Table 1.
Primary antibodies used in this study.
Fig 1.
Trx2 antibody validation in chick spinal cord.
(A-D) Immunofluorescence assay of E4.5 and E6.5 chick embryo spinal cords with anti-Trx2 (A, B) and anti-Trx2 preadsorbed with recombinant Trx2 (C, D). Colocalization of anti-Trx2 with mitochondrial marker ATPB (E-H). Scale bar = 50 μm (A-D) and 10 μm (E-H).
Fig 2.
Gallus gallus Trx2 cDNA and amino acid sequence.
(A) Sequence of Gallus gallus Trx2 PCR-amplified from total chick heart, liver, lung and muscle cDNA and predicted amino acid sequence. Amplification yielded a 642-bp amplicon instead of the 482-bp amplicon expected from the NCBI reference sequence (Accession number: NM_001031410.1). This difference is associated to a 160-bp sequence (indicated in yellow) located in the 3’-half of the whole Gallus gallus Trx2 cDNA. (B) Alignment of Gallus gallus Trx2 protein predicted from cDNA sequence with human (Accession number: Q99757) and mouse (Accession number: P97493) Trx2 amino acid sequences. Nucleotide sequences used to design forward and reverse primers for amplification are indicated by the black arrows. Mitochondrial targeting sequences (MTS) are indicated in green and conserved catalytic WCGPC motives are indicated in red.
Fig 3.
Trx2 colocalizes with NeuN-positive cells at E4.5 and Isl1/2 positive cells at E6.5.
Colocalization of Trx2 with NeuN at E4.5 (A-C) and with Isl1/2 at E6.5 (D-F) in the embryonic chick spinal cord. Scale bar = 50 μm.
Fig 4.
Trx2 overexpression and downregulation by in ovo electroporation.
(A-H) Immunofluorescence detection of Trx2 in E4.5 chick embryo spinal cords electroporated with Trx2 vector or MoTrx2. (I) Western blotting analysis of the contralateral (non-electroporated; left well) and ipsilateral (electroporated; right well) sides of dissected spinal cords of E4.5 chick embryos electroporated with empty vector, Trx2 vector, MoCTRL or MoTrx2. (J) Densitometric analysis of Trx2 expression, shown as the ratio of the ipsilateral measurement on the contralateral measurement. Scale bar = 50 μm.
Fig 5.
Overexpression or downregulation of Trx2 does not affect markers of neuronal populations in the spinal cord of E4.5 chick embryos.
E4.5 chick embryos electroporated with Trx2 vector (A-H) and MoTrx2 (I-P) were probed with several markers of neuronal subpopulations (NeuN, Isl1/2, Lhx3, Lhx1/5, Nkx2.2, Evx1) and a marker of apoptosis (cleaved Casp-3). Scale bar = 50 μm.
Fig 6.
Trx2 overexpression and downregulation affect neuron PCD in the E6.5 chick spinal cord.
(A-F) Immunofluorescence detection of motor neuronal marker Isl1/2 and apoptosis marker cleaved Casp-3 and (G-J) TUNEL staining in the spinal cord of E6.5 chick embryos electroporated with Trx2 vector or MoTrx2. (K) Comparison of the average TUNEL-positive cell count on the ipsi- and contralateral sides of spinal cord slices of E6.5 chick embryos electroporated with empty vector (N = 4), Trx2 vector (N = 9), MoCTRL (N = 4) or MoTrx2 (N = 5). Statistical significance is indicated as follows: ns = non-significant, * = p< 0.05, *** = p<0.0005.
Fig 7.
Trx2 downregulation increases neuron PCD in target-deprived spinal cord explants.
(A-F) Immunofluorescence detection of motor neuronal marker Isl1/2 and apoptosis marker cleaved Casp-3 and (G-J) TUNEL staining in E4.5 chick spinal cords electroporated with Trx2 vector and MoTrx2 after culture for 12h, (K) Comparison of the average TUNEL-positive cell count of the ipsi- and contralateral sides of E4.5 chick spinal cord explants electroporated with empty vector (N = 6), Trx2 vector (N = 6), MoCTRL (N = 6) or MoTrx2 (N = 5). Statistical significance is indicated as follows: ns = non-significant, * = p< 0.05.