Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis

Abstract

The mechanisms controlling axon guidance are of fundamental importance in understanding brain development. Growing corticospinal and somatosensory axons cross the midline in the medulla to reach their targets and thus form the basis of contralateral motor control and sensory input. The motor and sensory projections appeared uncrossed in patients with horizontal gaze palsy with progressive scoliosis (HGPPS). In patients affected with HGPPS, we identified mutations in the ROBO3 gene, which shares homology with roundabout genes important in axon guidance in developing Drosophila, zebrafish, and mouse. Like its murine homolog Rig1/Robo3, but unlike other Robo proteins, ROBO3 is required for hindbrain axon midline crossing.

Fig. 1

HGPPS clinical profile. (Top) Photographs of an affected member with HGPPS demonstrating absent horizontal eye movement on attempted gaze to right (left) or left (right) but normal vertical gaze upward (upper) and downward (lower) from the primary position (central). (Bottom) A coronal scout MR imaging of the thoracic and lumbar spine demonstrating profound scoliosis.

Fig. 2

Functional anatomical defects in HGPPS. Brain MR images of a normal subject (A to C) and an HGPPS patient (D to F) at comparable anatomical levels. (A and D) Sagittal view. Normal appearance of cortex and corpus callosum (cc) but dysmorphic hindbrain with enlarged fourth ventricle (*) in the patient. Dashed lines represent sections through pons (P) and medulla (M) shown in (B to F). (B and E) Axial view, caudal pons in box (see fig. S2 for details). Note hypoplasia, enlarged fourth ventricle, absent protrusions of the abducens nuclei (arrowheads) in the patient. (C and F) Axial view, medulla. Note flattened, butterfly-like appearance with deep midline cleft in the patient, compared with the rounded appearance in the control (open arrow). (G and H) Representative somatosensory and motor evoked-potential studies demonstrating sensorimotor nondecussation in an HGPPS patient (H) compared with control (G). ( Top) Bilateral peripheral nerve in the arms (Br, brachial-medial arm just above the antecubital fossa) and scalp recordings following left and then right median nerve stimulation. The N20 cortical sensory response was abnormally ipsilateral in patient. CP4, CPz, and CP3 are right, midline, and left centro-parietal scalp sites, respectively (mastoid reference). (Middle) Bilateral peripheral nerve in the legs (PF, popliteal fossa) and scalp recordings following left and then right tibial nerve stimulation. The cortical sensory response showed abnormally reversed P37/N37 lateralization in patient. (Bottom) Note abnormally ipsilateral hand (APB, abductor pollicis brevis) and foot (AH, abductor hallucis) muscle responses in patient compared with contralateral responses in control following transcranial electric stimulation of the left and then right hemisphere.

Fig. 3

(A) Physical map of the HGPPS region with annotated genes (UCSC Human Genome Browser July 2003 freeze) screened for mutations. (B) A GenScan-predicted hypothetical gene NT_033899.598 overlaps with AK056544 and BC008623 (also annotated as RBIG1). (C) Refinement of genomic structure of ROBO3; exons are represented by open boxes. (D) Predicted ROBO3 topology and locations of 10 homozygous mutations. The mutated residues are conserved, as demonstrated by aligning orthologous and paralogous sequences in human (h), mouse (m), zebrafish (z), Drosophila (d), and C. elegans (c). (E) In situ hybridization analysis of ROBO3, with intense signal from an antisense ROBO3 probe in the basis pontis of fetal human brain at 15 weeks (left) and 19 weeks (right).