Data from: RNA Polymerase III subunit Polr3a is required for craniofacial cartilage and bone development in zebrafish
Data files
May 27, 2026 version files 815.84 KB
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Fig_1.xlsx
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Fig_2.xlsx
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Fig_3.xlsx
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Fig_4.xlsx
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Fig_5.xlsx
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Fig_6.xlsx
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Fig_7.xlsx
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Fig_8.xlsx
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README.md
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S1_Fig.xlsx
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S10_Fig.xlsx
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S11_Fig.xlsx
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S12_Fig.xlsx
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S13_Fig.xlsx
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S14_Fig.xlsx
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S15_Fig.xlsx
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S2_Fig.xlsx
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S20_Fig.xlsx
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S21_Fig.xlsx
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S3_Fig.xlsx
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S4_Fig.xlsx
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S6_Fig.xlsx
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S8_Fig.xlsx
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S9_Fig.xlsx
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Abstract
Transcription by RNA Polymerase III (Pol III) is essential for ribosome biogenesis and translation in all cells, but pathogenic variants in genes encoding subunits of Pol III lead to tissue-specific phenotypes including craniofacial differences. To understand the function of Pol III in craniofacial development, we examined polr3a mutant zebrafish. These mutants display hypoplasia of the neural crest cell-derived craniofacial cartilage and bone but, surprisingly, no significant changes were observed in neural crest cell proliferation or survival during embryogenesis. At larval stages, increased cell death was observed throughout the head, including in the craniofacial cartilage. These changes coincide with reduced transcription of transfer RNAs and reduced ribosome biogenesis in polr3a mutant zebrafish. To determine tissue-specific transcriptional changes, we performed single-cell RNA-sequencing. Analysis revealed both global and cartilage-specific changes, including upregulation of tp53. However, Tp53 inhibition alone was not sufficient to rescue craniofacial cartilage and bone, indicating that additional factors are important to support cartilage and bone growth in polr3a mutants. Altogether, our study provides new mechanistic insights into the functions of Pol III in craniofacial development. Data deposited here are the values from image quantifications, qRT-PCR, and polysome profiling used to generate statistics and graphs.
Dataset DOI: 10.5061/dryad.nvx0k6f6z
Description of the data and file structure
Data here were collected to determine differences between control and polr3a mutant zebrafish. The numerical values used to calculate differences are organized in spreadsheets according to each figure as published in Lubash et al., 2026.
Files and variables
File: Fig_1.xlsx
Description: Figure 1. qRT-PCR data with processed values representing quantification of polr3a in controls and mutants relative to b-actin. Ct values for b-actin, canx, and polr3a are in the raw data tab
Variables
- wt (polr3a+/+)
- het (polr3asa907/+)
- mut (polr3asa907/sa907)
File: Fig_2.xlsx
Description: Figure 2. Quantification of sox9:gfp volume in Fig 2I with values in um3 and quantification of cell number in Meckel's cartilage based on sox9:gfp expression in Fig 2J.
Variables
- control (polr3a+/+ and polr3asa907/+)
- mut (polr3asa907/sa907)
File: Fig_3.xlsx
Description: Figure 3. Quantification of Alizarin red and sp7:gfp in control and polr3asa907/sa907 zebrafish at 4 dpf and 6 dpf as represented in Fig 3. Values represent volume in um3.
Variables
- control (polr3a+/+ and polr3asa907/+)
- mut (polr3asa907/sa907)
File: Fig_4.xlsx
Description: Figure 4. Quantification of TUNEL spots and pH3 spots within arch 1-2 derived cartilage labeled with sox10:gfp at 3 dpf, 4 dpf, and 5 dpf. Numerical values are number of spots/um3.
Variables
- control (polr3a+/+ and polr3asa907/+)
- mut (polr3asa907/sa907)
File: Fig_5.xlsx
Description: Figure 5. Quantification of TUNEL spots and pH3 spots within the whole head stained with DAPI at 3 dpf, 4 dpf, and 5 dpf. Numerical values are number of spots/um3.
Variables
- control (polr3a+/+ and polr3asa907/+)
- mut (polr3asa907/sa907)
File: Fig_6.xlsx
Description: Figure 6. Data associated with graphs presented in Fig 6. In 6A, the calculation and normalization of area under the curve to quantify tRNA and 5S rRNA relative to 5.8S rRNA from the small RNA chips on the Bioanalyzer. Fig 6C, Fig 6E analyzed and raw values for qRT-PCR data. Analyzed values represent relative expression normalized to b-actin for 7SL RNA, 5'ETS rRNA, tRNA-Arg-TCT-1-1, tRNA-Leu-CAA-1-2, tRNA-Ile-TAT-1-3. Raw values as well as values calculated from the area under the curve in ImageJ are included for polysome profiling as represented in Fig 6D.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: Fig_7.xlsx
Description: Figure 7. qRT-PCR data for genes in the integrated stress response pathway (atf4b, chac1, aars) expressed as relative values normalized to b-actin as in Fig 7D and qRT-PCR data for polr3a and genes in the Tp53 pathway (mdm2, tp53, cdkn1a) expressed as relative values normalized to b-actin as in Fig 7E. Raw data (Ct values) are also included.
