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Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention

¹ Committee on Developmental Biology, The University of Chicago, 1027 E 57th Street, Chicago, IL 60637, USA
² Department of Organismal Biology and Anatomy, The University of Chicago, 1027 E 57th Street, Chicago, IL 60637, USA
³ Committees on Neurobiology and Evolutionary Biology, The University of Chicago, 1027 E 57th Street, Chicago, IL 60637, USA

View larger version (79K): [in a new window]   Fig. 1. Loss-of-function of hoxb1a alters the disposition of r4-derived branchiomotor neurons. The disposition of the BM neurons is revealed by expression of islet1, either in live islet-GFP transgenic embryos (A-D,G-J) or by islet1 in situ hybridization (E,F). Uninjected (wild-type) control embryos are shown in left-hand panels and embryos injected with MOb1a at 1 mg/ml in right-hand panels. The BM neurons of the following cranial nerves are indicated: III, occulomotor; IV, trochlear; V, trigeminal; VII, facial; IX, glossopharyngeal; X – vagal. Rhombomeres (r) are numbered. (A-H) Embryos are dorsal-side uppermost with anterior towards the left. (A-D) islet-GFP transgenic embryos at 24 hours of development. (A,B) Merged bright-field and fluorescent images. o, otic vesicle; n, notochord. (C,D) Fluorescent images alone. In wild-type embryos (A,C), the Vth (trigeminal) nerve cell bodies lie in r2 (Va cluster; see E-J) and r3 (Vp cluster; see E-J); the VIIth (facial) nerve cell bodies migrate posteriorly, close to the floorplate, from r4 and r5, to ultimately reach r6 and r7. In MOb1a-injected embryos (B,D), the VIIth nerve cell bodies do not migrate, and instead lie in laterally positioned clusters similar to Vth nerve cell bodies. In both control and injected embryos, axons can be seen exiting the hindbrain at the r4 level and projecting towards the second pharyngeal arch. (E,F) islet1 in situ hybridization at 30 hours reveals the same neuronal disposition. Several islet1 expression sites additional to those in the GFP line can also be seen. These include the laterally located cranial ganglia, as well as the r6 and r7 located glossopharyngeal (IXth) nerve cell bodies. In the absence of r6/7-located VIIth nerve neurons, the IXth nerve neurons are revealed after MOb1a injection (F). These neurons express islet1 mRNA but are not labeled by the islet-GFP transgene (compare F with D). (G,H) Merged confocal images of 40 hour embryos. In wild-type specimens (G), Vth and VIIth nerve neurons have now reached their final locations. In MOb1a-injected embryos (H), the r4-derived neurons remain at the r4 level (VII) and show a similar mediolateral localization to r2-derived Vth nerve neurons. (I,J) Confocal analysis of 48 hour larvae in lateral view; anterior towards the left. In wild-type larvae (I), the VIIth nerve neurons are localized significantly posterior to the Vth nerve neurons (red labels); Vth nerve neurons project axons out of r2 to innervate the first pharyngeal arch; VIIth nerve neurons project axons anteriorly to exit the hindbrain in r4 and innervate the second arch; the Xth (vagal) nerve neurons innervate arches 4 through 7 (axons indicated by red arrows). The red arrowhead indicates VIIIth nerve/octavolateralis efferent (OLe) axons projecting into the otic region. In MOb1a-injected larvae (J), the projections into the pharyngeal arches are indistinguishable from normal (red arrows). However, the r4-derived cell bodies (VII) continue to be localized in r4, immediately posterior to Vth nerve cell bodies, and the VIIIth/OLe axon tract is absent (red asterisk). The BM neuron axons followed the same general pathways into the arches in all specimens analyzed, but we observed occasional individual stray axons that were not fasciculated with the main bundles in both wild-type and injected embryos.

View larger version (106K): [in a new window]   Fig. 2. Maintenance of zebrafish hoxb1a transcription requires Hoxb1a protein. Transcription of endogenous hoxb1a was assayed by whole-mount in situ hybridization in uninjected control embryos (left-hand panels) or in embryos injected with 1 mg/ml MOb1a (right-hand panels). (A,B) 24 hour embryos: (A) hoxb1a is expressed at high levels in r4 of control embryos; (B) in the MOb1a-injected embryos, there is a reduction in transcript levels in the medial part of the neural tube. (C,D) At 36 hours, loss of medial transcription in response to MOb1a is more severe. (E,F) Transverse sections (planes indicated by red arrows in C,D, dorsal towards the top) encompassing the whole of r4 were hand cut from 36 hour embryos post in situ.

View larger version (97K): [in a new window]   Fig. 3. Loss-of-function of hoxb1 duplicate genes causes alterations in segmental organization of the posterior hindbrain. (A-C) In situ hybridization with krox20 (a marker for r3 and r5) and hoxb4 (expressed in r7 and posterior) at 20 hours, anterior towards the top, rhombomeres r3-r6 and their AP extent are indicated (red double-headed arrows). (A) Wild-type control; (B) embryo injected with 4 mg/ml MOb1b, note reduction in AP extent of r4, r5 and r6 and expansion of r3; (C) Embryo co-injected with 1 mg/ml MOb1a + 4 mg/ml MOb1b, note exacerbation of reduced size of r4 and r6, and further expansion of r3 towards the posterior. (D-F) Bright-field lateral views of live embryos at the 24-hour stage, anterior towards the left. The AP extent of the otic vesicles is indicated by white bars. (D) Wild-type embryo; (E) MOb1b-injected embryo; (F) MOb1a+MOb1b-injected embryo. Arrows indicate otoliths.

