Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ( A ) Relative eye sizes from control (left) and CKO (right) embryos. ( B ) Comparative immunohistology for control (top row) and CKO eyes (bottom row) showing tissue organization with DAPI, the RPC marker and progenitor maintenance factor Hes1, the RPC marker Cyclin D1, the RGC marker Pou4f2, and the photoreceptor precursor marker Otx2. Although the retina is noticeably smaller in the CKO compared to control, only Hes1 shows a qualitative change in expression at E15.5, 4 days after tamoxifen treatment.

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: Control, Marker, Expressing

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ATAC-seq and Lhx2 ChIP-seq tracks from E14.5 RPCs. Differentially accessible chromatin regions (DARs) in Lhx2 CKO RPCs compared to control are indicated by red (open) and blue bars (closed). Lhx2 ChIP-seq peak calls are indicated by grey bars.

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: ChIP-sequencing, Control

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ATAC-seq and Lhx2 ChIP-seq tracks from E14.5 RPCs. Differentially accessible chromatin regions (DARs) in Lhx2 CKO RPCs compared to control are indicated by red (open) and blue bars (closed). Lhx2 ChIP-seq peak calls are indicated by grey bars.

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: ChIP-sequencing, Control

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ( A ) Schematic showing that addition of recombinant Shh-N to a confluent monolayer of responder cells activates nuclear mCitrine expression. ( B ) Dose response and time course for accumulation of mCitrine expression in open-loop responder cells. Fields were randomly selected from the same cultures for each timepoint. ( C ) Scatter plots showing the accumulation of mCitrine + cells over time for each dose tested as a function of average mean fluorescence intensity per cell. Means are shown for each condition (lines).

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: Recombinant, Expressing, Fluorescence

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: .

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques:

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ( A ) Control and ( B ) CKO explants treated with vehicle or 10 nM purmorphamine for 24 hr. Left column: tdTomato expression from heterozygous ai14 allele reveals CreER activity throughout the explants. Middle column: Caspase 3 immunofluorescence (green) reveals extent of apoptotic cells. Nuclei are stained with DAPI (blue). Right column: Pou4f2 immunofluorescence (green) marks RGCs. ( C ) Relative Gli1 expression in wild type E15.5 retinas treated with increasing concentrations of recombinant Shh-N. Data points are relative to the mean expression value from retinas harvested at the start of the experiment (t=0; orange line). Means and standard deviation are shown, and results are from a pilot experiment (n=3 for all conditions; One way ANOVA followed by Dunnett’s multiple comparisons test;see for statistics including p adj values for multiple comparisons).

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: Control, Expressing, Activity Assay, Immunofluorescence, Staining, Recombinant, Standard Deviation

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ( A ) Overview of genetics and tamoxifen treatment. ( B ) Schematic of Ptch1 locus encompassing the regions containing exons 2–6. Exon 2 forward (ex2F) and Exon 6 reverse (ex6R) were used for RT-PCR to detect unrecombined and recombined transcripts from the flox and CKO alleles, respectively. ( C ) Agarose gel shows most samples were recombined (CKO) although some unrecombined (flox) was detected. All samples were used for qPCR. Figure 6—figure supplement 1—source data 1. RT-PCR for non-deleted ( flox ) and deleted (Δ) Ptch1 mRNAs. File with the word ‘unedited’ shows the original gel capture. File with the word ‘labeled’ shows the fragment sizes of the DNA standards in base pairs (bp). The red box indicates the portion of the gel shown in . The sizes of the non-recombined and deleted Ptch1 mRNAs are shown in base pairs (bp).

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Labeling

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ( A ) Expression of Gas1, Cdon, and Gli1 at E15.5 after tamoxifen treatment at E11.5. Boxes in Gas1 and Cdon panels mark locations of insets, which correspond to the retinal periphery, where Gas1 and Cdon are expressed. Arrowheads in Ctrl panels show the restricted peripheral expression that is absent in the CKO. Bracket in CKO panel for Gli1 expression reveals low, but persistent, expression in the dorsal retina. ( B ) Temporal expression patterns of Gas1 , Cdon , Gli1 , and Atoh7 from E11.5 to E13.5. Dashed lines in E12.5 and E13.5 images show that temporal downregulation of Cdon mRNA has a tighter spatial complementarity with Atoh7 mRNA accumulation than Gli1 . ( C ) Expression of Vsx2 , Gli1 , Gas1 , and Cdon at E12.5 after tamoxifen treatment at E10.5. The presence of Gli1 in the periocular mesenchyme indicates that the staining protocol was sufficient to detect expression in other tissues. Circles in Gas1 images show regions of Gas1 expression in peripheral retina that is lost upon Lhx2 inactivation. Bracket in image of Cdon for the CKO image shows absence of Cdon in ventral domain, and arrowheads point to persistent Cdon expression. Abbreviations: c, cornea; cb, ciliary body; le, lens; onh, optic nerve head; nr, neural retina; pom, periocular mesenchyme.

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: Expressing, Staining

Journal: eLife

Article Title: Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

doi: 10.7554/eLife.78342

Figure Lengend Snippet: ( A ) Merged and single channel images for left panels in 8 C. ( B ) Merged and single channel images for right panels in 8 C. ( C ) Merged and single channel images for left panels in 8D. ( D ) Merged and single channel images for right panels in 8D. b-Gal expression in C and D detects LacZ expression from the Gli1 LacZ allele. ( E ) Merged and single channel images for representative control and CKO explants electroporated with pCIG and stained for GFP, b-Gal, and Lhx2. Zoomed in views for Lhx2 are shown to emphasize the loss of Lhx2 protein in the CKO. The low signal to noise ratio for Lhx2 is due to the use of a secondary antibody conjugated to fluorophore in the 405 nm channel, which yields weak signal and elevated tissue autofluorescence and was necessary to perform the co-expression analysis (tdTomato occupied the 568 nm channel).

Article Snippet: RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO 3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).

Techniques: Expressing, Control, Staining