Red nucleus and rubrospinal tract disorganization in the absence of Pou4f1

Abstract

The red nucleus (RN) is a neuronal population that plays an important role in forelimb motor control and locomotion. Histologically it is subdivided into two subpopulations, the parvocellular RN (pRN) located in the diencephalon and the magnocellular RN (mRN) in the mesencephalon. The RN integrates signals from motor cortex and cerebellum and projects to spinal cord interneurons and motor neurons through the rubrospinal tract (RST). Pou4f1 is a transcription factor highly expressed in this nucleus that has been related to its specification. Here we profoundly analyzed consequences of Pou4f1 loss-of-function in development, maturation and axonal projection of the RN. Surprisingly, RN neurons are specified and maintained in the mutant, no cell death was detected. Nevertheless, the nucleus appeared disorganized with a strong delay in radial migration and with a wider neuronal distribution; the neurons did not form a compacted population as they do in controls, Robo1 and Slit2 were miss-expressed. Cplx1 and Npas1, expressed in the RN, are transcription factors involved in neurotransmitter release, neuronal maturation and motor function processes among others. In our mutant mice, both transcription factors are lost, suggesting an abnormal maturation of the RN. The resulting altered nucleus occupied a wider territory. Finally, we examined RST development and found that the RN neurons were able to project to the spinal cord but their axons appeared defasciculated. These data suggest that Pou4f1 is necessary for the maturation of RN neurons but not for their specification and maintenance.

Keywords: Cplx1; Npas1; Pou4f1; development; maturation; midbrain; red nucleus; rubrospinal tract.

Figure 1

Development of RN and RST in absence of Pou4f1. Coronal mesencephalic sections in Pou4f^TauLacZ/+ (A–D) and Pou4f^{TauLacZ/TauLacZ} (E–H) embryos processed by immunohistochemistry. We have analyzed the embryonic stages: E12.5 (A,E,E’), E13.5 (B,F,F’), E14.5 (C,G) and E15.5 (D,H). We followed the normal generation of the RN and its projections in the control embryos. The RN development in the mutant embryos showed a clear delay in radial migration. The RN displayed certain spatial disorganization. Squares in (E) and (F) shows magnification of these areas in (E’) and (F’) respectively. The arrowhead points to the rubrospinal tract. The arrow points to migrating neurons. Abbreviations: EW, Edinger-Westphal nucleus; RN, red nucleus; preEW, pre-Edinger-Westphal; vtg, ventral tegmental decussation. Scale bars = 150 μm.

Figure 2

Disorganization of pRN in absence of Pou4f1. Coronal diencephalic sections in Pou4f^TauLacZ/+ (A–C, G–I) and Pou4f^{TauLacZ/TauLacZ} (D–F, J–L) embryos at E18.5 processed by immunohistochemistry (A,D,G,J) or in situ hybridization (B,C,E,F,H,I,K,L). The Pou4f ^{TauLacZ/TauLacZ} pRN showed a certain grade of disorganization in the diencephalic region as compared to the control (A,D). The Cplx1 expression was weakly detected in the control (B), while the Npas1 expression was detected in the pRN and the SNR (C). In Pou4f ^{TauLacZ/TauLacZ}, Cplx1 and Npas1 expression was completely absent in the pRN but remained unaffected in SNR (E, F). Squares shows overlapping and magnifications of these areas in (B’) and (E’). The TH distribution was used to identify the SNC (G,J). Gad2 and vGluT2 was used to identify GABAergic and glutamatergic neurons respectively (H,I,K,L) Abbreviations: Dk, Nucleus of Darkschewitsch; IP, Interpeduncular nucleus; RF, reticular formation; pRN, parvocellular red nucleus; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bar = 450 μm.

Figure 3

Disorganization of mRN in absence of Pou4f1. Coronal mesencephalic sections in Pou4f^TauLacZ/+ (A–C, G–I) and Pou4f^{TauLacZ/TauLacZ} (D–F, J–L) embryos at E18.5 processed by immunohistochemistry (A,D,G,J) or in situ hybridization (B,C,E,F,H,I,K,L). The Pou4f^{TauLacZ/TauLacZ} mRN exhibited a clear spatial disorganization while the III is completely normal (A,D). Cplx1 expression identified both mRN and III in Pou4f1 ^TauLacZ/+ (B). In the mutant, Cplx1 was exclusively expressed in the III (E). The Npas1 expression identified the same populations (C). Npas1 was lost in the mutant mRN (F). The TH distribution was used to identify the SNC (G,J). Gad2 and vGluT2 was used to identify GABAergic and glutamatergic neurons respectively (H,I,K,L). Abbreviations: III, oculomotor complex; mRN, magnocellular red nucleus; RF, mesencephalic reticular formation; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bar = 450 μm.

Figure 4

Neuronal migration defects and cell death. Coronal diencephalic (A–F) and mesencephalic (G–L) sections in Pou4f^TauLacZ/+ (A–C, G–I) and Pou4f^{TauLacZ/TauLacZ} (D–F, J–L) embryos at E18.5 processed by in situ hybridization (A,B,D,E,G,H,J,K) or immunohistochemistry (C,F,I,L). The Robo1 and Slit2 expression identified the pRN (A,B) and the mRN and III (G,H). The Robo1 and Slit2 expression is completely lost in the mutant in both pRN and mRN (D,E,J,K). Caspase3 do not show any difference between control and mutant (C,F,I,L). Abbreviations: III, oculomotor complex; mRN, magnocellular red nucleus; pRN, parvocellular red nucleus. Scale bar = 450 μm.

Figure 5

The RST in the spinal cord. Abnormal growth in absence of Pou4f1. Coronal sections of cervical spinal cord in Pou4f^TauLacZ/+ (A–C) and Pou4f^{TauLacZ/TauLacZ} (D–F) embryos processed by X-Gal staining. We have analyzed the embryonic stages: E15.5 (A,D), E16.5 (B,E) and E18.5 (C,F). The pioneer axons of the RST were detected for the first time at E15.5 (arrow in (A)). From E16.5 onwards, the RST was well established in the dorso-lateral region of the spinal cord (arrow in (B,C)). In the mutant the RST was also observed from E15.5 onwards but it occupied a wider region in the spinal cord (arrow in (D,E,F)). Abbreviations: DC, dorsal column. Scale bars = 150 μm.