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First published online 13 December 2006
doi: 10.1242/dev.02745

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Lmx1b is essential for Fgf8 and Wnt1 expression in the isthmic organizer during tectum and cerebellum development in mice

Chao Guo^1,*, Hai-Yan Qiu^2,*, Ying Huang¹, Haixu Chen³, Rong-Qiang Yang¹, Sheng-Di Chen², Randy L. Johnson³, Zhou-Feng Chen⁴ and Yu-Qiang Ding^1,

¹ Institute of Neuroscience and Key Laboratory of Neurobiology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
² Department of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
³ Department of Biochemistry and Molecular Biology, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA.
⁴ Departments of Anesthesiology, Psychiatry, Molecular Biology and Pharmacology, School of Medicine Pain Center, Washington University, St Louis, MO 63110, USA.

View larger version (111K): [in this window] [in a new window]   Fig. 1. Expression of Lmx1b in the developing embryo and its localization in the MHB. (A-C) Lmx1b expression revealed by whole-mount in situ hybridization. Expression of Lmx1b was first detected at E7.5 in the anterior embryo (A, arrow), appeared at the prospective MHB at E8.5 (B, arrow) and in the isthmus at E9.5 (C, arrow). (D-F) Expression of Wnt1 (D), Fgf8 (E) and Lmx1b (F) in the MHB at the 4-somite stage. Expression of Wnt1 and Fgf8 showed anteroposterior non-overlapping patterns (arrowheads), whereas the Lmx1b expression domain covered the majority of these two domains. (G,H) Lmx1b expression (G,H, red arrows) overlapped with Wnt1 expression (H, blue arrows) in the isthmus, as shown for the same embryo processed sequentially for the detection of Lmx1b and Wnt1 RNAs. Note that there was a Lmx1b+ domain located posterior to the Wnt1+ domain in the MHB. (I) Double in situ hybridization for Lmx1b and Fgf8. Note that the Lmx1b+ domain (red arrow) overlaps with the anterior portion of the Fgf8+ domain (blue arrow) in the isthmus.

View larger version (109K): [in this window] [in a new window]   Fig. 2. Lmx1b deletion results in a marked reduction in the size of the tectum and cerebellum. (A,B) Dorsal view of the mid/hindbrain region of P0 mouse brains. The medial portion of the cerebellum (Cb) was missing, and the remaining part of the cerebellum and the tectum were much smaller in the Lmx1b-/- brain (B) as compared with the wild-type control (A). (C,D) Parasagittal Nissl-stained sections of P0 brains. The inferior colliculus (IC) was missing and the reduced cerebellum fused with the superior colliculus (SC) in the Lmx1b-/- brain. (E,F) Double immunostaining of the brain sections for BRN3b (marking IC and laminated SC) and for calbindin (marking Purkinje cells in the cerebellum). cp, choroids plexus; Hy, hypothalamus; PT, pretectal region; Th, thalamus. Scale bars: 700 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 3. Early morphological changes and reduced cell proliferation in the Lmx1b-/- embryo. (A,B) Nissl-stained sagittal sections of embryos at E10.5, showing the reduced rhombomere 1 (r1) and less conspicuous isthmus in the mutant brain (B, arrow), as compared with the control brain (A, arrow). (C,D) Immunostaining of Ki67, showing a reduced number of proliferating cells in the tectum and r1 of Lmx1b-/- embryo (D) as compared with that in the control (C) at E10.5. (E-H) Sagittal sections of embryos at E12.5, showing the loss of the constriction between the midbrain and hindbrain (F, arrowhead) and reduced Math1 expression (E,F, arrows) in the presumptive cerebellum (H, arrow) in the mutant embryo, as compared with control brains (E,G). (I,J) Expression domain of Otx2 (arrow) did not show a caudal extension in Lmx1b-/- mutant embryos (J) as compared with wild-type (I) at E12.5. (K,L) Transverse sections of embryos at E12.5, showing reduction of Nurr1 expression in the ventral midbrain of the mutant embryo (L) as compared with the control (K). (M-P) Transverse sections of embryos at E15.5, showing the loss of the cerebellar vermis (N, arrow) and normal Brn3a expression in the presumptive red nucleus (P, arrow) in the mutant embryos as compared with control brains (M,O). (Q,R) Transverse sections of embryo at E15.5, showing the loss of tyrosine hydroxylase (TH) immunostaining in the tegmentum of the mutant (R) as compared with the wild-type embryo (Q). Aq, aqueduct; Cb, cerebellum. Scale bars: A-D, 1000 µm; E-L, 700 µm; M-R, 500 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 4. Analysis of Fgf8, Wnt1, En1 and Pax2 expression in the MHB of Lmx1b-/- embryos. (A-D) Whole-mount in situ hybridization showing the absence of Fgf8 expression in the MHB at the 4-somite stage (B, arrow) and in the isthmus at E9.5 (D, arrow) in the Lmx1b-/- embryo. Note that Fgf8 expression in other areas of the mutant embryo (B, arrowhead) was normal as compared with that in the wild-type embryo (A, arrowhead). (E-H) Lmx1b deletion resulted in reduced Wnt1 expression in the MHB at the 3-somite stage (F, arrow) and the loss of Wnt1 expression in the isthmus at E9.5 (H, arrow) as compared with that in the wild-type brain (E,G). Note that Wnt1 expression in the roof plate was maintained in the mutant brain at E9.5 (H). (I-L) Lmx1b deletion caused a marked reduction in En1 expression in the MHB at the 3-somite stage (J, arrow) and the loss of En1 expression at E9.5 (L, arrow), as compared with that in the control (I,K). (M-P) Lateral view of embryos showing similar changes in Pax2 expression (arrows) to those found for En1 (I-K).

