Integrin-α5 coordinates assembly of posterior cranial placodes in zebrafish and enhances Fgf-dependent regulation of otic/epibranchial cells

Abstract

Vertebrate sensory organs develop in part from cranial placodes, a series of ectodermal thickenings that coalesce from a common domain of preplacodal ectoderm. Mechanisms coordinating morphogenesis and differentiation of discrete placodes are still poorly understood. We have investigated whether placodal assembly in zebrafish requires Integrin- α5 (itga5), an extracellular matrix receptor initially expressed throughout the preplacodal ectoderm. Morpholino knockdown of itga5 had no detectable effect on anterior placodes (pituitary, nasal and lens), but posterior placodes developed abnormally, resulting in disorganization of trigeminal and epibranchial ganglia and reduction of the otic vesicle. Cell motion analysis in GFP-transgenic embryos showed that cell migration in itga5 morphants was highly erratic and unfocused, impairing convergence and blocking successive recruitment of new cells into these placodes. Further studies revealed genetic interactions between itga5 and Fgf signaling. First, itga5 morphants showed changes in gene expression mimicking modest reduction in Fgf signaling. Second, itga5 morphants showed elevated apoptosis in the otic/epibranchial domain, which was rescued by misexpression of Fgf8. Third, knockdown of the Fgf effector erm had no effect by itself but strongly enhanced defects in itga5 morphants. Finally, proper regulation of itga5 requires dlx3b/4b and pax8, which are themselves regulated by Fgf. These findings support a model in which itga5 coordinates cell migration into posterior placodes and augments Fgf signaling required for patterning of these tissues and cell survival in otic/epibranchial placodes.

Figure 1. Knockdown of itga5 impairs morphogenesis of posterior cranial placodes.

(A–D) pax2a expression at 12 and 14 hpf in the otic/epibranchial domain in control embryos (A, C) and itga5 morphants (B, D). Expression is normal at 12 hpf in itga5 morphants but the otic placode (o, brackets) is smaller than normal by 14 hpf. (E, F) fgf24 expression at 14 hpf in a control embryo (E) and itga5 morphant (F). (G, H) Otic vesicles at 24hpf in a control embryo (G) and itga5 morphant (H). (I, J) ngn1 expression at 11 hpf in a control embryo (I) and itga5 morphant (J). (K, L) neuroD expression at 14 hpf in a control embryo (K) and itga5 morphant (L). Precursors of the trigeminal ganglion (tg) and anterior lateral line (al) are indicated. (M, N) Anti-Isl-1 immunostaining at 24 hpf in a control embryo (M) and itga5 morphant (N). (O, P) phox2a expression in epibranchial ganglia at 30 hpf in a control embryo (O) and itga5 morphant (P). Facial (f), glossopharyngeal (g), and vagal ganglia (v1+v2) are indicated. A–E, I–K are dorsal views with anterior to the top; G, H, M–P are lateral views with anterior to the left. Scale bar, 50 µm.

Figure 2. Otic/epibranchial precursors show aberrant migration in itga5 morphants.

(A–D) Images from time-lapse movies showing transgenic expression of pax2a:GFP (green) and mosaic expression of cmv:RFP (red). The first frame (11.5 hpf) and final frame (14.5 hpf) of a control movie (A, B) and itga5 morphant movie (C, D) are shown. Arrows indicate cells that expressed both GFP and RFP. Blue arrows indicate cells that contributed to the otic domain, and white arrows indicate cells that contributed to non-otic domains. Red arrows indicate cells that lysed during the recording period (C, D). Positions of rhombomeres 3 and 5 (r3, r5) are indicated. (E, H) Maps showing the trajectories of all marked cells in the embryos recorded in A–D. Trajectories in red denote cells that lysed during recording (H). The origins of cell trajectories are marked with dots. The initial and final positions of the pax2a:GFP domain are indicated by purple and green boundaries, respectively. Final positions of the otic placode and non-otic domains are indicated. (F, I) Vector maps showing net displacement of all cells tracked in 5 control movies (F) and 4 itga5 morphant movies (I). Red arrows indicate cells that died during recording (I). (G, J) Summaries of average migration patterns of cells in different quadrants of the pax2a:GFP domain in control embryos (G) and itga5 morphants (J). Arrow length indicates the mean of the net displacement of cells in the indicated region, and colored cones represent the range of angle of net displacement. Quadrants 1 and 2 contained cells contributing to both otic and non-otic domains, which were grouped separately. All images depict the right half of the embryo with lateral to the right and anterior to the top. Scale bar, 50 µm.

Figure 3. Trajectories of non-otic cells tracked in reverse.

(A, C) Maps showing the trajectories of GFP positive cells pooled from the time-lapse movies in Fig. 2 of control embryos (A) and itga5 morphants (C). Trajectories in orange indicate cells originally tracked by coexpression of GFP and RFP. All other trajectories represent cells tracked retrospectively by GFP alone. Dots indicate origins of tracked cells. Trajectories in red denote cells that died during recording (C). (B, D) Summaries of average migration patterns on non-otic cells in different quadrants in control embryos (B) and itga5 morphants (D). The mean length of net displacement (arrows) and range of angle of net displacement (colored cones) are indicated. The dashed orange lines indicate regions from which non-otic cells originated. Initial and final positions of the pax2a:GFP domain are represented by the purple and green boundaries, respectively. The position of rhombomere 5 (r5) is indicated. Lateral is to the right and anterior is to the top. Scale bar, 50 µm.

