The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome

Abstract

Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention.

Fig 1. polr1c and polr1d are dynamically expressed during zebrafish embryogenesis.

polr1c and polr1d are maternally expressed at early stages (A,B, arrows) and ubiquitously expressed at 6 hpf (C,D) and 11 hpf (E,F) when the embryo surrounds the yolk (dashed lines). At 24 hpf, expression becomes enriched in regions such as the eye and midbrain-hindbrain boundary (G,H). Elevated levels of expression are evident in the pharyngeal arches (adjacent to curved line) at 36 hpf (I,J) whereas lower levels are observed throughout the embryo at 48 hpf and beyond (K,L) and beyond. Abbreviations: e, eye; mbhb, midbrain-hindbrain boundary; pa, pharyngeal arches; l, lens; t, tectum. Scale bar = 200 μm.

Fig 2. Mutations in polr1c and polr1d disrupt craniofacial development in zebrafish embryos.

24 hpf (A-D), 3 dpf (E-G) and 5 dpf (H-J) polr1c^-/- and polr1d^-/- zebrafish exhibit craniofacial defects, including smaller eyes, a disrupted midbrain-hindbrain boundary and cranial necrosis compared to controls. At 3 dpf, distinct craniofacial anomalies such as a smaller jaw and eyes become apparent. By 5 dpf, polr1c^-/- and polr1d^-/- mutants are distinguished from their control siblings by their smaller head, microphthalmia, jaw hypoplasia, and failure to inflate their swim bladder (E-G). Abbreviations: e, eye; mbhb, midbrain hindbrain boundary; j, jaw; h, heart; sb, swim bladder. Scale bar = 200 μm (A-G) and 500 μm (H-J).

Fig 3. Craniofacial cartilage development is disrupted in polr1c^-/- and polr1d^-/- mutant embryos.

(A-C) Alcian blue staining reveals cranial cartilage in 5 dpf polr1c^-/- and polr1d^-/- mutant embryos is hypoplastic compared to controls. (D-F) The jaws of mutant embryos are smaller overall, with noticeable differences in the size of Meckel’s cartilage, the palatoquadrate, and ceratohyal elements. (G-I) Staining of the viscerocranium reveals smaller cartilage elements derived from each of the pharyngeal arches in mutant embryos, most notably the ceratobranchials, as well as altered polarity of the ceratohyal. (J-L) Staining of the neurocranium reveals hypoplasia of the ethmoid plate. Abbreviations: M, Meckel’s cartilage; pq, palatoquadrate; ch, ceratohyal; cb, ceratobranchial; ep, ethmoid plate; pch, parachordal. Scale bar = 200 μm.

Fig 4. Analysis of NCC development in polr1c and polr1d mutant embryos.

(A-H) sox10 expression at 12 hpf and (I-P) foxd3 expression at 14 hpf reveal relatively normal patterns of early cranial NCC specification and migration in polr1c^-/- and polr1d^-/- embryos (black arrows). (Q-X) In contrast, dlx2 expression at 36 hpf reveals slightly diminished domains of activity in mutant embryos, particularly with respect to the posterior pharyngeal arches, which is suggestive of fewer mature NCC colonizing the pharyngeal arches. White arrows indicate pharyngeal arches 1 and 2. Scale bar = 200 μm.

Fig 5. Pharyngeal arch size is reduced in polr1c^-/- and polr1d^-/- mutants.

(A-C) Immunostaining of 36hpf fli1a:egfp labeled control, polr1c^-/- and polr1d^-/- mutant embryos with Zn-8 (red), which labels the endodermal pouches, revealed comparatively normal pharyngeal arch and pharyngeal pouch patterning. Pharyngeal arches 1–5 are indicated. (D-F) fli1a:egfp labeling of post-migratory NCC illustrates an overall reduction in pharyngeal arch size in polr1c^-/- and polr1d^-/- mutants. Pharyngeal arches 1 and 2 are outlined in red. (G-H) Quantification of pharyngeal arches 1 & 2 (red arrows, D-F) volume revealed a reduction in polr1c^-/- (G) and polr1d^-/- (H) mutants. Scale bar = 100 μm. * = p < 0.05 and error bars represent 95% confidence intervals.

Fig 6. Increased Tp53-dependent cell death within the neuroepithelium of polr1c^-/- and polr1d^-/- embryos.

(A-C) TUNEL staining (green fluorescence) revealed increased cell death in the cranial region and dorsal tissues of 24 hpf polr1c^-/- and polr1d^-/- mutant embryos. (D-F) Transverse sections revealed that cell death (green fluorescence) occurred primarily within the neuroepithelium. (G) qPCR (H) and Western blot analyses demonstrated the levels of tp53 and Tp53 were increased respectively in 36 hpf and 5 dpf polr1c^-/- and polr1d^-/- mutant embryos compared to controls. α-tubulin was used as a loading control. * = p < 0.05 and error bars represent 95% confidence intervals.

Fig 7. Ribosome biogenesis is disrupted in polr1c and polr1d mutant embryos.

(A, B) qPCR quantification of 47S rRNA production. (A) polr1c^-/- mutants exhibit reduced levels of 5’ETS (39%), ITS2 (23%) and 18S rRNA (58%) compared to controls. (B) polr1d^-/- mutants similarly exhibit reduced levels of 5’ETS (25%), ITS2 (39%), and 18S rRNA (32%) compared to controls. (C, D) Polysome profiling shows decreased 80S and polysome peaks in polr1c^-/- (C) and polr1d^-/- (D) mutant embryos.

Fig 8. tp53 inhibition ameliorates cartilage anomalies in polr1d^-/-mutant embryos in a dosage-dependent manner.

(A-D) Alcian blue staining of cartilage in an allelic series of polr1d and tp53 mutant embryos. Dosage-dependent improvement in cartilage development is particularly noticeable in the jaw (E-H), elements of the viscerocranium (I-L), and more specifically the ceratohyal (G-L). Abbreviations: M, Meckel’s cartilage; pq, palatoquadrate; ch, ceratohyal; cb, ceratobranchial. Scale bar = 200 μm.