Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements

Abstract

Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.

Figure 1. tri encodes a Stbm homologue

a, A schematic representation of Stbm, showing mutations in three tri alleles (TM, putative transmembrane domains; PDZ-DBM, putative PDZ-domain binding motif). b–g, Stbm controls convergence and extension cell movements. Wild-type (b) and tri^m209 (c) embryos at 1 day post-fertilization. Injection of stbm RNA partially suppresses the tri convergence and extension defect (d). Wild-type embryos injected with stbm RNA (e) or stbm MO (f) show inhibited convergence and extension. g, stbm MO enhances the convergence and extension defect in tri mutant embryos. h–j, stbm MO phenocopies tri neuronal migration phenotype. Embryos at 36 h post-fertilization with gfp expression in branchiomotor neurons under control of the islet1 promoter (dorsal views). Motor neurons (arrowheads) induced in rhombomere 4 (r4) migrate into r6 and r7 in wild-type (h) but remain in r4 in tri^m209 embryos (i) and wild-type embryos injected with stbm MO (j). k–m, Stbm suppresses tri neuronal migration defect. Embryos at 36 h post-fertilization with anti-Islet antibody-labelled branchiomotor neurons. Motor neurons migrated into r6 in wild-type (k) and tri^m209 embryos injected with stbm RNA (m), but not in tri^m209 (l) embryos. oto, otocyst.

Figure 2. tri functions autonomously and non-autonomously to control cell polarity

a, A schematic representation of the methods used to measure cell elongation (LWR) and mediolateral alignment (MLA) relative to the notochord (n). Wild-type (b) and tri (c) ectodermal cells labelled with membrane-localized GFP (green). d, LWRs of wild-type and tri paraxial ectodermal cells and donor cells (wild-type->wild-type; wild-type->tri; tri->wild-type) with their standard deviations. e, Cumulative percentage of mediolaterally aligned paraxial ectodermal cells as a function of their angle, relative to a line perpendicular to the notochord. Graph line colours correspond to bar graph colours in d. Dashed line indicates sector ± 20° perpendicular to the notochord. Rose diagrams (insets) depict cell orientations in wild-type and tri paraxial ectoderm of late gastrulae. The mediolateral axis corresponds to the horizontal plane (0°), perpendicular to the notochord, and the antero-posterior axis is aligned vertically (90°). Transplanted wild-type (f) and tri (g) donor-derived ectodermal cells (red) surrounded by host ectoderm (green) are shown. The dashed lines in b,c,f and g indicate notochord boundary. s, somite.

Figure 3. tri is required for the increased net speed of directed dorsal migration

a, Domains of convergence and extension cell movements in zebrafish gastrulae. Yellow arrows in dorsal region indicate strong extension movements with little convergence. Light and dark blue arrows indicate domains of slow and fast convergence and extension, respectively. b, LWR of lateral mesodermal cells in wild-type and tri embryos, and tri embryos injected with tri MO. c, Total and net dorsal migration speeds of lateral mesodermal cells at 80% epiboly, yolk-plug closure (YPC)-tailbud (TB), and TB-1 somite stages. d–g, Shape changes and dorsal migration trajectories of lateral mesodermal cells. Colours match domains of slow and fast convergence and extension movement in a. D, dorsal. V, ventral. Scale bars represent 10 μm.

Figure 4. tri functions autonomously and non-autonomously during tangential neuronal migration

a–c, Dorsal views of hindbrain in unlabelled host embryos containing transplanted rhodamine–dextran-labelled donor cells (red), some of which differentiated into GFP-expressing motor neurons (green/yellow). Wild-type motor neurons (arrowhead) migrated out of r4 and into r6 in a wild-type host (a), but not in a tri^tc240a host (b). tri^tc240a motor neurons (arrowhead) migrated into r6 in a wild-type host (c). d, A quantitative summary of the transplantation data. e,f, Representative behaviours of wild-type and tri^tc240a motor neurons during a 25-min time-lapse recording. Red indicates area of retraction from previous cell position (closed circle marks cell centre) and green indicates area of expansion in current cell position (open circle marks cell centre). Arrows point in direction of cell movement. r, rostral; c, caudal. e, Wild-type motor neurons show biased caudal migration. f, tri^tc240a motor neurons meander showing no directionality. g,h, Dorsal views of wild-type hindbrains labelled with an Islet antibody. Motor neurons migrated into r6, posterior to the otocyst (oto), in control wild-type embryos (inset; g) and in Xdd1-injected wild-type embryos (h) showing a strong convergence and extension defect (inset).