Abstract
rRNA transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies, however the cellular and molecular reasons for this are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. We discovered that Nucleolin is dynamically expressed during embryonic development with high enrichment in the craniofacial tissues. Consistent with this pattern of expression, ncl homozygous mutant (ncl-/-) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observe that ncl-/- mutants exhibit decreased rRNA synthesis and p53-dependent neuroepithelial cell death. In addition, the half-life of fgf8a mRNA is reduced in ncl-/- mutants, which perturbs Fgf signaling, resulting in misregulation of Sox9a mediated chondrogenesis and Runx2 mediated osteogenesis. Exogenous addition of human recombinant FGF8 to the mutant zebrafish significantly rescues the cranioskeletal phenotype, suggesting that Nucleolin regulates osteochondroprogenitor differentiation during craniofacial development by post-transcriptionally regulating Fgf signaling. Our work has therefore uncovered a novel tissue-specific function for Nucleolin in rRNA transcription and growth factor signaling during embryonic craniofacial development.
Introduction
The craniofacial complex consists of the primary sense organs, central and peripheral nervous systems, and musculoskeletal components of the head and neck. Craniofacial development is an intricate process that involves coordinated interaction of all three germ layers and is sensitive to environmental and genetic insults resulting in craniofacial disorders. Despite advances in sequencing, many affected individuals have an unknown genetic diagnosis. Therefore, it is necessary to identify novel genetic factors and understand the cellular and molecular mechanisms that regulate normal craniofacial development, which may also aid in identifying potential therapeutic targets to prevent and/or ameliorate craniofacial diseases.
Ribosome biogenesis is essential for cell growth and survival because ribosome quantity and quality dictate the translation of mRNA into proteins. Transcription of ribosomal DNA (rDNA) by RNA Polymerase (Pol) I in the nucleolus generates a 47S pre-ribosomal RNA (pre-rRNA). The 47S pre-rRNA is then cleaved and processed into 18S, 5.8S and 28S rRNAs. These rRNAs together with Pol III transcribed 5S rRNA associate with ribosomal proteins and accessory proteins to form ribosomes1. The transcription and processing of pre-rRNA requires RNA Pol I together with associated proteins such as UBTF and SL-12,3 as well as rRNA processing proteins including Tcof14, Nol115, Wrd436 and Fibrillarin7. Interestingly, when Pol I subunits or any of the associated factors are disrupted or mutated in zebrafish, xenopus or mice, it results in developmental defects that mostly affect craniofacial cartilage and bone differentiation6,8–12. This raises the question of why disruptions in these ubiquitously expressed genes, which are required in a global process, result in tissue specific craniofacial anomalies. One hypothesis is that neural crest cells (NCC), which are the progenitors of most of the craniofacial bone and cartilage, are more proliferative or metabolically active than non-neural crest cells. In addition, NCCs undergo major cytoskeletal changes during their epithelial-to-mesenchymal transition, which requires high levels of new protein synthesis and hence, more rRNA transcription. Another hypothesis is that RNA Pol I subunit, associated proteins and rRNA processing proteins have other non-ribosome functions, which together with the regulation of rRNA synthesis make the craniofacial skeleton more susceptible to disruption.
Nucleolin is a major nucleolar protein and rRNA processing protein as well as an mRNA and DNA binding protein13,14. Among the top ten highly enriched genes in neural crest cells, Ncl is the only one involved in rRNA transcription15. Therefore, in this study we explore the hypothesis that Nucleolin regulates rRNA transcription as well as craniofacial specific gene expression and function during embryogenesis. We show that Nucleolin is essential for embryo survival and is required for craniofacial bone and cartilage development. Nucleolin regulates rRNA transcriptionally, fgf8a mRNA post-transcriptionally and p53 protein post-translationally. Consistent with this model, we demonstrate that exogenous human recombinant FGF8 can ameliorate the cranioskeletal defects as well as recover rRNA transcription in ncl-/- mutant embryos. Our work, therefore, has uncovered novel tissue-specific functions for Nucleolin in craniofacial development through regulating rRNA transcription and FGF signaling during embryogenesis.
Results
Nucleolin is dynamically expressed during craniofacial development
To understand the function of Nucleolin in vertebrate development, we characterized its expression during zebrafish embryogenesis (Fig. 1). Nucleolin is maternally expressed at 1.5 hour post fertilization (hpf) and remains ubiquitously expressed through gastrulation (3 hpf), early neurulation (12 hpf) and axial segmentation (18 hpf) (Fig. 1A-D). At 24 hpf, the expression of Nucleolin is still ubiquitous, however it is enriched in the eye and midbrain-hindbrain boundary (MHB) (Fig. 1E). In 36 and 72 hpf zebrafish embryos, elevated expression of Nucleolin is observed within the eye, pharyngeal arches and brain (Fig. 1F,G). These expression analyses demonstrate that Nucleolin is dynamically expressed during embryogenesis with enriched expression in craniofacial tissues. Further, this suggests that Nucleolin may be required for proper craniofacial morphogenesis.
