An early broad competence of motoneurons to express ER81 is later sculpted by the periphery

Abstract

The ETS transcription factor ER81 is expressed in sensory neurons and motoneurons that innervate the adductor and femorotibialis muscles in chick hindlimb and is essential for the development of monosynaptic connections between these two populations of neurons. Neurons need a signal(s) from limb bud mesoderm to initiate ER81 expression. It is not known whether the mature expression pattern arises because adductor and femorotibialis motoneurons are uniquely competent to respond to peripheral signals and express ER81, or whether all motoneurons are competent to express ER81, but normally only adductor and femorotibialis motoneurons are exposed to the requisite activating signal. To investigate these possibilities, we examined ER81 expression in motoneurons that encountered limb tissue surgically mismatched with their target identity at stages after motor pool identities are established. We found that ER81 expression was not invariably linked to motor pool identity or target innervation and was more malleable in later-born femorotibialis motoneurons than in earlier-born adductor motoneurons. We also found that ER81 expression is regulated differently in sensory neurons and motoneurons. Most striking was the observation that motoneurons caudal to the normal adductor and femorotibialis pools could express ER81 when exposed to the appropriate peripheral signals, although this competence did not extend through the entire lumbosacral (LS) region. Thus, it appears that a prepattern of competence to express ER81 is established in early LS motoneurons, most likely in concert with their target identity, and that the expression domains of motoneurons are subsequently refined by peripheral signals at later stages.

Figure 2.

Comparison of motoneuron connectivity and ER81 expression in embryos with rostral limb shifts. Each pair of bars represents a single embryo, designated by the number on the left. A single representative control embryo (gray; top bars in each panel) is shown for comparison. Hatched bars represent the segmental levels at which stimulating the ventral spinal cord elicited e.m.g.s from the adductor and femorotibialis muscles (top and bottom panels, respectively). Solid bars represent the distribution of ER81-positive neurons in MLMC (top panel) and LLMC (bottom panel) motoneurons. The thinner solid bars in the bottom panel indicate that fewer than normal LLMC motoneurons expressed ER81. Note that MLMC and LLMC motoneurons in segments LS1-LS3, the normal location of adductor and femorotibialis motor pools, express ER81 when they innervate the adductor and femorotibialis muscles, respectively. In contrast, motoneurons in thoracic segments do not express ER81, even when they innervate these muscles. Activation of muscles in shifted limbs generally required a more intense stimulus than activation of control muscles, as if fewer motoneurons than normal innervated these muscles, which most likely accounts for the reduced expression of ER81 in LLMC motoneurons.

Figure 5.

Comparison of motoneuron connectivity and ER81 expression in embryos with A-P limb reversals. Each pair of bars represents a single embryo, designated by the number on the left. A single representative control embryo (gray; top bars in each panel) is shown for comparison. Hatched bars represent the segmental levels at which stimulating the ventral spinal cord elicited e.m.g.s from the adductor and femorotibialis muscles (top and bottom panels, respectively). Solid bars represent the distribution of ER81-positive neurons in MLMC (top panel) and LLMC (bottom panel) motoneurons. Note that ER81 expression and target innervation are not invariably linked. ER81 is expressed in rostral MLMC motoneurons that do not innervate the adductor muscle and is not expressed in motoneurons in segments LS5-7, even when they do innervate the adductor muscle. Moreover, whereas ER81 expression is generally correlated with innervation of the femorotibialis muscle in LLMC motoneurons in segments LS3-LS4 or LS5, motoneurons in more caudal segments that innervate the femorotibialis muscle do not express ER81.

Figure 1.

ER81 expression is reduced in femorotibialis motoneurons after rostral limb shifts. Left, Top, Ventral view of the spinal cord and DRGs from thoracic segment T7 through LS segment LS4 of a sham-operated embryo [St. 31 (E7.5)] in which one limb (represented on the right) was removed and replaced at St. 17. Left, Bottom, Ventral view of the spinal cord and DRGs from a St. 30 (E7) embryo in which one limb (represented on the right) was shifted two to three segments rostrally at St. 17. Right, Sections through the ventral spinal cord of another limb-shifted embryo, St. 31 (E7.5). In both embryos with shifted limbs, ER81 expression in MLMC (M) motoneurons that innervated the shifted limb is nearly identical to that of control adductor motoneurons (A). In contrast, very few LLMC (L; arrow) motoneurons expressed ER81 after limb shifts, although the lateral motor column (encircled by white dots in panels LS2 and LS3) is quite large. Note that ER81 is nearly identical in both motor pools after sham operations. F, Femorotibialis motoneurons.

