Prickle1 regulates differentiation of frontal bone osteoblasts

Wan, Yong; Lantz, Brandi; Cusack, Brian J.; Szabo-Rogers, Heather L.

doi:10.1038/s41598-018-36742-0

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Published: 21 December 2018

Prickle1 regulates differentiation of frontal bone osteoblasts

Yong Wan¹,
Brandi Lantz¹,
Brian J. Cusack¹ &
…
Heather L. Szabo-Rogers ORCID: orcid.org/0000-0002-7921-8031^1,2,3

Scientific Reports volume 8, Article number: 18021 (2018) Cite this article

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Abstract

Enlarged fontanelles and smaller frontal bones result in a mechanically compromised skull. Both phenotypes could develop from defective migration and differentiation of osteoblasts in the skull bone primordia. The Wnt/Planar cell polarity (Wnt/PCP) signaling pathway regulates cell migration and movement in other tissues and led us to test the role of Prickle1, a core component of the Wnt/PCP pathway, in the skull. For these studies, we used the missense allele of Prickle1 named Prickle1^Beetlejuice (Prickle1^Bj). The Prickle1^Bj/Bj mutants are microcephalic and develop enlarged fontanelles between insufficient frontal bones, while the parietal bones are normal. Prickle1^Bj/Bj mutants have several other craniofacial defects including a midline cleft lip, incompletely penetrant cleft palate, and decreased proximal-distal growth of the head. We observed decreased Wnt/β-catenin and Hedgehog signaling in the frontal bone condensations of the Prickle1^Bj/Bj mutants. Surprisingly, the smaller frontal bones do not result from defects in cell proliferation or death, but rather significantly delayed differentiation and decreased expression of migratory markers in the frontal bone osteoblast precursors. Our data suggests that Prickle1 protein function contributes to both the migration and differentiation of osteoblast precursors in the frontal bone.

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Introduction

The craniofacial complex can be divided into three distinct regions: the cranial base, the skull vault and the face. The cranial base is the floor of the braincase and is composed of the presphenoid, basisphenoid, and basioccipital bones. The skull vault is composed of the frontal, parietal and interparietal bones providing the roof of the braincase. The bones of the cranial base are formed by endochondral ossification, while osteogenesis in the skull vault occurs by intramembranous ossification. Both the skull vault and cranial base have a composite embryological origin. In the mouse, the anterior regions (frontal, ethmoid, presphenoid, basisphenoid bones) are mostly neural crest-derived, while the posterior regions (parietal, basioccipital bones) are mesodermally derived^1,2,3. In contrast, the facial skeleton is composed of intramembranous bones that are neural crest-derived¹.

The development of the frontal bone begins with the migration of neural crest cells from the dorsal edge of the neural tube into the region anterior to the eye^2,3. The cells settle there by E12.5 and express Alkaline phosphatase (ALP), and Runx2. Subsequently, a sub-population of these cells migrate anteriorly to insert into the growing osteogenic front^3,4 Heterozygote Twist1 mouse mutants develop coronal synostosis^5,6. The allelic series of Msx2 and Twist1 mutants suggests that these proteins interact cooperatively for osteoblast proliferation and differentiation in the frontal bone. The most severe phenotypes are observed in the Msx2^−/−; Twist1^+/− compound mutants⁶. Decreased Runx2 protein function causes cleidocranial dysplasia in both humans and mice^{7,8,9,10,11,12}. The causes of craniosynostosis and frontal bone insufficiency could result from early defects in setting up the initial frontal bone condensation, or later problems with the migratory populations of cells. The mesenchymal deletion (Dermo1-cre) of Wntless (Wls) decreases the apical extension of the frontal bone¹³. The extension of the frontal bone relies on both β-catenin and Twist1¹⁴. The combined loss of all Wnt ligands in the Wls mutants and the ability of Twist1 to direct cell movements in the absence of β-catenin imply that both Wnt/PCP and Wnt/β-catenin signaling may be required for apical extension of the frontal bone^13,14.

