Fgf8-Related Secondary Organizers Exert Different Polarizing Planar Instructions along the Mouse Anterior Neural Tube

Peer reviewe

Fat4-Dchs1 signalling controls cell proliferation in developing vertebrae

The protocadherins Fat4 and Dchs1 act as a receptor-ligand pair to regulate many developmental processes in mice and humans, including development of the vertebrae. Based on conservation of function between Drosophila and mammals, Fat4-Dchs1 signalling has been proposed to regulate planar cell polarity (PCP) and activity of the Hippo effectors Yap and Taz, which regulate cell proliferation, survival and differentiation. There is strong evidence for Fat regulation of PCP in mammals but the link with the Hippo pathway is unclear. In Fat4(−/−) and Dchs1(−/−) mice, many vertebrae are split along the midline and fused across the anterior-posterior axis, suggesting that these defects might arise due to altered cell polarity and/or changes in cell proliferation/differentiation. We show that the somite and sclerotome are specified appropriately, the transcriptional network that drives early chondrogenesis is intact, and that cell polarity within the sclerotome is unperturbed. We find that the key defect in Fat4 and Dchs1 mutant mice is decreased proliferation in the early sclerotome. This results in fewer chondrogenic cells within the developing vertebral body, which fail to condense appropriately along the midline. Analysis of Fat4;Yap and Fat4;Taz double mutants, and expression of their transcriptional target Ctgf, indicates that Fat4-Dchs1 regulates vertebral development independently of Yap and Taz. Thus, we have identified a new pathway crucial for the development of the vertebrae and our data indicate that novel mechanisms of Fat4-Dchs1 signalling have evolved to control cell proliferation within the developing vertebrae

Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis

Skeletal shape varies widely across species as adaptation to specialized modes of feeding and locomotion, but how skeletal shape is established is unknown. An example of extreme diversity in the shape of a skeletal structure can be seen in the sternum, which varies considerably across species. Here we show that the Dchs1–Fat4 planar cell polarity pathway controls cell orientation in the early skeletal condensation to define the shape and relative dimensions of the mouse sternum. These changes fit a model of cell intercalation along differential Dchs1–Fat4 activity that drives a simultaneous narrowing, thickening and elongation of the sternum. Our results identify the regulation of cellular polarity within the early pre-chondrogenic mesenchyme, when skeletal shape is established, and provide the first demonstration that Fat4 and Dchs1 establish polarized cell behaviour intrinsically within the mesenchyme. Our data also reveal the first indication that cell intercalation processes occur during ventral body wall elongation and closure

Brefeldin A (BFA) treatment inhibits ERK1/2 activity and modulates differentially Fgf8 negative feed-back regulators.

<p>A, B and C) are 12 µM cryostat transversal sections of mouse ONTCs to the isthmic constriction. A and B) are control example before BFA administration to the culture medium showing an ISH for <i>Fgf8</i> (A) and an Immunostaining for anti-FGF8 (B). Note the different domains of expression of the transcript (delineated by the solid line and arrows) and of the protein FGF8 (delineated by the red arrows). Note also that FGF8 immunodetection is detected both at basal and ventricular sides of the ONTCs (see black arrows in B). After 4 hours of BFA incubation (C-J) the mRNA of <i>Fgf8</i> was maintained at the IsO (D) while the FGF8 protein profile changed dramatically being accumulated only at the ventricular side (D) as small vesicle-like, (see arrows in the figure C and the magnified insert). Moreover, ERK1/2 activity disappears in Isthmic cells and nearby cells (E). Inside this negative gap, FGF8b beads still exerts polarizing ERK1/2 effects (F). Also inside this gap, genes such us <i>Mkp3</i> (G) and <i>Sef</i> (H) disappear in the mesencephalon while <i>Sprouty</i> family genes are maintained (I,J). K) represents the experiments and model by which FGF8 planar induction activity coming from mouse FGF8-related secondary organizers (IsO and anr) exerts a different tissue preferential signaling effects (based on the activation of ERK1/2). The direction of polarized ERK1/2 activity depends on the location of FGF8-related secondary organizers and the establishment of this positional information signaling is dependent mainly on FGF8 negative modulator system, particularly <i>Sprouty2</i> (blue gradient). Moreover, FGF8 morphogenetic planar instruction signals coming from rostral (anr) and caudal (IsO) diminish and loose their polarization effect at the diencephalic region (zli) resulting in an equilibrium state. Scale bars is 100 µm except for D,E, G-J which is 0,5 mm.</p

Initial FGF8 planar instruction effects in IsO after a short signaling deprivation.

