Hox paralogue group 1 genes: sequence and functional domains. (A) Clustal X (Thompson et al., 1997) alignment of amino acid sequences of zebrafish, mouse and amphioxus Hox paralogue group 1 genes. Conceptual translations of the four zebrafish Hox PG1 genes are compared with mouse Hoxa1, Hoxb1 and Hoxd1 and AmphiHox-1. Identical residues in red, conserved changes in blue. Hexapeptide and homeodomain are overlined in blue and green, respectively; note the unusually long linker region between the hexapeptide and homeodomain regions of hoxc1a. The diagnostic PG1 residues (Sharkey et al., 1997) are indicated with asterisks (below sequence). The 2/7 diagnostic residues not conserved in hoxc1a are indicated with black rather than red asterisks. (B) Schematic of intron/exon structure for the four zebrafish PG1 genes, drawn to scale; hexapeptide and homeodomain are indicated in blue and green, respectively, alternatively spliced exon is indicated in yellow. Based on comparison to genomic sequences (GenBank Accession Numbers AF071243, AF071251, U40995, AF071263). Note that hoxc1a has no intron, as confirmed by PCR on genomic DNA. Numbers indicate intron/exon boundaries with respect to the start of translation; the length of the primary intron is also indicated. The following coding sequences have been placed on the EMBL database: hoxc1a, Accession Number AJ306432; hoxb1b, Accession Number, AJ306433; and hoxa1a, Accession Numbers AJ306430 and AJ306431. (C) Neighbour-joining tree to show the phylogenetic relationships between Hox PG1 genes (based on Clustal X alignment in A; displayed using NJ-Plot) (Perriere and Gouy, 1996), bootstrap values based on 1000 replicates are shown; scale bar refers to branch lengths. The tree suggests that mouse Hoxd1 and zebrafish hoxc1a group together; however, the long branch lengths imply that these genes are more distantly related.

Whole-mount in situ hybridization analysis of zebrafish PG1 genes. Two-colour double in situ hybridization shows Hox genes in purple, plus krox-20 in red as a marker of r3 and r5. All embryos are mounted with dorsal side towards reader and anterior upwards. Rhombomere (r) numbers as indicated. (A-D) The hoxb1b and hoxb1a genes both have early expression domains in r4. (A) hoxb1b expression at 80% epiboly (8 hours, h) lies in bilateral epiblast domains above the margin. (B) At tailbud stage (10 h) hoxb1b expression is localized to r4 abutting early krox-20 expression in r3. (C) By the one-somite stage (10.5 h) hoxb1b expression has already started to retreat posteriorly and is absent from r4. (D) hoxb1a expression at the equivalent stage (one somite) is already upregulated in r4. (E-I) hoxa1a is expressed in an anterior subpopulation of neurones. (E) At 24 h hoxa1a expression is localized to discrete bilateral clusters of cells in the anterior hindbrain and ventral midbrain (arrowheads). (F) 36 h; expression is now localized to cell clusters in the midbrain, medial to the eyes, and r1 (arrowheads). (G) 3.5 μm transverse section (t.s.) through plane indicated in F. (H) HNK-1 antibody staining reveals cell bodies of the nMLF (arrowheads), the MLF axon tract and the trigeminal ganglia (TG) at 22 h. hoxa1a expression colocalizes to the nMLF. (I) hoxa1a-expressing cells continue to colocalize with HNK-1-positive neurones in the nMLF (arrowheads) at 28 h, arrows indicate hoxa1a-expressing cells in the anterior hindbrain. (J-M) hoxc1a expression: (J) at 12 h in notochord (n); (K) at 16.5 h in CNS (anterior limit at spinal cord/hindbrain junction); (L) at 24 h in bilateral cell clusters in the ventral midbrain (arrowhead) and Mauthner neurones (arrow); (M) at 36 h in cells medial to eyes (arrowhead), similar to hoxa1a.

Efficiency of Hox protein translation. (A) In vitro translation products of synthetic PG1 Hox gene mRNAs analyzed by SDS-PAGE electrophoresis. Protein products of the expected size are efficiently produced in vitro. Predicted sizes: long form of hoxa1a, 36 kDa; hoxb1a, 35 kDa; hoxb1b, 34 kDa; long form of hoxc1a, 34 kDa. Molecular weight marker sizes in kDa are indicated. (B) Western blot analysis of Myc-tagged Hox proteins synthesized in vivo after micro-injection of 50 ng/μl concentrations of each PG1 mRNA. Lysates of whole embryos were prepared at the 20-22 hour stage and 10 μg of total extracted protein electrophoresed and blotted. As expected, Myc-tagged proteins (six Myc-epitopes) are approximately 10 kDa larger that untagged versions. Molecular weight marker sizes in kDa are indicated.

Disposition of neuroanatomical and molecular markers reveals that mis-expression of hoxb1b transforms r2 to an r4 phenotype. (A-D) Retrograde labelling from the spinal cord of 5-day-old larvae reveals the disposition of the RS neurones (anterior to the top). (A) The subset of neurones labelled from the right hand side of the spinal cord only is schematized and labelled: M, Mauthner; nV, nucleus of the vestibular formation; MiM1, middle medial 1 cells; MiV1, middle ventral 1 cells; RoL2, rostral lateral 2 cells; llf, lateral longitudinal fascicle. Other abbreviations as previously designated (Hanneman et al., 1988). The locations of the rhombomeres relative to the RS neurones are indicated on the left-hand side. (B) Retrograde labelling of a normal 5-day-old larva. Mi, MiM1 plus MiV1 cells; rhombomeres numbered on right-hand side. Inset, 3A10 antibody staining reveals the r4 Mauthner neurones, M. (C,D) Mis-expression of hoxb1b leads to formation of the r4 characteristic M, Mi and nV cells at the r2 level; ectopic neurones are indicated by ′. These examples show unilateral duplications, less frequently bilateral duplications were observed, as shown in the inset in (D) by 3A10 antibody staining to reveal ectopic Mauthner neurones at the r2 level, M′. (E-J) Whole-mount in situ hybridization analysis of injected embryos, mounted with dorsal side uppermost and anterior to the left; in each case an unmanipulated control embryo is shown on the left-hand side and a hoxb1b-injected embryo at the same stage on the right-hand side. (E,F) 28 h; (G-J) 19 h; (E) islet1 expression labels cell bodies of the branchiomotor neurones. The trigeminal (Vth nerve) cell bodies have a lateral location, show intense labelling, and are subdivided into a major anterior (r2) population and a minor posterior (r3) population (white arrowheads). The facial (VIIth nerve) cell bodies lie more medially, show lower level expression, and form an anterior-posterior array through r4-r6 at this stage (bracket). (F) hoxb1b-injected embryo, islet1 expression shows medial facial-like neurones (VII′) at the level of r2 and r1, and possibly extending into the midbrain. O, otic vesicle. (G) In wild-type embryos, hoxb1a (blue) is expressed in r4 and krox-20 (red) in r3 and r5. (H) In hoxb1b-injected embryos there is ectopic hoxb1a (blue) expression at the r2 level, note expansion of the r3 krox-20 territory (red). (I) mar is expressed at elevated levels in rhombomere boundaries (arrowheads). (J) In hoxb1b-injected embryos, mar boundary staining reveals expansion of the r3 territory at the expense of the r2 territory.