Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1 - Isl1 transcriptional cascade

doi: 10.1007/s00018-024-05523-6

Figure Lengend Snippet: TRN neuronal identity is dependent on Isl1 function. (Top) Schematic showing the wild-type anatomy of the prethalamus. (A-N) In situ hybridization of prethalamic markers on control ( Dlx5/6-Cre; Isl1 F/+ ) and mutant ( Dlx5/6-Cre; Isl1 F/F ) coronal sections. The red boxed areas are magnified as indicated. The patterns of gene expression in the prethalamus at E14.5 were similar to those at E16.5. (A-B) Dlx1 was normally expressed in all prethalamic nuclei, including the rZI, TRN, and vLG, as well as the central diencephalic organizer zli. Dlx1 expression in the TRN of control embryos was high (A2, B2), while Isl1 -deficient embryos lost Dlx1 expression specifically in the TRN but not in the vLG, rZI or zli (A4, B4). (C-D) Within the prethalamus of control embryos, Meis2 expression was limited to the TRN (C2, D2). Loss of Isl1 resulted in the abrogation of Meis2 expression (C4, D4). (E-L) Arx , Pax6 , and Lhx1 were normally expressed in the rZI, zli, and rostroventral parts of the vLG but were not detectable in the TRN. Lhx5 is also weakly expressed in the rZI, zli, and vLG but not in the TRN. In the absence of Isl1 function, the expression of these genes was largely unaffected in the rZI, zli, and vLG but was ectopically induced in the TRN. (M , N) A GABAergic neuronal marker Gad1 was similarly expressed in control and Isl1 mutants. (O) Quantification of in situ hybridization. The pixel intensity values of TRN or vLG were grouped and averaged from three different sections per embryo. ( n = 5 embryos; Student’s t -test; ** p < 0.01, *** p < 0.001, **** p < 0.0001) (P) A schematic diagram indicating that Dlx1 + Meis2 + TRN cells lose their identity and instead acquire molecular features of the remainder of the prethalamus. Cx, cortex; Hyp, hypothalamus; Pt, prethalamus; rZI, rostral zona incerta; Th, thalamus; TRN, thalamic reticular nucleus; vLG, ventral lateral geniculate nucleus; zli, zona limitans intrathalamica. Scale bar, 200 μm

Article Snippet: The transfected plasmids (0.3 µg) consisted of a mixture of 0.1 µg of pCMV-tdTomato (a gift from Davidson M, Shaner N, and Tsien R; Addgene plasmid #54642) [ ] + 0.2 µg of pCMV-Isl1 (Sino Biological plasmid #MG56983-UT).

Techniques: In Situ Hybridization, Control, Mutagenesis, Expressing, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1 - Isl1 transcriptional cascade

doi: 10.1007/s00018-024-05523-6

Figure Lengend Snippet: Loss of Isl1 leads to TCA pathfinding defects in the prethalamus and thalamus. (A) Immunohistochemistry for GFP on coronal sections of control ( Gbx2 GFP ; Dlx5/6-Cre; Isl1 F/+ ) (A1-A6) and mutant ( Gbx2 GFP ; Dlx5/6-Cre; Isl1 F/F ) (A7-A12) embryos at E16.5. The red arrows in A10 and A11 indicate abnormal TCA projections in the prethalamus of Isl1 mutant embryos. White asterisks in A10, A11, and A12 indicate abnormally large bundles of TCAs crossing the prethalamus and entering the ventral telencephalon in Isl1 mutants compared to controls. GFP + TCAs misrouted caudally to the habenula (white arrows in A9). Red asterisks in A10-A12 mark abnormal fasciculation of TCAs within the thalamus. GFP + TCAs misrouted ventrally in the ventral telencephalon (A7-A11, red arrowheads) and hypothalamus (A12, yellow arrowhead). In the cortex, Isl1 mutant TCAs were shorter than those of control embryos (A7-A11, white arrowheads). (A13) Examples of the measurements taken for the quantification. Yellow dashed lines in A4, A5, A10, A11 indicate the position of the lines used to quantify the number of GFP + fluorescent signals crossing the prethalamus. (n = 5 embryos; Student’s t -test; * p < 0.001). (B) Double immunofluorescence for GFP and NF-M on sagittal sections of control and Isl1 mutant embryos at E14.5. Control TCAs extended in parallel and oriented ventrolaterally, forming a thin fasciculus in the thalamus and prethalamus, including the TRN. In contrast, Isl1 mutant TCAs formed an abnormally thick fasciculus and extended randomly in the thalamus (white arrowheads) and prethalamus (white arrows). (C) Double immunofluorescence for GFP and NF-M on sagittal sections of control and Isl1 mutant embryos at E18.5. In the thalamus, GFP + TCAs formed abnormally thick axon bundles orienting randomly and misrouting caudally into the pretectum (white arrowheads in C4’,C4”). Cx, cerebral cortex; Eth, epithalamus; Hyp, hypothalamus; Prt, pretectum; Pt, prethalamus; Th, thalamus. Scale bars, 200 μm

Article Snippet: The transfected plasmids (0.3 µg) consisted of a mixture of 0.1 µg of pCMV-tdTomato (a gift from Davidson M, Shaner N, and Tsien R; Addgene plasmid #54642) [ ] + 0.2 µg of pCMV-Isl1 (Sino Biological plasmid #MG56983-UT).

