Ancestry of motor innervation to pectoral fin and forelimb

Abstract

Motor innervation to the tetrapod forelimb and fish pectoral fin is assumed to share a conserved spinal cord origin, despite major structural and functional innovations of the appendage during the vertebrate water-to-land transition. In this paper, we present anatomical and embryological evidence showing that pectoral motoneurons also originate in the hindbrain among ray-finned fish. New and previous data for lobe-finned fish, a group that includes tetrapods, and more basal cartilaginous fish showed pectoral innervation that was consistent with a hindbrain-spinal origin of motoneurons. Together, these findings support a hindbrain-spinal phenotype as the ancestral vertebrate condition that originated as a postural adaptation for pectoral control of head orientation. A phylogenetic analysis indicated that Hox gene modules were shared in fish and tetrapod pectoral systems. We propose that evolutionary shifts in Hox gene expression along the body axis provided a transcriptional mechanism allowing eventual decoupling of pectoral motoneurons from the hindbrain much like their target appendage gained independence from the head.

Figure 1. Occipital region in actinopterygian fish.

(a) Cranio–vertebral junction (asterisk) in a juvenile midshipman stained with alcian blue and alizarin red. (b) Hindbrain–spinal cord boundary (yellow hatching) demarcated in the zebrafish hoxb4a enhancer trap line. (c) Pectoral fin innervation in juvenile midshipman. (d) Schematic drawing of c showing occipital nerves (Oc, red, orange and brown in d) exiting through occipital foramen (OcF) located anterior to the cranio–vertebral junction, and spinal nerves (Sp, black in d) exiting through vertebrae (V). (e) Embryonic alignment of caudal hindbrain, fourth ventricle, otic vesicle (OV) and myotomes (M) (includes blue lipophilic dye injection in fin buds; midshipman fish). The location of pectoral motoneurons is also indicated. NE, neuroepithelium. Images are dorsal (a, b, e) and ventral (c, d) views with anterior to the left. Scale bars are 1 cm (a), 200 μm (b), 500 μm (c, d) and 100 μm (e).

Figure 2. Embryonic alignment of pectoral and occipital motoneurons with nerves and myotomes in basal and derived actinopterygians.

(a–d) Location of pectoral motoneurons and nerves in actinopterygians revealed by lipophilic dye labelling from fin buds. The pectoral motor column began at the level of myotomes (M) 2–3 in all species studied (vertical hatching marks hindbrain–spinal boundary; also see Figure 1b,e). (e–h) Double labelling with fluorescent dextrans from fin buds and M1–3 showed that the occipital motor column began one myotomal segment anterior to pectoral motoneurons. Horizontal hatching marks midline in f–h. (i) Alignment of myotomes, nerves and motoneurons (pectoral/red and occipital/grey) with phylogenetic relationships of actinopterygians studied here (right). Paddlefish innervation pattern was deduced from juvenile gross anatomy (Supplementary Fig. S1, S2) as individual roots were not clearly visualized using retrograde labelling. All images are dorsal views with anterior to the left. Scale bars are 50 μm. Specimen stages: a (10 days postfertilization (dpf)/~5.5 mm), b (2 dpf/~3 mm), c (100 dpf/~10 mm), d (9 dpf/~13 mm), e (18 dpf/~11 mm), f (4 dpf/~4 mm), g (115 dpf/~12 mm), h (11 dpf/~16 mm).

Figure 3. Embryonic alignment of precerebellar, pectoral and other hindbrain neurons in transgenic zebrafish.