Mutations in zebrafish ncl result in craniofacial anomalies
To test our hypothesis that Nucleolin functions during craniofacial development, we characterized the phenotype of the ncl mutant zebrafish line, nclhi2078Tg. nclhi2078Tg was generated by insertional mutagenesis of exon 1, which disrupts ncl transcription16, dramatically reducing the levels of Nucleolin protein between 18 hpf and 24 hpf (Fig 1C’-G’), thereby establishing nclhi2078Tg zebrafish as ncl-/- mutants.
ncl-/- embryos are phenotypically distinguishable from their WT siblings at 24 hpf by the presence of necrosis in the craniofacial region (Fig. 2A-B). At 36hpf, ncl-/- embryos have a beak- like frontonasal prominence and misshapen midbrain-hindbrain boundary (MHB) (Fig. 2C-D’). By 3 days post fertilization (dpf), ncl-/- mutants display craniofacial anomalies such as mandibular hypoplasia in addition to a misshapen MHB (Fig. 2E-F’). 5dpf ncl-/- embryos continue to exhibit craniofacial anomalies and fail to inflate their swim bladders (Fig. S1A-A’), which collectively leads to their lethality between 6-10 dpf (Fig. S1 D-D’).
To further characterize the craniofacial defects in ncl-/- embryos, we performed skeletal staining with Alcian blue and Alizarin red for cartilage and bone, respectively. At 3 dpf, ncl-/- embryos display hypoplastic Meckel’s and ceratohyal cartilages compared to their ncl+/+ siblings. (Fig. S1B-B’). In 5 dpf ncl-/- embryos, the craniofacial cartilages are severely hypoplastic (Fig. 2G-M). In the neurocranium, the trabecula and parasphenoid, which forms the base of the skull17, are smaller in ncl-/- mutants compared to ncl+/+ siblings (Fig. 2I-J). In ncl+/+ larvae, both edge and medial chondrocyte populations are present and are demarcated by differential Alcian blue staining. However, in ncl-/- larvae, cells in the ethmoid plate exhibit uniform Alcian blue staining, suggesting that differentiation of medial chondrocytes in the ethmoid plate may be perturbed. In addition, the trabecula and parasphenoid are smaller in ncl-/- larvae compared to ncl+/+ larvae (Fig. 2K). In the viscerocranium, Meckel’s cartilage is misshapen and the ceratohyal exhibits reverse polarity while the basihyal is missing in ncl-/- larvae. Furthermore, the posterior pharyngeal arch derived ceratobranchials are hypoplastic (Fig. 2L-M) and osteogenesis of the teeth is incomplete in ncl-/- larvae. At 8 dpf and 10 dpf, the polarity of the ceratohyal is similar between ncl+/+ and ncl-/- larvae, suggesting that ncl-/- larvae may have delayed cranioskeletal development. However, Meckel’s cartilage remains misshapened and the ceratobranchials remain hypoplastic in 8 dpf ncl-/- mutant zebrafish (Fig. S1 B-C’ and E-F’). This establishes Nucleolin as being essential for proper cranioskeletal development, and ncl-/- mutant zebrafish as a new model for understanding the etiology and pathogenesis of craniofacial anomalies.
ncl-/- zebrafish have diminished rRNA synthesis
Molecularly, Nucleolin binds to and modifies histones on the promoter of rDNA, and thereby regulates rRNA transcription, which is a rate-limiting step of ribosome biogenesis18. In addition, Nucleolin is a component of the U3 snoRNA, and thus binds to pre-rRNA to process and splice it13,19,20. Therefore, we hypothesized that ncl loss-of-function would lead to diminished rRNA transcription and processing.
To validate our hypothesis, we quantified rRNA transcription by assaying for the intergenic regions of the 47S pre-rRNA - 5’ETS, ITS1 and ITS2 as well as 18S rRNA by qRT-PCR. We observed a significant reduction of all four amplicons in ncl-/- embryos compared to ncl+/+ siblings, suggesting that Nucleolin is necessary for rRNA transcription in zebrafish (Fig. 3A). We then performed RNA immunoprecipitation using a Nucleolin specific antibody and observed that Nucleolin binds to the 5’ETS and ITS1 but not to ITS2 and 18S regions of the 47S pre-RNA (Fig. 3B), consistent with the known function of Nucleolin in vitro18.
