Polarization Activity of Fgf8 in Mouse Brain BAF for 2h after an implantation of a FGF8b bead in mesencephalon (C-C’) or middle diencephalon (D-D’). Fgf8 mRNA (A) and Sprouty 2 (B) were maintained at anterior neural ridge (ANR), optic stalk (os) and branquial arches (ba) but they were absent in caudal regions of the ablated ONTCs. Bead implantations in mesencephalon modified ERK1/2 polarization towards caudal parts of the bead (C, C’; for comparison see Figure 4C). Bead implantations in the diencephalon maintained symmetric distribution of ERK1/2 activity around the bead (D,D’). In these experiments FgfR1 expression (G) was maintained in IsO ablations (H). E,F,F’’ and I show type 2 experimental manipulation assays in ONTCs where rostral forebrain (anr included) and hindbrain (type 1 experiment) were ablated. Under these conditions and following the BAF incubation protocol, the tissue left did not express any FGF8 downstream genes (Sprouty2; D) and the ectopic induction of ERK1/2 activity was found symmetrically distributed around the bead (F and F’) on FgfR1 positive domain (I). In fact the lack of FgfR1 in the midbrain and hindbrain region, does not disturb ERK1/2 polarizing effects on both brain regions (J). Scale bars are 0,5 mm except for C’, E’ that are 100 mm. doi:10.1371/journal.pone.0039977.g005 and pial sides of this pseudostratified neuroepithelium suggesting diffusion facilitation through basal lamina (Figure S2C,D) and [54]). Similar patterns of Fgf8 gene expression and FGF8 protein distribution were also observed in cryostat sections of wild-type ONTCs (n = 3/3; Figure 6A,B). We then treated the ONTCs with BFA at 25 mg/ml (Figure 6C-J; [55]) for a period of 4 hours of incubation. Under BFA treatment, a positive anti-FGF8b im- munolabeling was detected exclusively in the territory of the IsO and now restricted to the Fgf8 gene expression domain (n = 3/3; compare Figure 6A, C). Interestingly, BFA treatment revealed a more intense immunopositive reaction for FGF8 only at the ventricular surface of the pseudostratified isthmic neuroepithelium (absent in the basal lamina; asterisk in Figure 6C). Also, the FGF8 immunostaining was detected as bead-like structures, resembling exocytic vesicles and interestingly only concentrated close to the ventricular surface (Figure 6C insert). These data strongly suggest that FGF8-producing cells (Fgf8 mRNA positive) in the mouse IsO, secrete the morphogen near the lumen of the neural tube but the gradient of secreted Fgf8 protein forms a gradient along the basement membrane. More importantly, blockade of exocytosis caused a fast downregulation of ERK1/2 activity in the IsO domain (n = 4/5; Figures 6E and Figure 7A,B), while in territories further away, cells still remained dpERK positive. Thus, the sudden blockade of exocytosis of FGF8 protein secretion affected first the FGF8b producing cells (i.e. the isthmic organizers cell population) and gradually the neighboring territories away from them (see Figure 6E and Figure 7A,B). These results provide new cellular mechanistic information about the induction characteristics of the isthmic FGF8-expressing cells (see discussion). Therefore, these neuroepithelial cells required secretion of FGF8 to exert proper activation of FGF8 intracellular pathways. Interestingly, implan- tation of an ectopic FGF8b bead inside this negative phosphor- ylated ERK1/2 domain for (n = 4/5; Figure 6F) still exerted an asymmetric distribution of dpERK labeling after 2 hours of incubation (n = 2/2; Figure 6G). During this process of depriving the neural tube from extracellular FGF8 protein, Mkp3 was not detected inside the negative phosphorylated ERK1/2 domain (n = 3/4; Figure 6H and Figure 7B). Of the other known FGF8 modulators [12] we found that Sef expression was completely downregulated in the mesencephalon but not in the r1 ((n = 3/3; Figure 6I). Moreover, the Sprouty1 gene showed a similar pattern as Sef but with a less severe phenotype in the mesencephalon (n = 4/ 6; Figure 6J and Figure 7B). Finally, expression pattern of Sprouty2 was maintained both in mesencephalic and rhombencephalic territories as in control ONTCs (n = 4/4; Figure 6K, Figure 7B and Figure S1B’). In conclusion, the specification of early FGF8- related positional information signaling that will induce later differentially biological responses at different distances from the IsO seems to be controlled by graded expression of the FGF8 negative modulators. Our results strongly suggest requirement for mainly Sprouty1/2, but not of Mkp3 and most probably not of Sef, for the initial polarized response to FGF8 in the mouse midbrain. Discussion The reliable mapping of active ERK signaling domains in Drosophila [56] and diverse vertebrates such as xenopus, zebrafish, mouse and chick [37–39,57] have been obtained using antibodies specific to the di-phosphorylated forms of ERK1 and ERK2. We have demonstrated a close relationship between FGF8 signal activity coming from the isthmic region and ERK1/2 phosphorylation, using open-book E9.5 mouse neural tube organotypic tissue explants cultures (ONTCs; [42]), which also corroborated other in vivo studies [38,39]. Investigators working in other vertebrate brain regions such as mouse telencephalon [58,59], chick caudal hindbrain and spinal cord [60,61] have proposed also ERK1/2 phosphorylation immunostaining as a direct readout tool of FGF signal activity. Although other pathways, such as integrins, cytokines and G-protein-coupled- receptors, can also activate the RAS-MAP-ERK pathway [62], the majority of ERK1/2 activity domains correspond to FGF signaling domains [24,32,38,39,63]. Nevertheless, the isthmic region remains the most reliable and sensitive model system for understanding FGF8 function in neural tube development in vertebrates [23,41]. Here, we demonstrated that expression of neither negative feedback modulators of FGF8 signaling or phosphorylated forms of ERK1/2 were detected in the mid- hindbrain territories at E9.5 on Fgf8 hypomorph mutant mice [23,45] corroborating the tight close link between the morphogen FGF8 and ERK1/2 activity. FGF8 downstream negative modulators have been used as indirect markers for the study of FGF8 signal activity in the vertebrate IsO [12,16,22]. These genes are expressed in same regions as Fgf8 but in a wider and graded long-range pattern. In contrast, at E9.5, phosphorylated forms of ERK1/2 showed no clear graded patterns. In fact, the more homogeneous distribution of ERK1/2 labeling in the mid- hindbrain territories reached rostrally the diencephalic/mesencephalic boundary and caudally the rhombomere R1/R2 limit, unveiling the maximal long-range activity of endogenous FGF8 at this developmental stage. Other mouse IsO-related genes can reach similar neuroepithelial boundary limits before E9.5 such as Engrailed1/2, Pax2 and Sprouty1/2 but from E9.0 they become restricted closer to the isthmus [25,43,64,65]. Moreover, we have found that ERK1/2 activity is also the fastest readout of FGF8b morphogenetic activity in the mouse anterior neural tube. Already at 60 minutes, FGF8b soaked bead implantations in the mesencephalon of E9.5 mouse ONTCs caused ectopic ERK1/2 activation. That makes the detection of ERK1/2 phosphorylation form a convenient tool for understanding early FGF8b morphogenetic signal interpretation [38,39]. Similar experimental assays have been also described in the chick spinal cord at earlier stages of development [60]. The ability of a rapidly internalized receptor to signal after endocytosis is important to ensure the sufficient duration and intensity of signaling. However, this capacity requires receptors to remain active in endosomes and therefore able to di-phosphorylate ERK [47]. Several Receptor Tyrosine Kinases (RTKs), for PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e39977