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First published online January 16, 2004
doi: 10.1242/10.1242/dev.00954

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Has2 is required upstream of Rac1 to govern dorsal migration of lateral cells during zebrafish gastrulation

Jeroen Bakkers^1,*,, Carina Kramer¹, Joris Pothof², Nicolette E. M. Quaedvlieg², Herman P. Spaink² and Matthias Hammerschmidt^1,

¹ Max-Planck Institute for Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany
² Leiden Institute of Biology, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands

View larger version (82K): [in a new window]   Fig. 1. Zebrafish Has genes and their expression during embryogenesis. (A) Homology tree of Has1, Has2, and Has3 family members based on amino acid sequences. The GenBank accession numbers for the sequences listed above are: Xenopus laevis xHas1 (X52958), Xenopus laevis xHasrs (AF015780), mouse mHas3 (MMU86408), human hHAS2 (HSU54804), mouse mHas2 (MMU52524), human hHAS1 (HSU59269), mouse mHas1 (D82964). (B) RT-PCR analysis of zebrafish has1, has2 and has3 expression during different stages of development. (C-J) Wild-type embryos stained for has2 mRNA (blue), and No tail (Ntl) protein (brown; F-J). (C) Early gastrulation (shield stage), animal pole view. Arrowheads indicate the dorsal shield devoid of staining. (D) Midgastrulation (80% epiboly stage), dorsal view. (E) 90% epiboly stage, lateral view, dorsal to the right. (F) End of gastrulation (100% epiboly stage), dorsal view on flat-mounted embryo; anterior to the left. (G-J) 8-somite stage: (G) dorsal view on flat-mounted embryo, anterior to the left; (H-J) optical cross sections at levels indicated in G.

View larger version (89K): [in a new window]   Fig. 2. Presence of HA depends on Has2 activity and cannot be restored by constitutively active Rac1, whereas Fibronectin is unaffected in has2 morphants. (A-C) HA staining on transverse sections through the posterior region of 10-somite stage embryos. (A) Wild type; (B) embryo injected with has2 MO; (C) embryo co-injected with has2 MO and mRNA encoding constitutively active Rac1 (caRac1). At tailbud stage, the embryo in B was elongated, indicating convergence defects, the embryo in C was round like the wild-type control, indicating that convergence was rescued (compare with Fig. 4). Notochord and somites are outlined in B,C. (D,E) Anti-Fibronectin immunostaining. Section through posterior region of 3-somite stage embryos. (D) Wild type; (E) embryo injected with has2 MO. nc, notochord; nt, neural tube; s, somite.

View larger version (117K): [in a new window]   Fig. 3. has2 morphants are elongated, similar to Bmp mutants, but are not dorsalized. (A-D) Live embryos at tailbud stage, lateral view, dorsal to the right. (A) Wild type; (B) has2 morphant; (C) bmp2b/swr mutant; (D) embryo co-injected with has2 MO and has2 mRNA. (E,F) sox17 expression at tailbud stage, lateral views, dorsal to the right. (E) Wild type; (F) has2 morphant. Arrow (E) indicates forerunner cells; arrowhead (F) endodermal cells that have remained on the ventral side. (G-I) pax2.1 expression at the 4-somite stage, lateral view. Arrow (H) indicates midbrain-hindbrain boundary; arrowheads (G,H) indicate presumptive pronephric duct cells. (G) Wild type; (H) has2 morphant; (I) swr mutant. (J-L) pax2.1 (marked as `p'), myoD (`m'), hgg1 (`h') expression at the 10-somite stage; dorsal views on regular (K) or flat-mounted (J,L) embryos. (J) Wild type; (K) has2 morphant; (L) kny/glypican6 mutant. In J, midbrain-hindbrain boundary is marked by an arrow, pronephric ducts by arrowheads. (M,N) gata1 (`g') and myoD (`m') expression at the 10-somite stage; dorsal view on flat-mounted embryos. (M) Wild type; (N) has2 morphant. (O,P) krox20 (`k') and myoD (`m') expression at the 10-somite stage; dorsal view on flat-mounted embryos. (O) Wild type; (P) has2 morphant.