Decreased rRNA transcription has previously been shown in vitro to result in increased free ribosomal proteins in cells, which then interact with Mdm2, changing its conformation such that Mdm2 can no longer bind to p53 and ubiquitinate it for degradation. Consequently, p53 accumulates in the cell and results in p53 dependent cell death21. To test if this mechanism holds true in ncl-/- embryos, we first performed qPCR and western blot to assess for p53 expression. We observed that the p53 transcript level is not significantly changed in ncl-/- embryos compared to ncl+/+ siblings between 18 hpf and 36 hpf (Fig. 3C). In contrast, p53 protein levels are significantly upregulated at 24 hpf, however the difference between ncl+/+ and ncl-/- mutants subsides by 36 hpf (Fig. 3E, I). The downstream target of p53, p21 is initially upregulated between 24-30 hpf, but then is considerably downregulated by 36 hpf (Fig. 3C). Nucleolin regulates p53 expression by various mechanisms including increasing p53 mRNA and/or protein stability22,23. We observe that in wildtype zebrafish tissues, Nucleolin does not bind to zebrafish p53 mRNA (Fig. 3D). Instead, Nucleolin binds to p53 protein (Fig. 3F). Prior to examining the binding efficiency of Mdm2 and p53 in control and ncl-/- embryos, we confirmed that Nucleolin is indeed absent in ncl-/- embryos at 28hpf (Fig. 3G). We then performed immunoprecipitation for Mdm2 in control and ncl-/- embryos and immunoblotted for both Mdm2 and p53. We observe decreased pulldown of Mdm2 and p53 in ncl-/- embryos compared to control embryos indicating that the initial accumulation of p53 is a result of reduced interactions between Mdm2 and p53 (Fig. 3H-J). The downregulation of p53 at later stages might be due to a lack of Nucleolin stabilizing p53 protein to increase its half-life.
p53-dependent cell death is increased in ncl mutant embryos
To examine if increased p53 activation and accumulation results in cell death and therefore necrotic craniofacial tissue in the mutants (Fig. 2A), we performed TUNEL staining at 24 hpf and observed a general increase in apoptosis in ncl-/- embryos compared to ncl+/+ controls (Fig. 3K-K’). However, by 36 hpf, TUNEL positive cells were restricted to the MHB in ncl-/- embryos (Fig. 3L-L’), consistent with a reduction in p53 protein levels.
p53 activation can lead to increased apoptosis as well as decreased proliferation that collectively result in tissue hypoplasia. Therefore, we performed immunostaining with the G2/M phase marker phospho-histone H3 (pHH3) and observed no significant change in pHH3+ cells in 24 hpf ncl-/- mutants compared to controls (Fig S2 A-B). Consistent with the decrease in p53 and p21 expression at 36 hpf, the number of pHH3 positive cells is significantly increased in ncl-/- embryos especially at the MHB at 48 hpf (Fig. S2 C,D,G). We also performed EdU staining on ncl+/+ and ncl-/- zebrafish at 48 hpf to label cells in S-phase of the cell cycle and observed that the mutants have a higher number of EdU positive cells (Fig. S2 E,F,H). The increase in proliferation may explain how ncl-/- embryos survive beyond 24 hpf, even though they exhibit higher apoptosis.
We hypothesized that the increased apoptosis in ncl-/- embryos is p53 dependent and that genetically inhibiting p53 levels would suppress cell death and rescue the craniofacial anomalies in ncl-/- embryos. Therefore, we crossed the tp53M214K/M214K allele (hereafter referred to as p53-/-) into the background of ncl-/- mutant zebrafish to generate ncl-/-;p53-/- double mutants. Consistent with our hypothesis, TUNEL staining revealed a reduction in apoptotic cells in 24 hpf ncl-/-;p53-/- embryos compared to ncl-/- embryos (Fig 3L-L’). However, EdU labeling indicates that ncl-/-;p53-/- embryos have increased proliferation at 48 hpf similar to ncl-/- (Fig. S2 I-J), suggesting that the proliferation defects are not p53 dependent.
Removal of both the copies of p53 and knocking down p53 using morpholinos altered jaw morphology in ncl-/- embryos (Fig. S3). Although the ceratohyal remained smaller compared to control siblings, its polarity was restored to normal. The basihyal, which was absent in ncl-/- mutant embryos is formed in ncl-/-;p53-/- embryos. However, chondrogenesis of the ceratobranchials was not improved. In addition, chondrogenesis in the neurocranium was disrupted. ncl-/-; p53-/- mutant zebrafish died around 10-12 dpf due to these cranioskeletal defects and a failure to inflate their swim bladders which is similar to ncl-/- mutant zebrafish. This suggests that while p53 accumulation results in apoptosis during early development, the skeletal defects in ncl-/- mutant zebrafish are not p53 dependent.
