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Additional Supporting Information may be found in the online version of this article.

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DVDY_22094_sm_SuppFig1.tif764KSupporting Information Figure 1. Temporal regulation of Lrp5 and Lrp6 expression during midbrain development. A,B: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that Lrp5 (A) is expressed at constant levels in the developing ventral midbrain (VM), whereas Lrp6 (B) shows a peak of expression at embryonic day (E) 11.5. The dopaminergic (DA) neurogenic period in the rat ventral midbrain (VM) is highlighted with a box. (Error bars represent SEM of technical replicates as qRT-PCR was performed on pools of VMs). C: Detection of TH, Lrp5, and Lrp6 expression on sagittal sections of E11.5 wild-type mouse embryos by in situ hybridization. Lrp5 and Lrp6 are ubiquitously expressed in the brain, including the VM domain containing TH+ DA neurons. au, arbitrary units; te, telencephalon; ov, optic vesicle; di, diencephalon; mes, mesencephalon; hb, hindbrain.
DVDY_22094_sm_SuppFig2.tif3956KSupporting Information Figure 2. Lrp6−/− mice with exencephaly display normal ventral patterning, despite gross morphological dorsal defects. A,B: Shh (floor plate [FP] and basal plate [BP] marker), Lmx1b (FP and roof plate [RP] marker), and Wnt3a (RP marker) in situ hybridization of embryonic day (E) 9.5 (A) and E10.5 (B) wild-type (WT) and exencephalic Lrp6−/− mice revealed that patterning of the ventral domains is maintained, even though dorsal structures are grossly disrupted in the mutants. A: At E9.5, the ventricular invagination at the midline appears sharper and Shh and Lmx1b expression in the FP/BP might be broadened in Lrp6−/− mice. B: This difference is not seen in the mutants at E10.5.
DVDY_22094_sm_SuppFig3.tif2320KSupporting Information Figure 3. Proliferation, as assessed by BrdU (5-bromo-2-deoxyuridine) incorporation, is unchanged in Lrp6−/− mice. The proliferative capacity of embryonic day (E) 11.5 ventral midbrain (VM) precursors was not affected in Lrp6−/− VM as assessed by immunohistochemistry against BrdU in sagittal sections at E10.5, E11.5, E12.5, and E15.5.
DVDY_22094_sm_SuppFig4.tif1597KSupporting Information Figure 4. Cell death is not affected in Lrp6−/− mice. Cleaved caspase 3+ cells were virtually absent in wild-type and Lrp6−/− ventral midbrain (VM) at embryonic day (E) 11.5. All images were acquired with the same settings. Inset in each image is another region of the same brain showing positive staining.

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