<p>.Schematic representation of isthmic FGF8 feedback modulator genes regulation given by the isthmic gradiental FGF8 morphogen (and signaling) on the mouse mesencephalon. The graphic describes the distribution of dosage (activity) versus time and space of FGF8 and of <i>Fgf8</i> negative signal modulators. A) represents the normal FGF8 signal activity in the IsO (high levels of FGF8 protein; red solid wedge), during which FGF8 maintains at dose-dependent manner the different FGF negative modulators expression profile starting from <i>Mkp3</i> (purple solid curve), <i>Sef</i> (green) and finishing <i>Sprouty</i> genes (where low levels of FGF8 protein). The yellow background represents ERK activity. B) describes the presumed situation during Brefeldin a (BFA) treatment (4 hours) on the FGF8 morphogenetic activity. Thus, at isthmus the FGF8 protein level (a therefore morphogenetic activity: red solid bell-shaped curve) would be cero but some residual protein away from the source would still activate ERK (yellow solid slope curve). Inside this negative gap of ERK1/2 activity the expression of <i>Mkp3</i> (purple dashed curve line) and <i>Sef</i> (green dashed curved line) at the mesencephalon is completely absent. Nonetheless the residual FGF8 morphogen apart from the isthmus is enough to maintain <i>Sprouty1/2</i> expression in the mesencephalon in the absence of ERK activity (red asterisks’; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039977#pone.0039977-SuzukiHirano1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039977#pone.0039977-Suzuki1" target="_blank">[76]</a>).</p

Phosphorylation patterns of ERK1/2 (dpERK) enzymes in the anterior neural tube.

<p>Whole mount in situ hybridization (ISH) of E9.5 mouse embryo (A,B,B’) and corresponding organotypic neural tissue cultures of mouse E9.5 anterior neural tube (A’,B”,C-E”; ONTCs; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039977#pone.0039977-Echevarria1" target="_blank">[42]</a>) where it shows the maintenance of gene expression profiles such us <i>Fgf8</i>, <i>Tcf4</i> (A,A’) (used to delimit main brain subdivisions such the diencephalon (D; <i>Tcf4</i> positive) and the mesencephalic anlage (M; negative staining), <i>Pax6</i> D-D” (delimiting di-mesencephalic boundary and rhombomere 1–2 limits) and <i>En2</i> E-E”. In B-B”) are photomicrographs of E9.5 mouse embryo with <i>anti-</i>dpERK Immunohistochemistry (IHC) taken from lateral (B) and caudal (B’) sides and the corresponding IHC in ONTCs. (C) double staining procedure: ISH (in blue) for <i>Fgf8</i> and IHC for dpERK (dark brown) to localize inside the dpERK domain the position of the IsO, marked by the solid red line. (D-E”) photomicrographs of same ONTCs in which first a whole mount ISH for <i>Pax6</i> (D) <i>or Tcf4/En2</i> (E) were made and afterwards IHC against dpERK. (D’,E’ respectively). Dashed lines mark the main transversal (in black) and longitudinal (in red) brain subdivisions. These ONTCs were cut into transversal sections to the isthmic constriction (D”,E”) to proof that indeed dpERK expression reaches diencephalic anlage (D”; see asterisk; rostral is left) and has a wider expansion than <i>En2</i> expression (E”; see asterisk; rostral is left). anr is anterior neural ridge secondary organizer; ba is branchial arch; IsO is isthmic organizer; os is optic stalk; ov is otic vesicle; r is rhombomere; T is telencephalon, D, diencephalon, M, mesencephalon. Scale bars are 0,5 mm except in D”, E” they are 100 µm.</p

FGF8 planar induction from IsO has initial tissue preferences in mesencephalon.