NCC development is unaffected in ncl-/- mutant embryos
In vertebrates, NCC differentiate into most of the bones and cartilages of the craniofacial skeleton. To determine if defects in NCC induction and migration underlie the cranioskeletal malformations in ncl-/- mutants, we labeled NCC by crossing sox10:egfp transgenic zebrafish into ncl-/- mutant zebrafish. In addition, we immunostained ncl+/+ and ncl-/- embryos with Zn-8, which labels the endodermal pouches to demarcate individual pharyngeal arches24,25. sox10:egfp labeling of pre- and post-migratory NCC and volumetric rendering of the pharyngeal arches revealed no significant change in the size of the pharyngeal arches in ncl-/- mutant embryos compared to ncl+/+ embryos (Fig. S4 A-B). Comparison of sox10:egfp fluorescence intensity in the pharyngeal arches of ncl+/+ and ncl-/- embryos suggests that NCC induction and migration to the arches are unaffected in the ncl-/- mutant embryos (Fig. S4 C). However, the pineal gland as well as the nasopharyngeal regions of ncl-/- mutants have reduced sox10:egfp expression suggesting that while NCC migration into the arches is unaffected, migration to other cranial regions may be altered. We further validated sox10;gfp transgene expression data by assessing endogenous Sox10 protein expression in ncl+/+ and ncl-/- embryos. The number of Sox10+ cells in the pharyngeal region is similar between 36 hpf ncl+/+ and ncl-/- mutant embryos (Fig. S4 D), confirming that NCC induction and migration into the pharyngeal arches occurs normally in ncl-/- mutants. We observed no alteration in the formation or segregation of the endodermal pouches in 36 hpf ncl-/- mutants as evidenced by a normal pattern of Zn-8 expression (Fig. S4 A). This implied that the osteochondrogenic anomalies in ncl-/- mutant embryos were not the result of abnormal pharyngeal pouch development.
Chondrogenic and Osteogenic defects in ncl-/- mutants
Given the chondrogenic defects observed in ncl-/- mutant embryos, we next tested for altered chondrogenic markers in ncl+/+ and ncl-/- embryos. Sox9a is a transcriptional factor that promotes NCC differentiation into chondrocytes26. sox9a mRNA and its downstream target, col2a1a are reduced in ncl-/- mutant embryos (Fig. 4A). The overall expression of Sox9a protein is also reduced in ncl-/- mutants, but significantly in pharyngeal arches 2-5, which differentiate into the ceratohyal and ceratobranchial cartilages (Fig. 4B). This provides a molecular explanation for the cartilage hypoplasia in ncl-/- mutant embryos.
To determine the basis for the osteogenic defects in ncl-/- mutant embryos, we assessed for altered osteogenic markers in ncl+/+ and ncl-/- embryos. Runx2a and Runx2b are transcription factors that regulate NCC differentiation to bone27. runx2a is upregulated in the ncl-/- mutant embryos, as is runx2b, but runx2b upregulation is not statistically significant (Fig. 4C). We examined the expression of Runx2a and Runx2b in ncl-/- and ncl+/+ embryos using a pan-Runx2 antibody, which revealed that Runx2 protein is upregulated in craniofacial regions especially in the MHB and pharyngeal arches (Fig. 4D). Runx2 overexpression has been previously observed to result in premature osteoblast differentiation28, which leads to a reduced pool of osteoblasts. In agreement with this finding, the expression of Runx2a and Runx2b downstream targets, col1a2 and col10a1 are reduced in ncl-/- embryos compared to ncl+/+ embryos. Furthermore, we observe an increase in early osteoblast markers, spp1 (osteopontin) and sp7 and a concomitant decrease in late osteoblast marker, bglap (osteocalcin) in 36 hpf ncl-/- mutant embryos. To confirm the presence of prematurely differentiated osteoblasts, we stained ncl+/+ and ncl-/- mutant embryos for alkaline phosphatase activity, which is endogenously high in primary osteoblasts29. At 3 dpf (Fig. 4F) and 5 dpf (Fig. S5), alkaline phosphatase staining is significantly diminished in the lower jaw of ncl-/- embryos compared to ncl+/+ embryos. Interestingly, in the neurocranium, osteoblasts in the cranial sutures at both 3 dpf and 5 dpf stain for alkaline phosphatase, which reveals the shape of the sutures in ncl-/- mutant embryos is altered, consistent with the effects of Runx2 mutations in humans30,31.
Fgf8a expression is reduced in ncl-/- mutant embryos
In mice, Fgf8 signaling upregulates chondrogenic genes such as Sox9 and Col2a1 while inhibiting osteogenic genes including Runx232,33. Similarly, Fgf signaling regulates Sox9a in zebrafish34. Interestingly, fgf8a mutant zebrafish have craniofacial defects comparable to ncl-/- embryos, especially in the cartilages of the viscerocranium35, suggesting a potential link between Nucleolin and Fgf signaling during chondrogenesis. Therefore, we examined the expression of Fgf8a in ncl+/+ and ncl-/- embryos and observed a significant general downregulation of Fgf8a in ncl-/- mutant embryos (Fig. 5A). We examined the expression of fgf8a mRNA by qPCR at four stages between 18-36 hpf in ncl-/- mutant embryos and discovered that as the expression of ncl decreases in the mutants, the expression of fgf8a also declines (Fig. 5B). In silico analysis of fgf8a mRNA revealed that the fgf8a 5’UTR contains a Nucleolin consensus binding site – UCCCGA14, leading us to hypothesize that Nucleolin post-transcriptionally controls the expression of fgf8a. We tested for Nucleolin binding to fgf8a mRNA through RNA immunoprecipitation and observed that fgf8a mRNA is pulled down with Nucleolin while that of the housekeeping gene actb is not (Fig. 5C). This suggests that Nucleolin specifically binds to fgf8a mRNA and is required to maintain its stability.