Techniques: Immunohistochemistry, Control, Mutagenesis, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1 - Isl1 transcriptional cascade

doi: 10.1007/s00018-024-05523-6

Figure Lengend Snippet: Loss of Isl1 derepresses the expression of Efna5 , a repellent for TCAs, in the TRN. (A) Differential expression of Efna5 in the prethalamus of E13.5 mutant embryos ( Dlx5/6-Cre; Isl1 F/F ) compared with control embryos ( Dlx5/6-Cre; Isl1 F/+ ) based on qRT-PCR. Prethalamic Efna5 expression was significantly elevated in Isl1 mutant embryos (two-way ANOVA with Sidak’s multiple comparison test; **** p ≤ 0.001). (B) RNA in situ hybridization for Efna5 , Epha4 , and Epha7 on coronal sections of control and Isl1 mutants at E13.5 and E14.5. Loss of Isl1 resulted in significant upregulation of Efna5 in the TRN (red dashed areas), despite similar staining in other brain regions. Scale bar, 200 μm. (B13) Quantification of in situ hybridization. The pixel intensity values of TRN were grouped and averaged from three different sections per embryo. ( n = 3 embryos; Student’s t -test; ** p < 0.01) (C) Thalamic explants from control ( Gbx2 GFP ; Dlx5/6-Cre; Isl1 F/+ ) and mutant ( Gbx2 GFP ; Dlx5/6-Cre; Isl1 F/F ) embryos were cocultured with Efna5 -expressing cells (outlined by a white dashed line). Explants were photographed, and outgrowth from the distal and proximal sides of the explants was quantified by GFP fluorescence-occupied area. The values were averaged from two explants per embryo, and the averaged values from three independent experiments were plotted. A significant increase in the distal-proximal (D:P) fluorescence ratio was observed in the presence of Efna5 -expressing cells (two-way ANOVA with Sidak’s multiple comparison test; * p < 0.05). Pt, prethalamus; Th, thalamus; TRN, thalamic reticular nucleus

Article Snippet: The transfected plasmids (0.3 µg) consisted of a mixture of 0.1 µg of pCMV-tdTomato (a gift from Davidson M, Shaner N, and Tsien R; Addgene plasmid #54642) [ ] + 0.2 µg of pCMV-Isl1 (Sino Biological plasmid #MG56983-UT).

Techniques: Expressing, Mutagenesis, Control, Quantitative RT-PCR, Comparison, RNA In Situ Hybridization, Staining, In Situ Hybridization, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1 - Isl1 transcriptional cascade

doi: 10.1007/s00018-024-05523-6

Figure Lengend Snippet: Isl1 is required for the formation of prethalamo-thalamic pioneer axons. (A) Immunohistochemistry for tdTomato on coronal sections of Dlx5/6-Cre; R26-tdTomato embryos at E13.5 and E16.5. Prethalamo-thalamic axons (PTAs) labeled by tdTomato extended into the thalamus, forming ordered and parallel projections. (B) Costaining for tdTomato and GFP on coronal sections of Dlx5/6-Cre; R26-TdTomato; Gbx2 GFP embryos at E11.5. PTAs labeled with tdTomato extended into the thalamus, while GFP + thalamic axons had yet to enter the prethalamus. (C) Costaining for tdTomato and NF-M on coronal sections of control ( Dlx5/6-Cre; Isl1 F/+ : R26-tdTomato ) and mutant ( Dlx5/6-Cre; Isl1 F/F : R26-tdTomato ) embryos at E13.5 and E14.5. Loss of Isl1 caused a visible reduction in the number of tdTomato + axons projecting from the prethalamus to the thalamus in E13.5 and E14.5 mutant embryos. Control NF-M + thalamic axons formed ordered and parallel projections, but mutant NF-M + thalamic axons aggregated into thick bundles that ran laterally. (C13) Examples of the measurements taken for the quantification. Yellow dashed lines in C1, C4, C7, C10 indicate the position of the lines used to quantify the number of axon bundles crossing the thalamus. (two-way ANOVA with Sidak’s multiple comparison test; * p < 0.05, ** p < 0.01, *** p < 0.001). (D) Implanting the chemical tracer Neurovue into the prethalamus of control ( Dlx5/6-Cre; Isl1 F/+ ) at E13.5 visualized PTA projections across the thalamus. Coronal sections from five independent experiments revealed a defective pattern in the outgrowth of PTAs in mutant embryos ( Dlx5/6-Cre; Isl1 F/F ) similar to that shown by tdTomato immunohistochemistry in Dlx5/6-Cre; Isl1 F/F ; R26-TdTomato embryos. Pt, prethalamus; Th, thalamus. Scale bars: 200 μm

