Marsh-Armstrong N et al. (2004), Thyroid hormone controls the development of con...

XB-ART-4229

Proc Natl Acad Sci U S A 2004 Jan 06;1011:165-70. doi: 10.1073/pnas.2136755100.

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Thyroid hormone controls the development of connections between the spinal cord and limbs during Xenopus laevis metamorphosis.

Marsh-Armstrong N , Cai L , Brown DD .

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During premetamorphic stages, Xenopus laevis tadpoles expressing either a dominant-negative thyroid hormone (TH) receptor or a type-III iodothyronine deiodinase transgene in the nervous system have reduced TH-induced proliferation in the spinal cord and produce fewer hindlimb-innervating motorneurons. During prometamorphic stages, innervation of the hindlimbs is reduced, and few functional neuromuscular connections are formed. By metamorphic climax, limb movement is impaired, ranging from uncoordinated leg swimming to complete quadriplegia. This phenotype is due to transgene action in the tadpole spinal cord. The requirement of TH for neurogenesis during premetamorphosis is the earliest TH-regulated process reported to date in the sequence of metamorphic changes in anurans. The muscle formed during limb growth was previously shown to be a direct target of TH control. Here, we show that the same is true of the development of spinal cord cells that innervate the limbs.

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Species referenced: Xenopus laevis
Genes referenced: aldh1a2 trdn
Lines/Strains:

Xla.Tg(tubb2b:dnthra-GFP)Brown

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	Fig. 1. Transgenes that inhibit TH action in the nervous system produce quadriplegic tadpoles at the climax of metamorphosis. NF stage 62 control (A) and NβT:GFP-TRDN-transgenic (B) animals. Dissected brain and spinal cord of NF stage 60 control (C) and NβT:GFP-TRDN-transgenic (D) animals. (Scale bar for A, 1 cm.)
	Fig. 2. Transgenes inhibit expression of TH-inducible genes in the spinal cord. In situ hybridization of cross-sections of the lumbar spinal cord at NF stage 62. TH/bZIP (A and D), gene 12 (B and E), xBTEB (C and F), control (A), and NβT:GFP-TRDN-transgenic (D) animals. (Scale bar, 200 μm.)
	Fig. 3. Transgene expression only in the spinal cord affects hindlimb movement. (A) A pair of crossgrafted animals, half of which were derived from a CMV:GFP-expressing transgenic animal, at NF stage 25. (B) The same pair of crossgrafted animals at NF stage 46. (C) A limb-paralyzed animal with CMV:GFP expression in the head, and NβT:GFP-TRDN expression in the body, at NF stage 59. Arrow points to paralyzed hindlimb. The dashed line delimits the boundary of the graft as determined by the boundary of skin expression of the CMV:GFP transgene. (Scale bars, 100 μm in A and B; 1 mm in C.)
	Fig. 4. TH controls the early proliferation and production of limb motorneurons. (A) Lumbar spinal cords of 3-week-old control tadpoles. (A and B) Lateral view of whole mounts. (C and D) Transverse sections. (A and C) Controls. (B and D) Animals treated with 10 nM T3 during the third week. Limb motorneurons labeled with a RALDH-2 antibody are red and nuclei labeled by DAPI are blue. (Scale bars, 100 μmin A and B; 25 μmin C and D.) (E) The number of lumbar motorneurons (MNs), determined by counting RALDH-2-positive cells in whole-mount spinal cords. (F) The number of proliferating cells in the lumbar spinal cord, determined by counting PH3-positive cells in whole-mount spinal cords. The labeled groups (*) in E and F are significantly different from the other three groups (P < 0.001). (G) The number of lumbar motorneurons at various stages during metamorphosis in control animals (○), and at NF stage 54 in NβT:GFP-TRDN-transgenic animals (▴), determined by counting RALDH-2-positive cells in sections throughout the spinal cord. Values for control and NβT:GFP-TRDN-transgenic animals at stage 54 are significantly different (P = 0.005). Error bars in E represent the SEM.
	Fig. 5. Transgenic animals develop defective neuromuscular junctions. Dorsal view of hindlimb nerves, labeled with 3A10 antibody, at NF stage 54 in control (A) and NβT:GFP-TRDN-transgenic (B) animals. (C) The diameter of tibial nerves, at positions marked in red in A and B, in control and NβT:GFP-TRDN animals at NF stage 54. Values are statistically different (*, P = 0.0014). Nerves and muscle synapses at NF stage 56 in control (D) and NβT:GFP-TRDN-transgenic (E) animals seen in a ventral view of the hindlimb. Arrows point to calf and foot muscles. Nerves and muscle synapses in the tibialis anterior muscle at NF Stage 60 in control (F) and NβT:GFP-TRDN-transgenic (G) animals. In D, the nerves are labeled with 3A10 antibody (red), and the AChRs are labeled with α-bungarotoxin (green). (Scale bars, 500 μmin A and B and D and E; 250 μm in F.)

References [+] :

Beach, Patterns of cell proliferation in the retina of the clawed frog during development. 1979, Pubmed, Xenbase