View larger version (105K): [in this window] [in a new window]   Fig. 5. Analysis of Gbx2, Otx2, Hoxa2 and Pax6 expression and neuronal death in the MHB of Lmx1b-/- embryos. (A-F) Gbx2 expression was normal in mutant embryos at E7.75 (B), but was barely detectable in the mutant brain at the 4-somite stage (D, arrow) and lost altogether in the isthmus at E9.5 (F, arrow). Note that Gbx2 expression was normal in the caudal hindbrain (D, arrowhead) and otic vesicle (F, arrowhead), as compared with controls (C,E). (G-J) Expression of Otx2 was not changed in mutant embryos (H,J) as compared with wild-type controls (G,I). (K-N) Double in situ hybridization for Otx2 and Hoxa2 showing no changes in the distance (double-arrowed line) between Otx2+ and Hoxa2+ domains at the 5-somite stage (L) and a reduction in this distance in the mutant embryos at the 13-somite stage (N), as compared with controls (K,M). (O,P) Expression of Pax6 was not changed in the mutant embryos (P) as compared with wild type (O). Note that the rostral-caudal distance (double-arrowed line) marked by Pax6 was maintained in the Lmx1b-/- embryo. (Q-T) There was a slight increase in the number of TUNEL+ cells (arrow) in the MHB in the mutant embryo at the 7-somite stage (R), when marked downregulation of gene expression was observed. However, there was a significant increase in the number of TUNEL+ cells (arrow) in the MHB of the mutant embryo at the 13-somite stage (T), as compared with the wild-type embryo (S).

View larger version (106K): [in this window] [in a new window]   Fig. 6. Region-specific deletion of Lmx1b in the MHB by Wnt1-Cre conditional knockout and its effect on gene expression. (A,B) Whole-mount in situ hybridization showing inactivation of Lmx1b in the MHB at the 6-somite stage (B, arrow) by the Wnt1-Cre recombination method. (C-J) Expression (arrows) of Fgf8 (D), Wnt1 (F), Pax2 (H) and En1 (J) was lost in the isthmus of Lmx1bw CKO embryos at E9.5. Normal expression (arrows) patterns of each gene in the control (C,E,G,I) are shown for comparison. (K,L) Decreased expression of En2 in the MHB of the Wnt1 Lmx1bw CKO embryo at E9.5 (arrow, L) as compared with that in the control (arrow, K). (M,N) The expression of Gbx2 of the Lmx1bw CKO embryos was lost in the isthmus (N, arrow) but normal in the otic vesicle (N, arrowhead) as compared with the control embryo (M). (O,P) Otx2 expression and the rostral-caudal distance of the midbrain (double-arrowed line) were normal in the conditional knockout embryo (P) as compared with the control embryo (O).

View larger version (96K): [in this window] [in a new window]   Fig. 7. Impaired tectum and cerebellum development in Lmx1bw CKO mice. (A,B) Dorsal view of the mid/hindbrain region of intact P0 brains. A similar reduction in the size and displaced location of the tectum and cerebellum (Cb) was observed to that described for the conventional Lmx1b-/- mice (see Fig. 2A,B). (C,D) Parasagittal Nissl-stained section of P0 brains. Similar defects were seen to those found in the conditional knockout mice (see Fig. 2C,D). (E,F) Double immunostaining of BRN3b and calbindin, marking the tectum and cerebellar Purkinje cells, respectively, showing the same staining patterns as described for the conventional knockout mice (see Fig. 2E,F). cp, choroids plexus; Hy, hypothalamus; IC, inferior colliculus; PT, pretectal region; SC, superior colliculus; Th, thalamus. Scale bars: C,D, 700 µm; E,F, 250 µm.