View larger version (87K): [in a new window]   Fig. 5. Has2 and Rac1 act in a cell autonomous fashion, promoting lamellipodia formation. All panels show individual lateral mesodermal cells at the 80% epiboly stage, visualized with membrane-localized GFP. Dorsal is to the right, anterior to the top. (A) Wild type; (B) has2 morphant; (C) wild type injected with mRNA encoding dominant-negative Rac1 (dnRac1); (D) has2 morphant injected with mRNA encoding constitutively active Rac1 (caRac1); (E) wild-type cells transplanted into has2 morphant host; (F) has2 morphant cells transplanted into wild-type host; (G) wild-type cells transplanted into host expressing dnRac1; (H) dnRac1-expressing cells transplanted into wild-type host.

View larger version (81K): [in a new window]   Fig. 4. Loss of Has2 function leads to blockage of dorsal convergence, but not of extension, and can be compensated by constitutively active Rac1. (A-I) Distribution of labeled cell clusters at onset of gastrulation, mid-gastrulation and end of gastrulation; lateral views, dorsal to the right. (A-C) Wild type; (D-F) has2 morphant; (G-I) embryo co-injected with has2 MO and caRac1 RNA. (A,D,G) Shield (6 hpf), directly after labeling of cell clusters by fluorochrome uncaging; (B,E,H) 80% epiboly (8 hpf); (C,F,I) tailbud (10.5 hpf). (J) Graph showing angle between labeled cells and dorsal axis at different time points, measuring dorsal convergence (indicated in B by blue bar). All clones start at 90° from the shield. (K) Graph showing the anteroposterior extension of labeled lateral cell populations (indicated in B by red bar) at different time points. In J and K, 10 embryos per treatment were evaluated, and standard deviations are indicated.

View larger version (84K): [in a new window]   Fig. 6. Forced dorsal expression of has2 and constitutively active Rac1 lead to supernumerary lamellipodia, blocking axis extension. Extension movement of labeled populations of dorsal cells. (A-D) Wild-type control embryos; (E-H) embryos injected with has2 mRNA; (I-L) has2 morphant embryos; (M-P) embryos injected with caRac1 RNA; (R,S) embryos co-injected with has2 mRNA and dnRac1 mRNA. The first two columns show uncaging experiments. Lateral view of embryos at shield stage (6 hpf), directly after uncaging (A,E,I,M), or at tailbud stage (10.5 hpf; B,F,J,N). Columns 3 and 4 show morphology of clusters (C,G,K,O,R) or individual (D,H,L,P,S) dorsal cells labeled with membrane-localized GFP. Arrowhead (D) indicates single lamellipodium sometimes visible on narrow dorsal side of highly polarized axial cells of wild-type embryos. Arrowheads (H,P) indicate multiple lamellipodia in axial cells of has2 RNA- or caRac1 mRNA-injected embryos. (Q) Graph showing the amount of extension in the axis of wild-type, has2 MO-, has2 mRNA- or caRac1 mRNA-injected embryos. Per treatment, ten different embryos were evaluated, and standard deviations are indicated.

View larger version (85K): [in a new window]   Fig. 7. Has2 is required for migration of presumptive slow muscle and sclerotomal cells during morphogenesis of somites, and for the migration of primordial germ cells. (A-D) 30 hpf; smbpc in situ hybridization to mark slow muscle cells of trunk somites: (A,C) wild type; (B,D) has2 morphant. (A,B) Dorsal view, anterior left; (C,D) transverse sections. Arrows (B,D) mark smbpc-positive cells located within the presumptive fast muscle tissue. The lower side of the embryo in B is less affected, which might have been the reason for its survival. The effects are restricted to the first 5-8 somites, consistent with previous findings indicating different properties between such anterior and more posterior somites (see van Eeden et al., 1996). (E,F) twist in situ hybridization, at 24 hpf, to mark sclerotomal cells of trunk somites. Arrowheads indicate dorsally migrated sclerotomal cells in wild type (E), which remain ventral in has2 morphant embryos (F). (G,H) Distribution of vasa-positive primordial germ cells, at 24 hpf, in wild type (G) and has2 morphant (H). Arrowheads (H) point to ectopic PGCs outside the genital ridges. hm, horizontal myoseptum; nc, notochord; nt, neural tube; s, somite.