To examine if the phenotype of ncl-/- mutant embryos is a direct consequence of Fgf8a downregulation, we treated ncl+/+ and ncl-/- embryos at 18hpf with human recombinant FGF8, which has 76% identity to the zebrafish Fgf8a protein. Compared to untreated ncl-/- mutant embryos, we observe considerable rescue of the craniofacial cartilage phenotype, which progressively improves with higher concentrations of FGF8 treatment at 60 hpf (Fig. S6 A). The same is true for 5 dpf mutants, with restoration of ceratohyal polarity at 0.25 ug/ul FGF8 (Fig. 5D, E) and rescue of basihyal and ceratobranchial chondrogenesis at 1 ug/ul (Fig. 5D, F). In addition, the length of the trabecula and the parasphenoid is restored in FGF8 treated ncl-/- larvae. However, while osteogenesis of the tooth improves, the mutant embryos are still missing their 4V1 teeth, indicating that FGF8 partially rescues the ncl-/- phenotype. At 8 dpf, the craniofacial cartilages (Fig. S6 C) as well as the putative posterior swim bladder (Fig. S6 B) are rescued in FGF8 treated ncl-/- larvae. The FGF8 treated ncl-/- fry survive at least until 15 dpf, which is 5 days longer than untreated ncl-/- fry and all the craniofacial skeleton elements are rescued (Fig. S6 D-E). However, these fry are smaller than ncl+/+ fry and their anterior swim bladders fail to inflate. Nonetheless, our data indicates that exogenous FGF8 is sufficient to rescue the craniofacial skeleton defects and increase the lifespan of ncl-/- mutants.
FGF8 treatment restores rRNA synthesis in ncl-/- mutant embryos
Fgf8 is expressed in the endoderm-derived epithelium of the pharyngeal arches, while Fgf receptors are expressed in both pharyngeal endoderm as well as osteochondroprogenitors36–38. Therefore, Fgf8a could directly or indirectly regulate chondrogenic and osteogenic differentiation, suggesting that the FGF8 rescue of cranioskeletal defects of ncl-/- embryos could be a result of 1) Fgf signaling through FGF8 interaction with Fgf receptor on osteochondroprogenitors, 2) FGF8 could rescue rRNA transcription, or 3) FGF8 could trigger mesenchymal cell-autonomous pathway such as Bmp signaling.
To test if addition of FGF8 rescues rRNA synthesis, we examined rRNA transcription in FGF8 treated ncl-/- embryos and observe an upregulation in 47S rRNA in FGF8 treated ncl-/- embryos (Fig. 6A). This could result either by FGF8 directly interacting with rDNA or through Fgfr2, which has been shown to positively regulate rRNA transcription39. Consistent with rescued levels of rRNA, the FGF8 treated ncl-/- embryos also have fewer TUNEL positive cells compared to ncl+/+ embryos (Fig. 6B), indicating that FGF mediated rRNA transcription is important for cell survival.
It is well known that Fgf and Bmp signaling interact synergistically during craniofacial development40. More specifically, Fgf8 and Bmp2, respectively regulate osteochondroprogenitor differentiation by positively regulating Sox9 expression41,42. Therefore, we examined the expression of bmp2 in FGF8 untreated and treated ncl-/- embryos at 30 hpf. We observe that while bmp2 expression is downregulated in untreated ncl-/- embryos, its expression is increased in treated ncl+/+ and ncl-/- embryos, suggesting that addition of FGF8 rescues cartilage and bone development by stimulating Bmp signaling in osteochondroprogenitors (Fig. 6C). Taken together, our data suggests that Nucleolin regulates craniofacial development through two different pathways, one by controlling ribosomal RNA transcription and the other by regulating FGF signaling.
Discussion
Nucleolin is the most abundant phosphoprotein in the nucleolus43, however its localization is not limited to the nucleolus. Nucleolin is also observed in the nucleus, cytoplasm and plasma membrane where depending on the cell type and the environmental condition, Nucleolin performs various functions ranging from DNA replication and repair44, chromatin remodeling18, rRNA transcription and processing13,20, mRNA turnover and translation22,45–51 and viral entry and replication52–54. While the cellular and molecular function of Nucleolin has been previously studied in vitro, its role in vertebrate development remains to be explored. Here we show that Nucleolin is critical for zebrafish craniofacial development and specifically for proper differentiation of NCC into cartilage and bone.