Article Snippet: The transfected plasmids (0.3 µg) consisted of a mixture of 0.1 µg of pCMV-tdTomato (a gift from Davidson M, Shaner N, and Tsien R; Addgene plasmid #54642) [ ] + 0.2 µg of pCMV-Isl1 (Sino Biological plasmid #MG56983-UT).

Techniques: Immunohistochemistry, Labeling, Control, Mutagenesis, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1 - Isl1 transcriptional cascade

doi: 10.1007/s00018-024-05523-6

Figure Lengend Snippet: Isl1 continues to regulate TRN identity during late embryogenesis (A-L) In situ hybridization for prethalamic markers on control ( SBR-Cre; Isl1 F/+ ) and mutant ( SBR-Cre; Isl1 F/F ) coronal sections at E16.5. Dlx1 expression in the TRN but not in other prethalamic nuclei was lost in Isl1 mutant embryos (G). Meis2 expression, which was restricted to the TRN of control embryos, was abrogated in mutant embryos (H). Loss of Isl1 resulted in ectopic induction of Arx , Pax6 , and Lhx1/5 in the TRN (I-L). (M) Quantification of in situ hybridization. The pixel intensity values of TRN or vLG were grouped and averaged from three different sections per embryo ( n = 3 embryos; Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Scale bars, 200 μm

Article Snippet: The transfected plasmids (0.3 µg) consisted of a mixture of 0.1 µg of pCMV-tdTomato (a gift from Davidson M, Shaner N, and Tsien R; Addgene plasmid #54642) [ ] + 0.2 µg of pCMV-Isl1 (Sino Biological plasmid #MG56983-UT).

Techniques: In Situ Hybridization, Control, Mutagenesis, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1 - Isl1 transcriptional cascade

doi: 10.1007/s00018-024-05523-6

Figure Lengend Snippet: Ascl1 controls TRN cell fate and PTA outgrowth and is essential for Isl1 expression in the prethalamus. (A) In situ hybridization for prethalamic markers on control ( Ascl1 KI/+ ) and mutant ( Ascl1 KI/KI ) coronal sections at E14.5 and E16.5. In Ascl1 mutants, Dlx1 expression was lost in the TRN as well as in the rZI, vLG, and zli (A6, A16). Loss of Ascl1 resulted in severe downregulation of Meis2 expression in the TRN (A7, A17). Arx , Pax6 , or Lhx1 expression in the vLG and zli of Ascl1 KI/KI mutants was downregulated but ectopically induced in the TRN (A8-A10, A18-A20). (A21) Quantification of in situ hybridization. The pixel intensity values of TRN or vLG were grouped and averaged from three different sections per embryo ( n = 5 embryos; Student’s t -test; ** p < 0.01, *** p < 0.001, **** p < 0.0001). (B) Immunohistochemistry for NF-M on coronal sections of control ( Ascl1 KI/+ ) and mutant ( Ascl1 KI/KI ) embryos at E14.5 and E16.5. In control embryos, NF-M + thalamic axons extended in ordered and parallel arrays that were less fasciculated in the thalamus and prethalamus (B1,B2) than in mutant embryos (B3,B4). Moreover, in Ascl1 mutants, thick NF-M + bundles crossed each other with abnormal overlapping course of axons (arrows in B3,B4). (C) Immunohistochemistry for tdTomato on coronal sections of control ( Dlx5/6-Cre; R26-TdTomato; Ascl1 KI/+ ) and mutant ( Dlx5/6-Cre; R26-TdTomato; Ascl1 KI/KI ) embryos at E13.5 and E14.5. The control prethalamus formed ordered and parallel tdTomato + projections to the thalamus (C1,C3), while very few PTAs were detected in the mutant embryos (C2,C4). (C5) Examples of the measurements taken for the quantification. Yellow dashed lines in C1-C4 indicate the position of the lines used to quantify the number of axon bundles crossing the thalamus. ( n = 3 embryos; two-way ANOVA with Sidak’s multiple comparison test; ** p < 0.01, *** p < 0.001, **** p < 0.0001). (D) Implanting the chemical tracer Neurovue into the prethalamus of control ( Ascl1 KI/+ ) and mutant embryos ( Ascl1 KI/KI ) at E13.5. Compared to those in controls (D1), no Neurovue + PTAs were labeled in the thalamus in the absence of Ascl1 (D2). (E) In situ hybridization for Isl1 and Shh on control ( Ascl1 KI/+ ) and mutant ( Ascl1 KI/KI ) coronal sections at E12.5. Shh is a specific marker for the zli. Loss of Ascl1 abrogated Isl1 expression in the TRN. (E5) Quantification of in situ hybridization. The pixel intensity values of TRN were grouped and averaged from four different sections per embryo ( n = 4 embryos; Student’s t -test; **** p < 0.0001).Pt, prethalamus: Hyp, hypothalamus; Pt, prethalamus; rZI, rostral zona incerta; Th, thalamus; TRN, thalamic reticular nucleus; vLG, ventral lateral geniculate; zli, zona limitans intrathalamica. Scale bars, 200 μm

