown of Robo3 results in a scattered LRN, suggesting that the Slit-Robo pathway controls the settling of LRN neurons (C). Control constructs had no effect on the LRNc (C9). To determine whether a local or a longitudinal distribution of guidance cues regulates the settling of LRN neurons into two sub-nuclei transplantation experiments were performed. After electroporation brainstem cultures were cut along the midline and the contralateral side was shifted to a posterior direction (D). The positions of the inferior olive (IO) and the hypoglossal nuclei (nXII) are indicated. The extent of the shift was determined by Etv1 in situ hybridization which specifically labels IO neurons (open arrows in F). (G) In a typical experiment in which the shift was larger than 500 mm neurons settled randomly in the contralateral LRN, suggesting, that cues close to the inferior olive, e.g. the hypoglossal nuclei, serve for the correct guidance of LRNc neurons. In control experiments in which there was no shift (E), LRNc neurons settled in a wt pattern and formed two groups of neurons (H). (Scale bar is 1.3 mm in [A,A9,B,B9]; 0,7 mm in [C,C9]; 0.6 mm in [F]; 0.44 mm in [G,H].) doi:10.1371/journal.pgen.1002099.g004 Pax6 Controls Cell Migration in the Brainstem (64.6%, n = 3) of E14.5 and 23.3% (66.2%, n = 3) of E15.5 wt mms neurons but was completely lost in the Pax6Sey/Sey mms (Figure 5A, 5A9, 5B, 5B9). Pou4f1 (also: Brn3a) was expressed between E13.5 and E15.5 in a subset of mms neurons, but was up- regulated in the E14.5 and E15.5 Pax6Sey/Sey mms (Figure 5C, 5C9). Expression of Pou4f1 and Pou4f2 in Pax6Sey/Sey IO neurons was unaltered (Figure 5A, 5A9, 5C, 5C9). Pou4f2 plays several roles in specifying and guiding retinal ganglion cells and their axons. We therefore asked whether Pou4f2 may accomplish similar tasks in rhombic lip derived neurons. Pou4f2 was only expressed in a subset of wt mms neurons. We therefore over-expressed Pou4f2 in all migrating mms neurons. Remarkably, growth cones of all Pou4f2 over-expressing neurons were arrested at the midline for about 1.5 days (60.5 days, n = 17), whereas the majority axons in control cultures crossed the midline instantly (Figure 5D, 5F). Interesting- ly, in control cultures the growth cones of some neurons also appeared to be arrested at the midline: 5% (63%) at 1DIV, 15% (66%) at 2DIV, 25% (65%) at 3DIV, and 6% (63%) at 4DIV (n = 11). This correlates well to the peak of Pou4f2 expression at E14.4 and E15.5 (in cultures: 2DIV and 3DIV). Over-expression of Pou4f2 had also a noticeable effect on the settling behavior of LRN neurons. Quantification of LRN neurons at 5DIV revealed that Pou4f2 expressing LRN neurons preferably settled at the ispilateral side (LRNc/LRNi = 0.8 6 0.1, n = 17; Figure 5G, 5M) compared to control cultures in which the majority of LRN neurons settled at the contralateral side (LRNc/LRNi = 2.5 60.1, n = 50; Figure 5E, 5M). Similar relations were obtained at 6DIV and 8DIV suggesting that Pou4f2 over-expression altered the migration behavior of mms neurons and did not cause a delayed settlement of these neurons. The effect was specific to Pou4f2 and could not be mimicked by over-expression of Pou4f1, Pou4f3 or Pou6f1 (Figure 5M and data not shown). Together these data suggest that Pou4f2 acts through a novel mechanism which induces an arrest of growth cones at the midline to regulate the ratio of ipsilaterally versus contralaterally settling neurons. We altered expression levels of about 25 potential Pou4f2 retinal target genes [38–40] and of these two showed an effect on the migration behavior of mms neurons. Over-expression of Gfi1, a zinc finger transcription factor, reduced the contra-/ipsi-lateral ratio of LRN neurons (Figure 5M). In contrast, the down- regulation of Gap43 by shRNA constructs caused a higher contra-/ ipsi-lateral ratio of LRN neurons (Figure 5M and Figure S4). Gap43 is slightly reduced in the Pax6Sey/Sey mms (Figure S5). In addition, mis-expression of Pou4f2 resulted in a massive down- regulation of Pou4f1 in transfected, but not in control, rhombic lips (Figure 5N), suggesting that the loss of Pou4f2 in Pax6Sey/Sey mms neurons leads to an up-regulation of Pou4f1 (Figure 5C, 5C9). Pou4f1 over-expression or down-regulation, however, did not alter migration behavior of mms neurons (Figure 5M). Pou4f2 is expressed only in a subset of Pax6 positive mms neurons suggesting that other factors together with Pax6 may co-regulate Pou4f2. In the developing retina Pou4f2 expression depends on two transcription factors: the bHLH protein Math5 and the zinc finger gene Wt1 [41–44]. Wt1 was found to be expressed in the rhombic lip, though, in a region just dorsally to the Pax6 positive domain (Figure 5J). Math5 was neither expressed in the rhombic lip nor in migrating mms neurons, however, a close homologue, Math1, was expressed in neuronal precursors at the rhombic lip and in a subset of early migrating mms neurons [22,23] (Figure 5K). Thus, Math1, but neither Math5 nor Wt1, was the most likely candidate to regulate Pou4f2 or Pou4f1 expression in mms neurons. Consistent with this, mis-expression of Math1, but not of Wt1 (+ and – KTS splice variants) or Math5, led to a midline arrest of migrating mms neurons and a reversed settling behavior of LRN neurons PLoS Genetics | www.plosgenetics.org 7 June 2011 | Volume 7 | Issue 6 | e1002099