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Differential expression of a transcription regulatory factor, the LIM domain only 4 protein Lmo4, in muscle sensory neurons

¹ Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15261, USA
² Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA

View larger version (32K): [in a new window]   Fig. 1. Identification of Lmo4 as a differentially expressed transcript in adductor but not sartorius sensory neurons by single-cell RT-PCR. PCR using Lmo4-specific oligonucleotide primers amplified a 544 bp fragment in single cell cDNAs derived from five out of nine adductor neurons but none out of eight sartorius neurons at stage 39. The PCR product was detected by Southern hybridization using the Lmo4 cDNA. trkC cDNA was PCR-amplified as a positive control from all cells.

View larger version (45K): [in a new window]   Fig. 2. Sequence analysis of Lmo proteins from human, mouse and chicken sequences. (A) The non-mouse non-human expressed sequence tag (EST) database was searched with mouse amino acid sequences using the tBLASTn algorithm to identify cDNA sequences of chicken Lmo1 (GenBank Accession Number, AL587905) and Lmo2 (GenBank Accession Number, BI067394). Sequences are aligned to show the high degree of homology within Lmo family members of different species. Dots indicate missing sequence. (B) Sequence alignment of chicken Lmo1, Lmo2 and Lmo4 reveals high divergence overall; only three out of 13 amino acids are shared between Lmo1, Lmo2 and Lmo4 in the region (amino acids 145-157, underlined in red) used to make the Lmo4-specific c15 antibody. Lim1 and Lim2 domains are underlined in black.

View larger version (154K): [in a new window]   Fig. 3. Lmo4 is expressed in trkC-positive proprioceptive sensory neurons in muscle-pool-specific patterns. Lmo4 expression in nuclei of DRG sensory afferents was detected with an Lmo4-specific antibody (red in all panels). At stage 35, trkC- and trkA-specific antibodies stain (green) cytoplasm of large-diameter trkC-positive proprioceptive sensory neurons located ventrolaterally (upper right-hand corner in A) and small-diameter trkA-positive cutaneous sensory neurons located dorsomedially (lower left-hand corner in B) in the DRG, respectively. Most (83%) trkC-positive sensory neurons are Lmo4 positive, whereas almost none of the trkA-positive sensory neurons express Lmo4. (C-G) Lmo4 expression in sensory neurons supplying individual muscles was determined by retrograde labeling with green fluorescent dye at stage 39. Lmo4 is expressed in most adductor (79%, D) and external femorotibialis (73%, E) sensory neurons, but few sartorius sensory neurons are Lmo4-positive (13%, C) (one Lmo4-positive cell is indicated with arrow). Sensory neurons supplying the internal femorotibialis (F) and posterior iliotibialis (G) muscles are Lmo4 negative. Scale bar: 150 µm.

View larger version (158K): [in a new window]   Fig. 4. Lmo4 is expressed in subsets of LMC motoneurons in the lumbosacral spinal cord (stage 39). (A-C) Isl1/Isl2 (green) is expressed in all LMC neurons, whereas Lmo4 (red) is expressed in some of these cells. (D-H) Lmo4 expression in motoneurons supplying individual muscles was determined by retrogradely labeling with green fluorescent dye at stage 39. Lmo4 (red) is expressed in most adductor (84%, E), external femorotibialis (92%, F) and posterior iliotibialis (95%, H) motoneurons. Few sartorius motoneurons express Lmo4 (15%, D) and about half of the internal femorotibialis motoneurons are Lmo4 positive (40%, G). Only the LMC is shown; lateral is towards the left. Scale bar: 100 µm.

View larger version (140K): [in a new window]   Fig. 5. Co-expression of Lmo4 and LIM homeodomain proteins by neurons at earlier developmental stages. Localization of Lmo4 (red) and the LIM homeodomain proteins Isl1/Isl2 and Lim1/Lim2 (green) was determined by double-label immunohistochemistry in DRG sensory neurons (A,B) and in lumbosacral spinal motoneurons (C). At stage 26, Lmo4 is co-expressed with Isl1/Isl2 in DRG neurons (A). At stage 28, Lmo4 is excluded from DM cutaneous sensory neurons (B, dotted outline indicates the major region of non-overlap of Lmo4 and Isl1/Isl2). (C) At stage 26, Lmo4 is expressed in a subset of Lim1/Lim2-positive (1) LMC motoneurons at LS2 (arrows). The dorsomedial region of DRGs is located in the upper right in A,B. Lateral is towards the right in C. Scale bars: 25 µm in A,C; 50 µm in B.

View larger version (128K): [in a new window]   Fig. 6. Comparison of Lmo4 (red in all panels) with the ETS gene Pea3 (green in A,C,E) and ER81 (green in D,F) in sensory and motoneurons. (A) At stage 27, most Pea3-positive (green) sensory neurons are Lmo4 positive (yellow cells), but a small subset (arrows) is Lmo4 negative. (B) By stage 35, when trkC expression (green) serves as a good marker for muscle proprioceptive sensory neurons, a small proportion of trkC-positive cells remain Lmo4 negative. At this stage, Lmo4 is expressed in many, but not all, Pea3-positive (C) and ER81-positive(D) sensory neurons. In the LS2 spinal cord, Lmo4 is expressed in many, but not all, Pea3-positive (E) and ER81-positive (F) LMC motoneurons. See also Table 2. Scale bars: 25 µm in A; 100 µm in B-F.

View larger version (72K): [in a new window]   Fig. 7. Limb ablation does not alter Lmo4 expression. Limb ablations were performed unilaterally at stage 15 and analyzed at stage 28. Lmo4 expression appeared unchanged in DRG (A) and in the spinal cord at LS2 (D) after limb ablation. By contrast, Pea3 expression was lost after limb ablation (B,E). (C,F) Sections adjacent to the dualstained A,B,D,E sections were stained for Isl1/Isl2 expression to estimate numbers of surviving neurons. Scale bar: 85 µm.