The Bj mutants have a non-synonymous point mutation in Prickle1 (C161F), a core component of the non-canonical Wnt/planar cell polarity (PCP) pathway^{15,16,17,18,19,20}. Prickle1 is a cytoplasmic protein with a single PET domain, three LIM domains and C-terminal Prickle homologous (PKH) domain. The Bj C161F mutation in Prickle1 occurs in the first cysteine knot of the first LIM domain and is predicted to be deleterious to protein function²⁰. Prickle1 is required for epiblast apical-basal polarity and neuronal morphogenesis in mice^21,22. Mutations in the human Prickle1 are associated with familial epilepsy^23,24. Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome¹⁹. The Bj mutant mice phenotype is consistent with another independent point mutation of Prickle1, C251X, including stunted limbs and cleft palate^20,25,26.

Prickle1^Bj/Bj mutants develop cardiac outflow tract misalignment and cleft palate which both contribute to the perinatal death of the mutant mice²⁰. Prickle1 is widely expressed in the cytoplasm of the epiblast, as well as limb, but little is known about the role of Prickle1 in craniofacial osteogenesis. We hypothesize that the insufficient frontal bones result from the delayed differentiation and migration of osteoblast precursors cells.

Results

Craniofacial defects in the Prickle1 ^Bj/Bj fetuses

We have briefly described the microcephalic phenotype in the Prickle1^Bj/Bj mutants previously²⁰. Here we present a detailed analysis of the skull vault. To begin to understand the underlying craniofacial phenotypes in the Prickle1^Bj/Bj mice, we performed an analysis of the morphology of the bones and cartilages of the head using alcian blue and alizarin red respectively. We noticed that the Prickle1^Bj/Bj skulls are smaller, and the proximal-distal length of the head is reduced with a concomitant increase in the medial-lateral width of the skull (Fig. 1b,c,f,g). The Prickle1^Bj/Bj viscerocranium, including the nasal, premaxillary, and maxillary bones, develops normal morphology, but these features are compressed proximally-distally (Fig. 1e,f,g). In the skull vault, we found that the interfrontal suture is enlarged in the Prickle1^Bj/Bj mutants while the sagittal suture is unaffected (Fig. 1g). We also observe that the mesodermally-derived skull is unaffected, including parietal, occipital and basioccipital bones (Fig. 1f,g). We measured the proximal-distal length of the frontal, parietal, and nasal bones and normalized them to the length of the skull vault. We found a statistically significant decrease in the contribution of the nasal bone to the total length of the skull vault. In contrast, the contribution of the frontal bone to the total length is increased, while the proportion of the parietal bone is unchanged in the Prickle1^Bj/Bj mutants (n = 3; Fig. 1h). Taken together, these results suggest that Prickle1 protein function is required for all stages of frontal bone development.

In addition to the microcephalic phenotype type (Figs 1e and 2c,d), we also observed several other craniofacial anomalies in the Bj mutants. All late stage fetuses developed a median cleft lip (Fig. 2c, n = 31/31). We observed a cleft palate between the palatine processes of the maxillary bones (Fig. 2d). The cleft palate is partially penetrant with 52% (n = 16/31) of Prickle1^Bj/Bj affected. As expected, with the cleft palate phenotype, the maxillary bones failed to meet in the midline (Fig. 2d). In agreement with Yang et. al.²⁵, the cleft extends through the entire proximal-distal axis of the secondary palate (Fig. 2d).

The frontal bone osteoblasts express Prickle1 and show decreased apical mineralization in Prickle1 ^Bj/Bj

We have chosen to focus our analysis on the function of Prickle1 in the developing skull vault and examined the tissue distribution of Prickle1 protein in the skull vault of wildtype embryos. At E12.5 we found that Prickle1 protein is expressed in a subpopulation of frontal bone osteoblasts (Fig. 3a,b). Later, Prickle1 protein is found in the frontal and parietal osteoblasts at E15.5 (Fig. 3c–f). We tested whether the frontal bones had a more severe phenotype because they differentially express Prickle1 mRNA, however using qPCR, we observed that the frontal and parietal bones express similar level of Prickle1 mRNA (Fig. 3g).

