Peripheral target regulation of the development and survival of spinal sensory and motor neurons in the chick embryo

Abstract

Unilateral limb-bud removal (LBR) before the outgrowth of sensory or motor neurons to the leg of chick embryos was used to examine the role of limb (target)-derived signals in the development and survival of lumbar motoneurons and sensory neurons in the dorsal root ganglia (DRG). After LBR, motor and sensory neurons underwent normal initial histological differentiation, and cell growth in both populations was unaffected. Before their death, target-deprived motoneurons also expressed a cell-specific marker, the homeodomain protein islet-1. Proliferation of sensory and motor precursor cells was also unaffected by LBR, and the migration of neural crest cells to the DRG and of motoneurons into the ventral horn occurred normally. During the normal period of programmed cell death (PCD), increased numbers of both sensory and motor neurons degenerated after LBR. However, whereas motoneuron loss increased by 40-50% (90% total), only approximately 25% more sensory neurons degenerated after LBR. A significant number of the surviving sensory neurons projected to aberrant targets in the tail after LBR, and many of these were lost after ablation of both the limb and tail. Treatment with neurotrophic factors (or muscle extract) rescued sensory and motor neurons from cell death after LBR without affecting precursor proliferation of either population. Activity blockade with curare failed to rescue motoneurons after LBR, and combined treatment with curare plus muscle extract was no more effective than muscle extract alone. Treatment with the antioxidant N-acetylcysteine rescued motoneurons from normal cell death but not after LBR. Two specific inhibitors of the interleukin beta1 converting enzyme (ICE) family of cysteine proteases also failed to prevent motoneuron death after LBR. Taken together these data provide definitive evidence that the loss of spinal neurons after LBR cannot be attributed to altered proliferation, migration, or differentiation. Rather, in the absence of limb-derived trophic signals, the affected neurons fail to survive and undergo PCD. Although normal cell death and cell death after target deprivation share many features in common, the intracellular pathways of cell death in the two may be distinct.

Fig. 1.

Transverse sections of the L3 spinal cord on E6 ipsilateral (A) and contralateral (B) to LBR. c, Central canal;DRG, dorsal root ganglion; LMC, lateral motor column; N, notochord. Note the almost complete absence of the LMC in A. Scale bar, 25 μm.C, Transverse section of E8 lumbar region. Note the complete absence of limb muscle (asterisk) ipsilateral to LBR. Scale bar, 100 μm. D, E, The L3 DRG on E8 ipsilateral (D) and contralateral to LBR. A comparison of D and E (also see Fig.2D,E) shows survival of many sensory neurons. Scale bar, 20 μm.

Fig. 2.

Transverse sections of the lumbarlmc on E18 ipsilateral (A) and contralateral (B) to LBR. Scale bar, 24 μm.C, Low-power photomicrograph of E18 lumbar spinal cord after LBR. Scale bar, 80 μm. D, E, The L3 DRG on E18 ipsilateral (D) and contralateral (E) to LBR. Scale bar (shown in Dfor D and E): 30 μm.

Fig. 3.

Transverse section (6 μm) of the L3 DRG ipsilateral to LBR on E5 (A). vl, Ventral-lateral; dm, dorsal-medial; dr, dorsal root; arrows, pyknotic cells in thevl region. Asterisks delineate the boundary between vl and dm. Scale bar, 10 μm. B, Islet-1 immunoreactivity of lumbar MNs on E4.5 after LBR (left side). A few immunopositive cells (interneurons?) are located in dorsal spinal cord (arrows). n, Notochord; c, central canal. Scale bar, 30 μm. C, BrdU immunoreactivity on E4.5 in lumbar spinal cord and DRG (asterisks) after LBR (left side). Note the larger number of immunopositive cells in the dorsal half of spinal cord. lmc, Lateral motor column. Scale bar, 30 μm.

Fig. 4.

Lumbar motoneuron numbers in segments L1–8 (mean ± SD) ipsilateral (LBR, solid circle) and contralateral (open circle) to LBR. *p < 0.01; ** p < 0.001;t tests. Inset, Pyknotic lumbar motoneurons (mean ± SD) ipsilateral (LBR, solid circle) and contralateral (open circle) to LBR. * p < 0.05; ** p < 0.001;t tests. The SDs that were smaller than the symbols are not shown.

Fig. 5.