Similar to other rRNA modifying proteins such as Nol11, Wrd43 and Fibrillarin whose absence results in chondrogenesis defects in zebrafish and frog6,11,12, it is interesting that perturbation of a global process such as ribosome biogenesis results in craniofacial defects. One possible reason for this tissue specificity could be that genes required for rRNA transcription such as subunits of RNA Pol I, fbl and ncl are highly expressed in NCC and craniofacial tissues, making them more susceptible to disruptions in rRNA transcription9–11,55. rRNA transcription is essential not only because it forms the catalytic core of ribosomes, but also because in its absence cells undergo proteotoxic stress as a result of ribosomal protein accumulation56,57. Free ribosomal proteins bind to Mdm2 resulting in the accumulation of p53, and consequently cell cycle arrest and cell death21,58 (Fig. 7A, B).
Contradicting previous in vitro studies in human cell lines59, we observe that Nucleolin does not bind to p53 mRNA in zebrafish, most likely due to the differences in the 5’ and 3’ UTR sequences of human and zebrafish p53. However, Nucleolin and p53 proteins bind to each other suggesting that Nucleolin is required to stabilize p53 protein23. In the absence of Nucleolin, p53 accumulates temporarily and apoptosis is initiated. However, the effect is not sustained and it does not result in lethality. This is consistent with an initial upregulation of p21 mRNA expression from 18-24 hpf, followed by a steep decrease by 36 hpf, and is indicative of a momentary surge in the transcriptional activity of p53. p21 is a cell cycle inhibitor, which when significantly reduced releases cell cycle arrest60,61, resulting in higher proliferation in ncl-/- embryos compared to ncl+/+ embryos. This increase in proliferation in the absence of Nucleolin is contrary to published literature, which suggests that Nucleolin promotes proliferation in various cell types62,63. This indicates that a different mechanism regulates proliferation in the absence of Nucleolin in mammalian cells and developing zebrafish. Over time, apoptosis decreases, and proliferation increases in ncl-/- embryos, which results in embryo survival until 5 dpf.
The chondrogenic hypoplasia observed in ncl-/- mutants can be linked to Sox9a downregulation, while the osteogenic defects arise due to the higher expression of Runx2. This leads to premature osteoblast differentiation, which reduces the pool of cells that can subsequently differentiate into osteoblasts. Thus, early osteoblast markers such as spp1 and sp7 are more highly expressed in ncl-/- embryos compared to ncl+/+ siblings, while late osteoblast markers such as bglap are downregulated. However, both Sox9a and Runx2 are indirect targets of Nucleolin based on in silico analysis of their 5’ and 3’ UTRs, which do not contain a Nucleolin consensus binding site. Fgf8a, which is also downregulated in ncl-/- mutants and is required for osteochondroprogenitor differentiation, does contain a Nucleolin binding site in its 5’ UTR. The 5’ UTR of an mRNA is responsible for both stability and translation of mRNA64. Given that fgf8a mRNA decreases over time together with reductions in Nucleolin, this suggests that Nucleolin is responsible for stabilizing fgf8a mRNA by binding to its 5’ UTR. In addition, the maternal expression of Nucleolin in zebrafish until 18 hpf possibly prevents early embryonic phenotypes such as agenesis of the cerebellum and MHB organizer which have previously been associated with Fgf8a downregulation65. This is further corroborated by the restoration of osteogenesis and chondrogenesis in ncl-/- mutants with exogenous FGF8, suggesting that the critical time for Fgf8a function in ncl-/- zebrafish is around 18 hpf (Fig. 7C). FGF8 treatment increased the survival of ncl-/- larvae by 5 days, suggesting continued treatment may further augment lifespan and this will be tested in the future.
In mice Fgf8 has been shown to preferentially bind to Fgfr166. However, in zebrafish Fgf receptors are functionally redundant with respect to Fgf8a ligand binding67. Therefore, it is likely that Fgf8a could activate Fgfr2. Fgfr2 is known to bind to the promoter of rDNA and result in histone modification, which activates rRNA transcription similar to Nucleolin39. This suggests that upon FGF8 treatment, either FGF8 or Fgfr2 translocates to the nucleolus and changes the state of the rDNA chromatin from closed to open (Fig. 7C). However, given that the upstream core elements of rDNA that RNA Pol I and UBTF bind to are as yet unannotated in zebrafish, further work is required to test our hypothesis. This involves identifying the upstream core elements using a combination of ATAC-seq and ChIP-seq followed by elucidating the mechanism of Fgf8 and Fgfr2 regulation of rRNA transcription in the absence of Nucleolin.
Fgf8 has been previously shown to regulate variance in facial shape in a dose dependent manner68. Furthermore, rDNA transcription is known to be essential for craniofacial development69,70 and variation71. Therefore, as an upstream regulator of Fgf8, and with a role in rDNA transcription, by extrapolation, Nucleolin may also play an important role in determining facial shape. Overall, our work uncovered that Nucleolin regulates Fgf8 signaling as well as rRNA transcription, making craniofacial development especially susceptible to Nucleolin loss-of-function.