View larger version (109K): [in a new window]   Fig. 4. The hoxb1 duplicates have redundant functions in RS neuron specification. Retrograde labeling of 5-day larvae, merged confocal images, anterior towards the top, rhombomeres are numbered. (A) Wild-type control, note the bilateral r4-specific Mauthner neurons (M), the more lateral vestibular neurons (nV), and the smaller, laterally located r2-specific rol2 neurons. (B-D) Larvae injected with MOb1a at 1 mg/ml and MOb1b at 4 mg/ml. Note loss of Mauthners (*) and their replacement with smaller more medially located cells. In B, a unilateral Mauthner remains, although it is displaced posteriorly (M'). In C, ectopic lateral cells at the r5/6 level are indicated (red arrowhead).

View larger version (113K): [in a new window]   Fig. 5. Morpholino generated loss-of-function phenotypes are rescued by ectopic protein. (A-D) Rescue of MOb1a phenotype, disposition of BM neurons is revealed with islet1: (A,B) islet-GFP transgenics; (C,D) islet1 in situ hybridization at 28 hours, anterior towards the left. (A,C) Embryos injected with MOb1a alone at 1 mg/ml, note lack of migration of r4-derived BM neurons (VII). (B,D) Embryos co-injected with MOb1a at 1 mg/ml and hoxb1a mRNA at 15 µg/ml, note rescue of migration of r4-derived neurons (VII), plus posteriorizing transformation of r2-derived neurons (VII’). (E-H) Rescue of MOb1b phenotype, embryos at 20 hours, anterior towards the top, krox20 in situ hybridization. (E) Uninjected wild-type control, note approximately equal AP extents of r3, r4 and r5; r4 size indicated with double-headed arrow. (F) hoxb1b mRNA injected (20 µg/ml) embryo, note no change in r4 size but increase in r3 AP extent, as we have previously described for gain-of-function experiments (McClintock et al., 2001). (G) MOb1b injected (4 mg/ml) embryo, note shift in r3/4 boundary towards the posterior, resulting in a significant reduction in AP extent of r4, together with increase in AP extent of r3. (H) Embryo co-injected with hoxb1b mRNA plus MOb1b, note rescue of r4 AP extent to wild-type proportions, together with increased size of r3 AP extent as seen with RNA alone.

View larger version (79K): [in a new window]   Fig. 6. The hoxb1a and hoxb1b genes have non-identical biochemical functions. BM neurons are revealed with islet1. (A,B) islet-GFP transgenics, (C,D) islet1 in situ hybridization, embryos at 28 hours with anterior towards left. (A,C) Embryos injected with hoxb1b mRNA at 20 µg/ml show posteriorizing transformations at the r2 level (V neurons are transformed to VII'); (B,D) embryos co-injected with MOb1a (1 mg/ml) and hoxb1b mRNA do not show either rescue of the hoxb1a loss-of-function phenotype, or any gain-of-function phenotype. o, otic vesicle.

View larger version (20K): [in a new window]   Fig. 7. Comparison of hoxb1a and hoxb1b regulatory sequences reveals mutations in defined regulatory elements. Zebrafish genomic sequences lying 5' and 3' of the hoxb1 duplicate genes were produced by the Zebrafish Sequencing Group at the Sanger Institute and can be obtained under Accession Number AL645782 (hoxb1a) and at http://www.sanger.ac.uk/cgi-bin/nph-getblast?humpub/zebrafish_all+dZ227H09 (hoxb1b). (A) Zebrafish hoxb1 gene upstream sequences are compared with the equivalent sequences upstream of mouse and chick Hoxb1 (Pöpperl et al., 1995). The Hox/Pbx-binding sites, repeats 1-3, are indicated in yellow, with changes from the consensus indicated in green. (B) PIPmaker plot (Schwartz et al., 2000) comparing hoxb1a regulatory elements (upper strand) with hoxb1b regulatory elements. There is an AT-rich region of homology lying approximately 4 kb downstream of the translational start site of each gene. These regions do not contain RAREs, or other regulatory elements known to be involved in Hoxb1 regulation. A 5' homology region overlaps the Hox/Pbx-binding repeats (indicated by gray shading).

View larger version (16K): [in a new window]   Fig. 8. Model outlining the evolutionary mechanism of Hox PG1 gene ‘function shuffling’. The cis-regulatory elements characterized for the mouse and human Hoxa1 and Hoxb1 genes [3' RAREs (blue), Hox/Pbx binding sites (red)] are assumed to be present in the ancestral, pre-’third’-duplication, condition. We also postulate the presence of a regulatory domain directing midbrain expression of Hoxa1 (purple), although no such domain has yet been characterized. The duplication event in the lineage leading to teleosts produced redundant copies of both Hoxa1 and Hoxb1 in an ancestor of the zebrafish. The hoxa1b duplicate was eventually lost by accumulation of deleterious mutations (‘non-functionalization’) as predicted by classical models. By contrast, the hoxb1a and hoxb1b genes accumulated complementary degenerative changes in their cis-regulatory elements, such that hoxb1a lost early RARE-mediated expression and hoxb1b lost autoregulation. This led to retention of the duplicate genes, as both were required to maintain the expression pattern and function of the single Hoxb1 ancestral gene (sub-functionalization), as predicted by the DDC model. As hoxa1a and hoxb1b shared similar coding sequences and expression patterns, these two genes were now functionally redundant with respect to a role during gastrulation in setting up segmental organization of the hindbrain. These non-orthologous genes were thus able to go through another ‘sub-functionalization’ event, such that hoxa1a lost its early RARE-mediated expression, which was retained by hoxb1b. Thus, hoxb1b became essential for proper hindbrain segmentation, the role played in the ancestral state by Hoxa1. Retention of the hoxa1a gene in the lineage leading to zebrafish was presumably dependent on a function that was not redundant with hoxb1b, possibly a role in midbrain patterning. We term this rearrangement of PG1 gene roles ‘function shuffling’.