<p>Classical FGF8 soaked bead implantation in mesencephalon induces ERK1/2 activity before earliest induction of mRNA (<i>Mkp3</i>) could be detected (D). Interestingly asymmetric distribution is detected during the first two hours after incubation (A, A’ and B, B’). This asymmetry is lost from 3 hours onwards after bead implantation (C, C’). In cryostat sections a mesencephalic bead implantation induced highly intense ERK1/2 activation as observed at the rostral side of the bead after 1 hour of incubation (A’). After 2 hours, ERK phosphorylation is detected caudal to the bead (B’; see small asterisk). Finally ERK activity is homogeneously distributed at both rostral and caudal cells after 4 hours (C’). Red asterisks indicate an FGF8 soaked bead; blue asterisk indicate a PBS bead. Scale bars are 0,5 mm except for A’, B’, C’ that are 50 µm, in D’ is 0,25 mm.</p

The position of FGF8- related secondary organizers determines the polarity of ERK1/2 activation.

<p>.A-D’) show the type 1 experimental manipulation in ONTCs where a dissection of the IsO region was made, left for 24 hours <i>in vitro</i> and thereafter it was incubated with BAF for 2h after an implantation of a FGF8b bead in mesencephalon (C-C’) or middle diencephalon (D-D’). <i>Fgf8</i> mRNA (A) and <i>Sprouty 2</i> (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039977#pone-0039977-g004" target="_blank">Figure 4C</a>). Bead implantations in the diencephalon maintained symmetric distribution of ERK1/2 activity around the bead (D,D’). In these experiments <i>FgfR1</i> 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 (<i>Sprouty2</i>; D) and the ectopic induction of ERK1/2 activity was found symmetrically distributed around the bead (F and F’) on <i>FgfR1</i> positive domain (I). In fact the lack of <i>FgfR1</i> 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 µm.</p

Low threshold of FGF8 protein levels disrupts ERK1/2 phosphorylation patterns.

<p>.DpERK immunodetection was absent in the isthmic domain using ONTCs severe hypomorphic mouse mutant (A, B; <i>Fgf8 ^neo/null</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039977#pone.0039977-Meyers1" target="_blank">[45]</a>). Yet a small tip of <i>En2</i> (B) positive expression was visible at the most dorsal parts, probably by the maintenance of <i>Fgf8</i> (C) expression. Under these mutant conditions, none of the FGF8 signal negative modulators <i>Mkp3</i> (D), <i>Sef</i> (E) <i>Sprouty2</i> (F) were observed at IsO. Asterisks indicate the position of abolished isthmic region and solid line the boundary between diencephalon/mesencephalon. Scale bar in C is 0,5 mm for all images.</p

Bafilomycin A1 (BAF) treatment demonstrates the polarization of ERK activity by FGF8 signal activity along the neural tube.

<p>A-C) BAF amplifies ectopic ERK1/2 activity-related FGF8 induction revealing a clear polarized distribution of ERK1/2 activity around FGF8 soaked beads depending of the implanted bead; rostral to IsO (C,D,G), or caudal to IsO (C,E). Nonetheles, isthmic organizer morphogenetic activity seems unaffected for <i>Fgf8</i> (A) and negative regulator <i>Sprouty 2</i> (B) expressions. Note that PBS bead implantation in control side (blue asterisk in C and F) did not show any ectopic induction. Two hours after bead implantation a clear amplified and almost non-homogeneous ERK1/2 activity was detected rostrally in the mesencephalon (rostral to the IsO), which was detected caudally when bead was placed in hindbrain (caudal to the IsO) territories (E). In telencephalic vesicles, caudal to the anr (H) the polarity of ERK activation was reversed. This polarized dpERK detection around the bead is lost at the zli (zona limitans intrathalamica) region (I). Similar symmetric ERK-related FGF8 signal found in zli was seen when placing a FGF8 bead in the midbrain of <i>Fgf8</i> hypomorphic mice (J,K). Importantly FGF8b protein distribution (M) was observed apparently in equal intensity and range at rostral (N) and caudal (O) sides of the bead (for comparison with PBS bead in panel L). Scale bars are 0,5 mm in A, B, C, H, I, 200 µm in D, E, J, 100 µm in F, G, K, L, M, and 50 µm in N, O.</p