Materials and Methods
Zebrafish
Adult zebrafish (Danio rerio) were housed and maintained in the Stowers Institute Zebrafish Facility according to an IACUC approved protocol (# 2021-124). Zebrafish embryos were raised at 28.5°C and staged using standard procedures72. To prevent pigment development for immunostaining experiments 0.002% 1-Phenyl-2-thiourea was added to the embryo media. nclhi2078Tg zebrafish were obtained through ZIRC and maintained as heterozygotes on the AB/TU background and incrossed to generate homozygous mutant embryos. PCR was used to detect for the presence or absence of the insertional mutation. The ncl wild type allele was detected using the following primers: forward 5’-TTACATGTGGTGAGAAGGCCC-3’ and reverse 5’-AACACCTCCCCTGGGTTTAT-3’. The ncl mutant allele was detected using the following primers: forward 5’-TTACATGTGGTGAGAAGGCCC-3’ and reverse 5’-GCTAGCTTGCCAAACCTACAGGT-3’. The ncl heterozygous mutant lines were crossed with reporter line Tg(7.2kb-sox10:gfp), referred to as sox10:egfp, as well as the tp53M214K line.
Live imaging
Embryos were anaesthetized with MS-222 and mounted in 2% methyl cellulose while submerged in E2 media. Embryos were imaged using a Leica MZ16 microscope equipped with a Nikon DS-Ri1 camera and NIS Elements BR 3.2 imaging software. When appropriate, manual Z stacks were taken and the images were assembled using Helicon Focus software.
Skeletal stain
Alcian blue and Alizarin red staining to label cartilage and bone, respectively, was performed according to Walker and Kimmel72. Embryos were cleared in glycerol and potassium hydroxide and dissected for neurocranium and viscerocranium images. Imaging was performed as described above.
Immunostaining, EdU and TUNEL
Whole-mount immunostaining was performed according to standard protocols73 using primary antibodies against Nucleolin (1:500, abcam #ab22758), GFP (1:500, Life Technologies #A6455), Zn-8 (1:250, DSHB), Sox10 (1:500, GeneTex # GTX128374), Sox9a (1:500, GeneTex # GTX128370), Runx2 (1:500, Abcam # ab23981), Fgf8a (1:500, GeneTex # GTX128126) and pHH3 (1:2000, Millipore # 06-570). Fluorescent secondary antibodies, either Alexa-488 or Alexa-546 (1:500, Invitrogen) were used for detection. TUNEL and EdU Click-IT assay were performed according to the manufacturer’s instructions with slight modifications. Embryos were incubated for 1 hour on ice and 1 hour at 37°C in the reaction buffer. Embryos were imaged using a Zeiss upright 700 confocal microscope and images were captured and processed using Zen software. ImageJ software was used to quantify the fluorescence intensity and area. The Student’s t-test was used to determine statistical significance.
Alkaline Phosphatase staining
Fixed embryos were washed in Tris-Buffer Saline and NTMT buffer (100mM NaCl, 100mM Tris-HCl, pH9.5, 50mM MgCl2, 1% Tween20), followed by incubation with NBT (3.5µl) and BCIP (5µl) in NTMT for the desired color revelation time. The images were collected using a Nikon DS-Ri1 camera.
Western blot
Protein samples consisting of 5 fish/sample were collected at appropriate stages. Embryos were homogenized and suspended in sample buffer containing Tris pH 8.0, Sodium Chloride, SDS, Sodium deoxycholate, NP-40 and protease inhibitor and used for Western blotting according to standard protocols74. Protein quantity was estimated via a BCA assay. Primary antibodies used were γ-tubulin (1:1000, Sigma), α-tubulin (1:10000, Sigma), p53 (1:500, Cell Signaling Technology, #2524S) and Nucleolin (1:500, Abcam #ab22758). Western blots were imaged and quantified using a CLx-Scanner (Li-COR) and Odyssey Software. For quantification, band intensities for p53 were compared to housekeeping control ɣ-Tubulin. The Student’s t-test was performed for statistical analysis.
Immunoprecipitation
Protein lysates from 28 hpf control and ncl-/- zebrafish were used (n = 25 per biological replicate; total three biological replicates) for pre-conjugation with Mdm2 antibody (Cell Signaling Technology, #86934) and IgG with magnetic beads. Because the embryos were group and lysed immediately after collection, genotyping by conventional methods could not be performed. Instead, the lysate was used in a western blot for Nucleolin to substitute as genotyping. Following immunoprecipitation, the samples were used in western blotting and immunoblotted for both Mdm2 and p53.