Figure 3.

ER81 is expressed in more rostral DRGs than normal after limb shifts. Images are through the approximate midsection of representative DRGs of a single St. 31 (E7.5) embryo with one limb shifted rostrally; lateral is toward the outer edge of each panel. Note that the pattern of ER81 expression is shifted rostrally in DRGs serving the operated limb, and in some DRGs the labeled cells are more widely scattered than normal.

Figure 4.

A-P limb reversals reveal a broad competence of MLMC and LLMC neurons to express ER81. Left, Ventral view of the spinal cord and DRGs from segments LS1-LS5 of two St. 31 (E7.5) embryos in which one limb (represented on the right) was reversed 180° along the A-P axis at St. 17, leaving the dorsoventral axis intact. Right, Sections through the ventral spinal cord of a St. 33 (E8) embryo with an A-P reversed limb. The control LS3 spinal cord was distorted by the suction electrode used to stimulate motoneurons. In all three embryos with A-P-reversed limbs, ER81 expression in MLMC (M) motoneurons that innervated the reversed limb extended up to one segment more caudal than the normal adductor pool (A). More strikingly, ER81 expression in LLMC (L) motoneurons that innervated the reversed limb extended as much as two segments more caudal than the normal femorotibialis pool (F).

Figure 6.

Innervation of the femorotibialis muscle and expression of ER81 are not invariably linked in LLMC motoneurons. A, ER81 expression (left panels) and HRP labeling (right panels) in adjacent sections of the LMC innervating an A-P-reversed limb. HRP was injected into the femorotibialis muscle in the reversed limb at St. 32. The LMC in each left panel is outlined by dots; lateral is to the right in each panel. Note that ER81 is expressed in LLMC (L) motoneurons in LS4, one segment more caudal than normal. In contrast, motoneurons in segment LS6 innervated the femorotibialis muscle but did not express ER81. M, ER81-positive MLMC neurons. B, DiI-labeled motoneurons innervating femorotibialis muscles in a St. 32 embryo with an A-P reversed limb (right side); uncleared whole-mount view of the ventral spinal cord (SC). The control femorotibialis muscle is innervated by motoneurons in segments LS2 and LS3, with a small contribution from LS4, whereas the femorotibialis muscle in the reversed limb is innervated by motoneurons in segments LS4-LS8, with a small contribution from LS3, shown in the inset. Inset is a higher-magnification view of the boxed area. In other embryos with A-P limb reversals (n = 19), ER81 was always expressed in LLMC motoneurons in segment LS4 and often in LS5 but never in more caudal motoneurons, although neurons in these caudal segments innervated the femorotibialis muscle, as shown here.

Figure 7.

ER81 expression in motoneurons after heterotopic neural tube transplantation at stages after motor pool identity is established. The diagrams at the top of each column depict the operation that was done in the embryo shown in the images directly below. In each diagram, the shaded box in the left panel shows the normal pattern of ER81 expression in adductor and femorotibialis motoneurons and the region of neural tube that was excised from the donor embryo. The shaded box in the right panel shows the location of the transplant in the host embryo and the pattern of ER81 expression expected if the donor cord expressed ER81 in accord with its original location. Photographs show sections of ventral spinal cord of operated embryos at St. 31-32. Designations in the margins indicate the segmental levels of the host embryo. Images through the donor spinal cord are outlined in black; the approximate segment of origin of each image is shown in the inset. A, Caudal-to-rostral transplantation. Donor neural tube from progressively more caudal regions was transplanted into segments LS1-LS3 of host embryos at St. 16 (n = 7). Note that ER81 was expressed in the LLMC of donor neural tube taken from segments LS3-5 but not in neurons derived from more caudal tissue. Thus, LLMC motoneurons in segments LS4 and LS5, but not more caudal segments, can express ER81 when exposed to appropriate peripheral signals. B, Rostral-to-caudal transplantation. Note that ER81 was expressed in MLMC neurons in donor tissue transplanted to caudal segments (LS4) in which ER81 is normally never expressed (n = 5), suggesting that expression was already fixed in rostral motoneurons at the time of transplantation.

Figure 8.

ER81 is expressed in some adductor motoneurons after early limb bud removal. Prospective limb tissue was removed unilaterally at St. 15, and ER81 expression was examined at St. 29, before extensive cell death. ER81 was expressed in a few adductor (A) motoneurons on the operated side. F, Femorotibialis motoneurons.