Sensory neuron numbers (mean ± SD) in the L3 DRG ipsilateral (LBR, solid circle) and contralateral (open circle) to LBR. * p < 0.01; ** p < 0.001; t tests.Inset, Pyknotic neurons (mean ± SD) in the L3 DRG ipsilateral (LBR, solid circle) and contralateral (open circle) to LBR. * p < 0.01; ** p < 0.001; t tests.

Fig. 6.

BrdU-labeled cells (mean ± SD) in the lumbar spinal cord ipsilateral (LBR) and contralateral (CON) to LBR.

Fig. 7.

BrdU-labeled cells (mean ± SD) in the L3 DRG ipsilateral (LBR) and contralateral (CON) to LBR. * p < 0.01;t test.

Fig. 8.

Peripheral nerve patterns in whole mounts of normal and experimental limbs as revealed by staining with the TUJ1 antibody. A and B are focused on the medial aspect of each preparation to show nerve projection patterns;C and D are focused on the lateral aspect to show skin innervation in the same limbs. A, Normal pattern of peripheral nerve projections. Lumbar segments L1–L3 largely contribute axons to the crural plexus (c), which gives rise to many peripheral nerves of the limb including the prominent obturator nerve (o). Lumbar segments L3–L8 all contribute axons to the sciatic plexus (s). The more caudal segments contribute axons to the pudendal plexus (p). f, Femur.B, After limb-bud deletion, neither the crural plexus nor the sciatic plexus develops. L1 and L2 typically project axons anteriorly toward thoracic segments (arrowheads). L2–L8 all project axons posteriorly toward the tail, where they form a novel plexus (arrows) and contribute to the pudendal plexus (double arrowhead). Dashed lines indicate connections broken during processing; dr indicates dorsal roots. The spinal cord and sympathetic chains were removed for clarity. C, Normal patterns of skin innervation on the lateral surface of the thigh. The crural plexus (C) gives rise to the lateral femoral cutaneous nerve (lfc), which branches (arrowheads) to provide much of the cutaneous innervation of the lateral thigh. The more proximal skin receives axons from the dorsal rami (d) of each spinal nerve. f, Femur. D, After limb-bud deletion, the lateral femoral cutaneous nerve does not develop, and the remaining skin is innervated by branches from dorsal rami (d). Some axons also reach the skin over the deletion site from the novel plexus in the tail region (arrow). Scale bar, 1 mm.

Fig. 9.

The number of surviving motoneurons (MNs) and sensory neurons (mean ± SD) in L3 DRG on E9.5 after LBR (L), tail ablation (T), or L + T. *p< 0.01 (L + T vs L).Numbers in bars are sample size.

Fig. 10.

Lumbar motoneuron numbers (mean ± SD) on E10 after daily treatment of normal control embryos with saline (SAL) or different trophic agents from E6 to E9. *p < 0.001; t test (vsSAL). Numbers in bars = sample size (embryos). See Materials and Methods and Results.

Fig. 11.

Lumbar motoneurons numbers (mean ± SD) on E7.5 ipsilateral (LBR) and contralateral (CON) to LBR after daily treatment with trophic factors from E4 to E7. *p < 0.05; **p < 0.001 (vs saline LBR); ***p < 0.0025 (vs saline CON); ttests. See Materials and Methods and Results.

Fig. 12.

The number (mean ± SD) of lumbar motoneurons on E7.5 ipsilateral (LBR) and contralateral (C) to LBR along the rostral-caudal axis after daily treatment with muscle extract (CMX) or saline (SAL) from E4 to E7. *p < 0.05; **p < 0.01; t tests. See Materials and Methods and Results.

Fig. 13.

The numbers of sensory neurons in the L3 DRG on E9.5 ipsilateral (LBR) and contralateral (CON) to LBR after daily treatment from E4 to E9 with neurotrophins. ¹p < 0.001LBR versus CON;²p < 0.01 NGFversus SAL;³p < 0.01NGF versus SAL;⁴p < 0.01BDNF versus SAL;⁵p < 0.05BDNF versus SAL;⁶p < 0.05NT3 versus SAL;⁷p < 0.001COMBO versus SAL andp < 0.01 COMBO versusNGF, BDNF, or NT3;⁸p < 0.01COMBO versus NGF, BDNF, NT3. See Materials and Methods and Results.

Fig. 14.

Pyknotic motoneurons (mean ± SD) on E6.5–E7.0 ipsilateral and contralateral to LBR after treatment withNAC or saline (SAL) on E5 and E6 (A). *p < 0.01.B, Surviving healthy motoneurons (mean ± SD) on E6.5–E7.0 after NAC or saline treatment as noted above. *p < 0.01; t test. See Materials and Methods and Results.