(A) Phylogenetic tree of zebrafish Alk8 (AF292028), zebrafish Alk3/BmpRIA (AB011826), zebrafish Alk6/BmpRIB (AB020758), human ALK2/ActRIA (Z22534), human ALK1 (Z22533), Drosophila Saxophone (U11441) and Drosophila Thick veins (U11442), calculated according to the J. Hein method (DNAstar software). (GenBank Accession Numbers are given in brackets.) (B) Temporal expression profile of zebrafish alk8, BR1a (alk3), BR1b (alk6) and, as control, ef1α (Nordness et al., 1994), determined via RT-PCR.

alk8 is implicated in ventral specification. All embryos, unless stated otherwise, are genetically wild type. Embryos in A-D are at 36 hours post fertilization, lateral view, head to the left. (A) Uninjected control. (B) Strongly ventralized embryo (V3) after injection of alk8CA mRNA. Arrowhead indicates absent head region, arrow indicates enlarged blood islands. (C,D) Strongly dorsalized embryo (C4) after injection of alk8(K232R) mRNA (C) or alk8 antisense morpholino oligonucleotide alk8morph2 (D). Arrows indicate wound-up trunk. Note that the phenotype is much stronger than that of the laf mutant (see Fig. 3B). (E)Wild-type looking embryo after injection of the alk8 four-mismatch control antisense morpholino oligonucleotide 4mm-alk8morph2. (F-H) In situ hybridization for krox20 mRNA (Oxtoby and Jowett, 1993), staining rhombomeres 3 and 5; five-somite stage, lateral view, anterior towards the left, dorsal upwards. (E) Uninjected control; (F) injected with alk8morph2; (G) swirl/bmp2b mutant swr^ta72. alk8morph2-injected and bmp2b mutant embryo display a ventral fusion of both krox20 stripes (arrows), indicative of C5 dorsalization (compare with Dick et al., 2000).

alk8 acts downstream of bmp2b and bmp7 and upstream of smad5. All embryos are shown at 36 hours post-fertilization, lateral view, head towards the left. The laf, swr and snh embryos shown in panels C-F,K,L were genotyped after photography. (A) Wild-type sibling. (B) laf^tm110 mutant. Arrows indicate absent ventral tail fin and enlarged pericardial cavity. (C) Rescued laf^tm110 mutant, injected with alk8 mRNA. (D) Rescued laf^tm110 mutant, injected with smad5 mRNA; arrow indicates starting swelling of the precardial cavity. (E) laf^tm110 mutant, injected with bmp2b mRNA. (F) laf^tm110 mutant, injected with bmp7 mRNA. Arrowheads indicate small head and enlarged blood islands, both signs for ventralization; arrows indicate absent ventral tail fin and enlarged heart cavity, both laf-characteristic features (compare with B). (G-J) Genetically wild-type embryos after injection with bmp2b mRNA (G), co-injection of bmp2b mRNA and the alk8 antisense morpholino oligonucleotide alk8morph2 (H), injection of smad5 mRNA (I), or co-injection of smad5 mRNA and alk8morph2 (J). The embryos in G,I,J display strong ventralization (V3; arrowheads indicate absent head and enlarged blood island); the embryo in H shows strong dorsalization (C4; arrow indicates wound-up trunk), similar to the alk8morph2-injected embryo shown in Fig. 2D. (K) Rescued bmp2b mutant swr^ta72, injected with alk8CA mRNA. (L) Rescued bmp7 mutant snh^ty68, injected with alk8CA mRNA. Arrows in K,L indicate reduced or absent ventral tail fin, indicating mild dorsalization (C1); arrowheads indicate smaller head, indicating weak ventralization. (M,N) Sibling embryos from sbn^dtc24 heterozygous mother; displaying C4 dorsalization, characterized by wound-up tail and trunk (arrows); (M) uninjected embryo; (N) alk8CA mRNA-injected embryo.

lost-a-fin is alk8. (A) Genomic position of the zebrafish alk8 gene and the lost-a-fin mutation laf^tm110. (B) Alk8 protein; the mutations found in laf^tm110 and laf^m100 mutant Alk8 are indicated with arrowheads. TM, transmembrane domain; GS, type I receptor-specific GS domain. (C,D) alk8 expression in laf^m100 mutant and wild-type sibling embryos at 36 hours post-fertilization, detected via whole-mount in situ hybridization (C), and via RT-PCR analysis, with ef1a as control (D).