Variables
- control (polr3a+/+)
- mut (polr3asa907/sa907)
File: Fig_8.xlsx
Description: Figure 8. Quantification of TUNEL and pH3 spots in the whole head stained with DAPI in control and polr3a; tp53 zebrafish at 3 dpf as in Fig 8D. Values represent number of spots/DAPI volume in um3. Quantification of Alizarin red volume in the opercle at 6 dpf as in Fig 8H. Values represent volume in um3. qRT-PCR data for tp53 and cdkn1a as in Fig 8I expressed as values relative to b-actin expression. Ct values are in the raw data tab. qRT-PCR data for polr3a and tRNA-Leu-CAA-1 as in Fig 8 with values representing expression relative to b-actin. Ct values are in the raw data tab.
Variables
- control (consisting of polr3a+/+; tp53+/+, polr3asa907/+; tp53+/+, polr3a+/+; tp53+/- and polr3a+/+; tp53-/- samples)
- polr3asa907/sa907; tp53+/+
- polr3asa907/sa907; tp53+/-
- polr3asa907/sa907; tp53-/-
File: S1_Fig.xlsx
Description: S1 Fig. Western blot quantification of Polr3a protein relative to a-tubulin completed in ImageJ. Values represent adjusted relative density for Polr3a based on a-tubulin.
Variables
- wt control (polr3a+/+)
- het (polr3asa907/+)
- mut (polr3asa907/sa907)
File: S2_Fig.xlsx
Description: S2 Fig. Measurements of 6 dpf skeletal elements in control and polr3a mutant zebrafish as measured in ImageJ. Values represent length in um.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S3_Fig.xlsx
Description: S3 Fig. Measurements of 8 dpf skeletal elements in control and polr3a mutant zebrafish as measured in ImageJ. Values represent length in um.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S4_Fig.xlsx
Description: S4 Fig. Values from quantification of arch 1 and 2 volume based on sox10:egfp expression at 36 hpf and 48 hpf in control and polr3asa907/sa907 embryos. Values represent volume in um3.
Variables
- control (polr3a+/+ and polr3asa907/+)
- mut (polr3asa907/sa907)
File: S6_Fig.xlsx
Description: S6 Fig. Numerical values from CellProfiler analysis of cell area and eccentricity of Meckel's cartilage at 5 dpf. The average area per cell is represented in pixels2 while eccentricity are values between 0 and 1.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S8_Fig.xlsx
Description: S8 Fig. Values from quantification of sp7:gfp and Alizarin red volume at 3 dpf. Values represent volume in um3, with each value representing an individual zebrafish.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S9_Fig.xlsx
Description: S9 Fig. Quantification of Alizarin red volume of bone elements in control and polr3a mutant zebrafish at 8 dpf. Values represent volume in um3 as quantified in Imaris.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S10_Fig.xlsx
Description: S10 Fig. Volume of cdh16 expression at 5 dpf from quantification in Imaris in um3. Values for startle response as represented in S10 Fig, representing percent responding at stimulus intensity of 0.537g, 1.279g, 2.5588g, 5.11g, 15.35g, and 51.177g.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S11_Fig.xlsx
Description: S11 Fig. qRT-PCR data of polr3a expression relative to b-actin in wild-type embryos over multiple stages of development. Data were normalized to b-actin expression.
Variables
- 4 cell stage (3 samples)
- 6 hpf (3 samples)
- 12 hpf (3 samples)
- 24 hpf (3 samples)
- 48 hpf (3 samples)
- 72 hpf (3 samples)
- 4 dpf (3 samples)
- 5 dpf (3 samples)
File: S12_Fig.xlsx
Description: S12 Fig. Quantification of mbp:tagRFP volume in 6 dpf zebrafish in Imaris. Values represent volume in um3.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S13_Fig.xlsx
Description: S13 Fig. Values from quantification of TUNEL and pH3 within arch 1 and 2 labeled with sox10:gfp at 2 dpf in control and mutant zebrafish. Values represent number of spots/arch 1 and 2 volume in um3.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S14_Fig.xlsx
Description: S14 Fig. Quantification of TUNEL and pH3 throughout the head (stained with DAPI) at 48 hpf in control and polr3a mutant zebrafish. Values represent number of spots/DAPI volume in um3.
Variables
- control (polr3a+/+ and polr3asa907/+)
- polr3asa907/sa907
File: S15_Fig.xlsx
Description: S15 Fig. qRT-PCR data evaluating relative expression of polr3a, 5'ETS, 7SL, and tRNAs at 2 dpf. Values were calculated relative to b-actin.
Variables
- control (polr3a+/+)
- polr3asa907/sa907
File: S20_Fig.xlsx
Description: S20 Fig. Values from quantification of pH3 within the head (stained with DAPI) at 3 dpf in control and polr3a; tp53 mutant zebrafish. Values represent number of spots/volume in um3.
Variables
- control
- polr3asa907/sa907; tp53+/+
- polr3asa907/sa907; tp53+/-
- polr3asa907/sa907; tp53-/-
File: S21_Fig.xlsx
Description: S21 Fig. Measurements of skeletal elements in 6 dpf control and polr3a; tp53 mutant zebrafish and calculations of length relative to controls.
Variables
- control (polr3a+/+; tp53+/+, polr3a+/+; tp53+/-, and polr3a+/+; tp53-/-)
- polr3asa907/sa907; tp53+/+
- polr3asa907/sa907; tp53+/-
- polr3asa907/sa907; tp53-/-
Code/software
All files are .xlsx
Access information
Other publicly accessible locations of the data:
- Raw data associated with single cell RNA-seq in this study was deposited in GEO under accession number GSE315895.