Figure 4. Cell-autonomous requirement for Itga5 in otic/epibranchial cells.

(A) Trajectories of cells transplanted from a wild type donor into a wild type host and tracked by time-lapse from 11.5–14.5 hpf. Data show tracks of 7 cells from a single embryo. (B) Trajectories of cells transplanted from itga5 morphant donors into wild type host embryos and tracked by time-lapse from 11.5–14.5 hpf. A total of 16 cells from 3 embryos were tracked, though some were not included on the map to avoid confusion. Red tracks represent cells that underwent lysis during the time-lapse. The purple and green boundaries represent the initial and final pax2a domain during time-lapse recording. Dots represent the initial positions of cells. Images show dorsal views with anterior to the top. Scale bar, 50 µm.

Figure 5. Convergence of trigeminal precursors is impaired in itga5 morphants.

(A, B, D, E) Images of time-lapse movies showing transgenic neuroD:EGFP expression in the first (11.5 hpf) and final (14 hpf) frames of a control movie (A, B) and itga5 morphant movie (D, E). Positions of precursors of the trigeminal ganglion (tg) and anterior lateral line (al) are indicated. (C, F) Maps showing the trajectories of individual trigeminal precursors in the control embryo (C) and itga5 morphant (F). Black and red boundaries mark the initial and final distribution, respectively, of neuroD:EGFP-positive trigeminal precursors. Black dots represent the initial and final positions, respectively, of individual cells. Images show dorsal views with anterior to the top, and summary figures show the right trigeminal field of each embryo, with lateral to the right. Scale bar, 50 µm.

Figure 6. Elevated cell death in itga5 morphants is rescued by hs:fgf8.

(A–D) Transgenic pax2a:GFP embryos immunostained for GFP (green) and Caspase3 (red). Images show dorsal views (anterior up) of the right otic/epibranchial domain in a control embryo (A), itga5 morphant (B), hs:fgf8/+ embryo (C) and hs:fgf8/+ embryo injected with itga5-MO (D). White arrows mark apoptotic cells. All embryos were heat shocked at 39°C for 30 minutes beginning at 11.5hpf and fixed at 13.5 hpf. Scale bar, 50 µm. (E) Mean number of Caspase3-positive cells in the otic/epibranchial domain in each of the four groups of embryos. Error bars indicate S.E.M.

Figure 7. Similar effects of Itga5 and Fgf on sox3 expression.

(A–H) sox3 expression at 12.5 hpf in a control embryo (A), itga5 morphant (B), Tg(hs:fgf8/+) heat shocked at 37°C alone (C) or with itga5 morpholino (D), Tg(hs:dnfgfr1/+) heat shocked at 39°C (E) or 35°C (F), erm morphant (G) and itga5-erm double morphant (H). The otic region where sox3 normally downregulates is indicated. Scale bar, 50 µm.

Figure 8. itga5 and erm interact during otic and epibranchial development.

(A–D) Otic/epibranchial expression of pax2a at 13 hpf. (E–H) Otic vesicle morphology in at 27 hpf. (I–L) phox2a expression in epibranchial ganglia at 30 hpf. Positions of facial (f), glossopharyngeal (g) and vagal (v1 and v2) ganglia are indicated. All images show lateral views with dorsal up and anterior to the left. Scale bar, 50 µm.

Figure 9. Differential spatial regulation of itga5 by dlx3b/4b and pax8.

(A–F) itga5 expression at 11 hpf and 13 hpf in control embryos (A, B), dlx3b-dlx4b double morphants at (C, D), and pax8 morphants (E, F). Regions where expression normally upregulates in precursors of anterior placodes (arrows) and otic/epibranchial precursors (arrowheads) are indicated. Images show dorsal views with anterior to the top.

Figure 10. Model for regulation of posterior placode development by itga5.

(A) Summary of cell migration during morphogenesis of trigeminal (t), epibranchial (e) and otic (o) placodes. Arrows indicate migration routes of cells tracked in neuroD:EGFP and pax2a:GFP expression domains (green). Dashed circles indicate the general areas from which trigeminal and epibranchial precursors were tracked. Most epibranchial precursors were not detected until relatively late (12.5–13.2 hpf) when they first activated pax2:GFP in a scattered pattern while still lateral to the contiguous domain of expression. We infer these cells originated from more lateral positions within the zone of recruitment (dashed tracks). RFP-positive otic cells were observed to migrate into the pax2a:GFP domain from nearby in the zone of recruitment (red tracks) between 11.7–12.5 hpf. Positions of the midbrain-hindbrain border (MHB) and rhombomere 4 (r4) are indicated. (B) A model for recruitment of otic/epibranchial cells. An initial otic domain (green) is induced by dorsally expressed Fgfs. Subsequently, itga5-dependent medial migration drives convergence of the otic field and draws new cells into range of inductive signaling. (C) Model for itga5 in reinforcing Fgf signaling. Erm helps mediate Fgf signaling, which begets more erm expression. Fgf also activates pax8, which stimulates upregulation of itga5 in the otic/epibranchial domain, further reinforcing Fgf signaling. Fgf acts primarily through the MAPK and PI3K pathways, and Itga5 facilitates these pathways through a variety of mechanisms (see text for details). Together these functional interactions control directed cell migration, cell survival, and gene expression within the otic/epibranchial field.