Article Snippet: The transfected plasmids (0.3 µg) consisted of a mixture of 0.1 µg of pCMV-tdTomato (a gift from Davidson M, Shaner N, and Tsien R; Addgene plasmid #54642) [ ] + 0.2 µg of pCMV-Isl1 (Sino Biological plasmid #MG56983-UT).

Techniques: Expressing, In Situ Hybridization, Control, Mutagenesis, Immunohistochemistry, Comparison, Labeling, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1 - Isl1 transcriptional cascade

doi: 10.1007/s00018-024-05523-6

Figure Lengend Snippet: Ascl1 requires Isl1 to promote TRN neuronal specification and neurite outgrowth in cultured neurons. (A , B) Isolation and primary culture of TRN cells from E13.5 control ( Ascl1 KI/+ ) and Ascl1 KI/KI mutant embryonic brains. TRN cells were cultured on coverslips coated with L-ornithine and laminin in 24-well plates. TRN cell identity was verified by immunocytochemistry of DIV2 cells using antibodies against Isl1, Dlx1 and Pax6. Consistent with the gene expression profiles of the embryos, most of the control cultured cells derived from Ascl1 KI/+ TRN were Isl1 + , Dlx1 + , and Pax6 − , while many of the Ascl1 KI/KI cells were Isl1 − , Dlx1 − , and Pax6 + . A total of 100 DAPI + cells were quantified for each field, and three measurements were averaged for each coverslip. The average values from six coverslips prepared from three embryos were plotted for each genotype (Student’s t -test; ** p ≤ 0.01). (C , D) Tuj1 immunostaining was performed at DIV2 and DIV6 to determine the number of neurites, the length of the longest neurite, and the total length of neurites. Quantification of the number of neurites, length of the longest neurite, and total length of neurites were achieved from 20 neurons for each field, and three measurements were averaged for each coverslip. The average values from ten coverslips prepared from five embryos were plotted for each genotype (two-way ANOVA with Sidak’s multiple comparison test; ** p < 0.01, **** p < 0.0001). (E-H) TRN cells isolated from Ascl1 KI/KI mutant E13.5 brains were cultured for 1 DIV and transfected with an Isl1 expression construct ( pCMV-Isl1 ) or an empty vector ( pCMV ). The reporter expression vector ( pCMV-tdTomato ) was cotransfected to identify the transfected cells. (E , F) Quantification was achieved from transfected Ascl1 KI/KI mutant cells cultured for 5 additional DIV by counting Dlx1 + or Pax6 + cells among 100 tdTomato + cells for each field. Three measurements were averaged for each coverslip, and the average values from eight coverslips prepared from four embryos were plotted for each genotype (Student’s t -test; ** p < 0.01). (G , H) The number of neurites, length of the longest neurite, and total length of neurites were measured for each tdTomato + neuron cultured for 5 additional DIV following transfection. Quantification was achieved from 20 tdTomato + - transfected cells for each field. Three measurements were averaged for each coverslip, and the average values from ten coverslips prepared from five embryos were plotted for each genotype (Student’s t -test; * p < 0.05, ** p ≤ 0.01)

Article Snippet: The transfected plasmids (0.3 µg) consisted of a mixture of 0.1 µg of pCMV-tdTomato (a gift from Davidson M, Shaner N, and Tsien R; Addgene plasmid #54642) [ ] + 0.2 µg of pCMV-Isl1 (Sino Biological plasmid #MG56983-UT).

Techniques: Cell Culture, Isolation, Control, Mutagenesis, Immunocytochemistry, Expressing, Derivative Assay, Immunostaining, Comparison, Transfection, Construct, Plasmid Preparation