(a) Rhombomere (r) 7–8 YFP expression in hoxb4a enhancer trap line. (b) Reticular (labelled from the spinal cord; red) and YFP (green) neurons showed hindbrain–spinal cord boundary between myotomes (M) 3–4. (c) Half of the pectoral column (labelled from fin bud; red) was within the hindbrain. (d) Occipital motor column (labelled from M1–3/occipital) extended to mid r8, one segment rostral to pectoral motoneurons. (e–g) Dorsal composites (e) and selected confocal planes (f, g) of pectoral (labelled from fin buds; red), inferior olive (IO) and Area II (AII) (labelled from the cerebellum; magenta) neurons in islet-GFP background (green), showing the relative position of pectoral motoneurons with major neuronal subgroups. GFP in this line is expressed in all hindbrain motoneurons, except abducens and pectoral. (h) Vagal (X) and more ventral occipital (Oc) motor columns that extended from spinal cord into the hindbrain (also see f, g). (i) Alignment of pectoral motoneurons (Pec) with other neuronal and anatomical landmarks. Pectoral motoneurons in zebrafish were located across the hindbrain–spinal cord boundary at the level of M3–5 (see Figure 2). Hindbrain motoneurons are located immediately caudal to the inferior olive, below the vagal nucleus (X) and hindbrain commissure (HB com). They are part of the occipital motor column (Oc Mns) at the level of two fibre tracts, the medial longitudinal fasciculus (mlf) and the lateral longitudinal fasciculus (llf). Other abbreviations: Mi2, Mi3 and Ca, reticulospinal neurons. Images are dorsal (b, c, e–g, i), ventral (d) and lateral (a, h) views with anterior to the left. Scale bars are 200 μm (a), 50 μm (b–h). Specimen stages: a, c, d (4 dpf), b (2 dpf), e–h (5 dpf).

Figure 4. Origin and maintenance of pectoral motoneurons across the hindbrain–spinal cord boundary.

(a) Future hindbrain–spinal cord region in a 12 h postfertilization (hpf) embryo showing somites 1–6. (b) Transiently expressed kaede protein was photoconverted from green to red at the level of somites 4–5. Yellow dashed line in (a) marks the area of photoconversion using a laser of 405 nm. (c) The same embryo at 2 days postfertilization (dpf) showed minimal, if any, anterior–posterior migration, with labelled neurons remaining tightly clustered at the level of myotomes (M) 4–5. Arrows point to neurons that were displaced 2–3 cell diameters posteriorly. (d, e) High magnification single plane images showed photoconverted kaede to be absent in pectoral motoneurons (white) at the level of M3 (d), but present in those at the level of M4 (e). (f–k) Ontogeny of pectoral motoneurons. At 2 dpf, pectoral motoneurons were labelled with lipophilic dye from fin bud (red) and appeared as a single column at the level of M3–4 (f) where they overlapped with the hoxb4a-YFP (green) expression domain (g, shown at a higher magnification in h). At 20 dpf, pectoral motoneurons increased in number and developed into paired ventrolateral and dorsomedial columns (i). The motoneurons remained across the hindbrain–spinal cord boundary (yellow dashed line in j) demarcated by YFP expression (shown at a higher magnification in k) and exhibited a progressive downregulation of hoxb4a activity (arrows; h, k). Images are dorsal (a–h) and ventral (i–k) views with anterior to the left. Scale bars are 50 μm (a–c, f, g, i, j) and 10 μm (d, e, h, k). Specimen stages: a, b (12 hpf), c–h (2 dpf) and i–k (20 dpf).

Figure 5. Pectoral innervation in Chondricthyes (ratfish) and Dipnoi (lungfish).

(a) Pectoral innervation in ratfish (H. colliei) included four occipital (Oc1–4) and 11 spinal nerves (Sp1–11). Inset shows the branching of Oc1–4 contributing to both hypobranchial nerve and the pectoral plexus. (b) Pectoral innervation in spotted African lungfish (P. dolloi) included one occipital (Oc3) and three spinal nerves (Sp1–3). Oc3 dorsal (DR) and ventral (VR) roots emerged at the caudal end of the hindbrain. Inset shows the ventral view of Oc1–3 and Sp1. Scale bars are 5 mm.

Figure 6. Evolution of pectoral innervation.

(a) Cladogram of living jawed vertebrates, with vignettes showing innervation patterns of pectoral appendages. Occipital (pectoral, red; hypobranchial, grey) and spinal (blue) nerves are illustrated schematically. (b) Summary of key Hox genes expressed in neuronal (top) and mesodermal (bottom) compartments along the anterior–posterior axis in fish and tetrapods.