qPCR
RNA was collected from zebrafish embryos using the Qiagen miRNeasy Micro Kit and tested for quality on an Agilent 2100 Bioanalyzer. The Superscript III kit (Invitrogen) was used to synthesize cDNA for qPCR using random hexamer primers. The following primers were used: ncl forward 5’-ATATCGAGGGCAGGAGTATT-3’ and reverse 5’-GTTTTCGTAGGTCCAGAGTT-3’; tp53 forward 5’-CGAGCCACTGCCATCTATAAG-3’ and reverse 5’-TGCCCTCCACTCTTATCAAATG-3’, p21 forward 5’-GACCAACATCACAGATTTCTAC-3’ and reverse 5’-TGTCAATAACGCTGCTACG-3’, sox9a forward 5’-GGAGCTCAGCAAAACTCTGG-3’ and reverse 5’-AGTCGGGGTGATCTTTCTTG-3’; col2a1 forward 5’-GCGACTTTCACCCCTTAGGA-3’ and reverse 5’-TGCATACTGCTGGCCATCTT-3’; runx2a forward 5’-AACTTTCTGTGCTCGGTGCT-3’ and reverse 5’-AACTTTCTGTGCTCGGTGCT-3’; runx2b forward 5’-CAAACACCCAGACCCTCACT-3’ and reverse 5’-GTATGACCATGGTGGGGAAG-3’; col1a2 forward 5’-CTGGCATGAAGGGACACAG-3’ and reverse 5’-GGGGTTCCATTTGATCCAG-3’; col10a1 forward 5’-CCTGTCTGGCTCATACCACA-3’ and reverse 5’-AAGGCCACCAGGAGAAGAAG-3’; fgf8a forward 5’-GCCGTAGACTAATCCGGACC-3’ and reverse 5’-TTGTTGGCCAGAACTTGCAC-3’; osteocalcin forward 5’-TGAGTGCTGCAGAATCTCCTAA-3’ and reverse 5’-GTCAGGTCTCCAGGTGCAGT-3’; osteopontin forward 5’-TGAAACAGATGAGAAGGAAGAGG-3’ and reverse 5’-GGGTAGCCCAAACTGTCTCC-3’; sp7 forward 5’-GGATACGCCGCTGGGTCTA-3’ and reverse 5’-TCCTGACAATTCGGGCAATC-3’; bmp2 forward 5’-TCCATCACGAAGAAGCCGTGG-3’ and reverse 5’-TGAGAAACTCGTCACTGGGGA-3’; actb forward 5’-TTCCTTCCTGGGTATGGAATC-3’ and reverse 5’-GCACTGTGTTGGCATACAGG-3’; canx forward 5’-ACGATACCGCAGAGAATGGAGACA-3’ and reverse 5’-TCCTGTTTCTGGGAGACCTCCTCA-3’. Previously published rRNA primer sequences were used for qPCR75. Power Sybr (Life Technologies) reaction mix and the ABI 7900HT real time PCR cycler was used measure cDNA amplification. Three biological replicates were run in technical triplicate for each experiment. No template and no reverse-transcriptase samples were run as negative controls.
RNA immunoprecipitation
Lysates from 28hpf wild-type zebrafish were used (n = 100 per biological replicate; total three biological replicates) for pre-conjugation with Nucleolin antibody (Abcam, # ab22758) and IgG with magnetic beads. Immunoprecipitation was performed using manufacturer’s instructions for the EZ-Nuclear RIP Kit (EMD Millipore, #17-10 523), followed by cDNA synthesis and qRT-PCR for 5’ETS, ITS1, ITS2 and 18S rRNA, fgf8a, p53 and actb.
FGF8 treatment
ncl+/+ and ncl-/- embryos were treated with 0.0625, 0.25 and 1 ng/ul human recombinant FGF8 (Thermofisher scientific, # PHG0184) diluted in E2 media by immersion starting at 18hpf. The FGF8 in media was replaced every 24 hours until 5 dpf following which the larvae were added to the tank system and allowed to develop until 15 dpf.
Morpholino knockdown
1-2 cell stage ncl+/+ and ncl-/- embryos were injected with 5–10 ng of morpholino (MO) targeting the p53 AUG start site or a control MO (Gene Tools, LLC). The sequences of the MOs used were as follows: p53 MO 5’-GCGCCATTGCTTTGCAAGAATTG-3’ and control MO 5’-CCTCTTACCTCAGTTACAATTTATA-3’. Following injection, embryos were raised at 28°C to 5 dpf for skeletal staining.
Acknowledgements
We are thankful to all members of the Trainor lab for their input and suggestions during the course of this work. We thank Dr. Tom Schilling for the gift of the sox10:egfp zebrafish, Dr. Tatjana Piotrowski for the gift of the tp53 zebrafish and the SIMR Aquatics staff, especially Carrie Carmichael for zebrafish care and maintenance. We also thank Mark Miller for illustrating Figure 7 and Dr. Cathy McKinney for imaging support. This work was supported by the Stowers Institute for Medical Research (P.A.T) and a postdoctoral fellowship from American Association for Anatomy (S.D). Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1658.