Overlapping Function of Lmx1a and Lmx1b in Anterior Hindbrain Roof Plate Formation and Cerebellar Growth

Development of the hindbrain roof plate is dependent on overlapping functions of Lmx1a and Lmx1b. A–H, Dorsal view of whole-mount in situ hybridization of E10.5 embryos using probes for Gdf7 and Wnt1. The brackets in A and B indicate the position of rh1 and the future cerebellum. The line in B specifies the level of the transverse section in I. C, D, In Lmx1b^−/− embryos, the fourth ventricle roof plate was nearly normal despite the loss of the IsO. E, F, The fourth ventricle roof plate was reduced in Lmx1a^−/− embryos. G, H, Lmx1a^−/−; Lmx1b^−/− embryos had little or no residual fourth ventricle roof plate (red arrow). The line in H indicates the transverse section in K. I, Schematic of a transverse section of the fourth ventricle. The boxed region indicates equivalent region in J and K. J, In wild-type embryos, the roof plate covered the fourth ventricle. K, This region was lost in Lmx1a^−/−; Lmx1b^−/− embryos, although residual, lateral non-roof plate Wnt1 expression remained. IV, Fourth ventricle.

Math1+ rhombic lip cells are lost in Lmx1a^−/−; Lmx1b^−/− embryos. A–H, Dorsal view of whole-mount in situ hybridization of subdissected E12.5 brains using probes for Math1 (A–D) and Ptf1a (E–H). Brackets in A and E indicate the developing cerebellum. A–C, Math1 expression, induced by roof plate signaling, was retained in Lmx1b^−/− (B) and Lmx1a^−/− (C) embryos. However, Math1 expression was completely lost in Lmx1a^−/−; Lmx1b^−/− embryos (arrow in D), confirming complete loss of roof plate function in rh1. E–H, Ptf1a expression is independent of roof plate signaling, and was present in all genotypes, although the domain of expression was reduced in Lmx1a^−/−; Lmx1b^−/− mutants (arrow in H). I–L, Otp expression was not disturbed in Lmx1a^−/−; Lmx1b^−/− mutants (arrow in L) compared with the wild-type (I) or single-mutant (J, K) embryos, indicating that the alar/basal boundary is not perturbed.

A conditional knock-out strategy to delete Lmx1b only in the roof plate, where it is coexpressed with Lmx1a. A, Schematic diagram demonstrating the Lmx1b conditional knock-out strategy. Lmx1a-cre transgenic mice were bred to mice carrying a floxed allele of Lmx1b (Lmx1b^f), leading to excision of Lmx1b^f only within the Lmx1a expression domain in the roof plate. B, C, β-Gal activity in the progeny of Lmx1a-cre mice mated to ROSA26 floxed LacZ reporter mice. B, Only a few LacZ-positive cells are visible in the fourth ventricle at E9.0. C, LacZ expression is clearly visible at the fourth ventricle at E9.5. D–L, In situ hybridization using the Lmx1b homeodomain (HD) probe indicated in A. D, E, Whole mount at E10.5 of wild-type (D) and Lmx1b^cko/− (E) embryos. Lmx1b HD expression was reduced in the fourth ventricle roof plate but remained intact in the IsO in Lmx1b^cko/− embryos. F, Schematic of a transverse section of the fourth ventricle at E10.5. The boxed region indicates the equivalent region in G, H, K, and L. G, H, Transverse sections at E10.5 showed that Lmx1b HD expression is present in the wild-type (G) but lost in Lmx1b^cko/− embryos (H). Arrowheads indicate Lmx1b HD expression in the wild type. I, J, Whole mount at E9.5 showed that Lmx1b HD is expressed in both the wild-type (I) and Lmx1a^−/−; Lmx1b^cko/− (J) embryos. K, L, Transverse sections through the fourth ventricle at E9.5 showed that Lmx1b HD expression is present in both the wild-type (K) and Lmx1a^−/−; Lmx1b^cko/− (L) embryos. Arrowheads indicate Lmx1b HD expression.

An intermediate roof plate phenotype is observed in conditional double-knock-out embryos. A–C, Dorsal view of E10.5 embryos hybridized with a Gdf7 in situ probe. B, Lmx1a^−/− embryos had a small fourth ventricle roof plate compared with the wild type. C, Lmx1a^−/−; Lmx1b^cko/− retained the roof plate (unlike the double-null embryos) but developed an even smaller fourth ventricle roof plate compared with Lmx1a^−/− embryos (red arrow). In addition, Gdf7 expression was patchy (arrowheads). Insets in B and C show roof plate at E9.5 in Lmx1a^−/− embryos (B′) and Lmx1a^−/−; Lmx1b^cko/− embryos (C′). D, Quantification of fourth ventricle roof plate area in wt, Lmx1a^−/−, and Lmx1a^−/−; Lmx1b^cko/− embryos at E10.5 (n = 3). The y-axis is normalized to wt. *p < 0.01; **p < 0.001. Error bars indicate SE. E, Lateral view of E10.5 embryo stained with Wnt1 in situ probe. IsO is normally present in Lmx1a^−/−; Lmx1b^cko/− embryos (arrow). F, G, Dorsal view of E12.5 Lmx1a^−/−; Lmx1b^cko/− embryos stained with Math1 (F) and Ptf1a (G) in situ probes. Variability of these markers was not detected, despite reduction in size of the fourth ventricle roof plate. Bracket in F indicates rh1, and arrowhead in G indicates Ptf1a domain in rh1.

Cerebellar morphogenesis is dependent on Lmx1-dependent roof plate signaling. A–F, Whole mount (A–C) and Nissl-stained midsagittal sections of P20 cerebella (D–F) demonstrating that loss of both Lmx1b and Lmx1a had significant effects on cerebellar development. The cerebella of Lmx1a^−/− mice were small, but those of Lmx1a^−/−; Lmx1b^cko/− mice were even smaller. G, Quantification of midsagittal sections of P20 cerebella (n = 4). Area of wild type is indicated as 100%. *p < 0.01; **p < 0.001. Error bars indicate SE. H–J, Ectopic cerebellar cells were observed in n = 2/8 Lmx1a^−/−; Lmx1b^cko/− cerebella in the midbrain, evident in Nissl-stained sections. I, Higher-magnification of the boxed region in H showing ectopic cerebellar cells in the midbrain. J, White arrows indicate calbindin-positive Purkinje cells present in the midbrain (MB), and cerebellar (Cb) Purkinje cells are indicated by *, in similar sections. K, An equivalent wild-type calbindin-stained section. Scale bars: A–C, 1 mm; D–F, 500 μm; H, 500 μm; I–K, 250 μm.