We confirmed the wholemount skeletal findings by performing a histological analysis of the Prickle1^Bj/Bj and their littermate controls. We sectioned the samples coronally and stained them with Von Kossa to determine the mineralization status in these animals. We found that the craniofacial bones have begun mineralization by E15.5, which continues through E18.5 as evidenced by von Kossa staining (Fig. 4d–f). At E15.5, and E18.5, we found that the Prickle1^Bj/Bj frontal bones did not extend as far apically as their littermate controls (Fig. 4d–f). As expected from the alizarin stained specimens, we observed that the interfrontal suture is expanded mediolaterally with the loss of anterior-posterior growth of the sutural blastema (Fig. 4e, arrows, and bracket). In the nasal septum, the posterior regions of the vomer flare laterally away from each other (Fig. 4f).

Proliferation and apoptosis are unaffected while differentiation is delayed in Bj mutants

Frontal bone insufficiency can result from defects in proliferation and cell death²⁷. We undertook these studies at E12.5 because this is a critical stage for setting up the frontal bone condensation. Using H&E staining we observed the frontal bone condensation in wildtype and Bj frontal bone primordia (Fig. 5a,f). We found very few apoptotic (TUNEL-positive) cells in the frontal bone primordia in either genotype (Fig. 5b,g). We looked at the number of cells in S-phase using a BrdU pulse-chase labeling experiment. We counted the number of BrdU-positive cells and total number of nuclei, and found no difference in the ratio of proliferating cells between the Bj mutants and their littermate controls (Fig. 5c,h,e). We next tested the number of actively dividing cells using phospho-histone H3 immunohistochemistry. We found no differences in the number of dividing cells in the mutant frontal bone compared with the wild-type littermates (Fig. 5d,i,j).

Since we observed no changes in cell death or proliferation in the frontal bones, we determined whether osteogenic differentiation was occurring correctly. In the frontal bones, both Alkaline phosphatase (ALP) and Osterix (Osx) are expressed in pre-osteoblasts and osteoblasts. Runx2 expression is an early marker of osteoblast commitment in the skull, while ALP is a marker of more mature osteoblasts. At E12.5 we observed that Runx2 is expressed in the frontal bone primordium of both the Prickle1^Bj/Bj and littermate controls (Fig. 6a,d). However, the domain of expression in the Prickle1^Bj/Bj was much smaller, but the level of expression of Runx2 was similar in both genotypes (Fig. 6a,d). The expression domains of ALP and Osterix were significantly smaller in size and signal intensity at E12.5 (Fig. 6e,f). At E15.5 we observed that the expression of both Runx2, ALP and Osx was decreased in the ectocranial layer of the mutant frontal bones (Supp Fig. 1) compared with the wild-type littermate. From this analysis we conclude that that intramembranous ossification is delayed in frontal bone.

HH and Wnt signaling is decreased in the Prickle1 ^Bj/Bj frontal bones

To determine if there is a defect in signaling that is leading to the delay in frontal bone osteogenesis, we sought to determine the level of canonical Wnt signaling and Hedgehog signaling in the frontal bones in the context of the Prickle1^Bj missense allele. As Prickle1 is a negative regulator of Wnt/β-catenin signaling pathway²⁸, we tested the level of active β-catenin in the frontal bone primordium and observed decreased active β-catenin in Prickle1^Bj/Bj mutant frontal bone primordia (Fig. 7a,e). In addition, we determined that downstream Wnt/β-catenin signaling is decreased as evidenced by the reduced Lef1 expression in the Prickle1^Bj/Bj frontal bone (Fig. 7b,f).

The Prickle1^Bj/Bj animals showed defects in primary and motile cilia²⁰ and several ciliopathy mutants have defects in frontal bone formation^29,30. These defects suggest that the level of Hedgehog signaling could be defective in the Prickle1^Bj/Bj animals. In addition, HH signaling is required for the cranial bone development^31,32,33. We tested the level of Hedgehog signaling via the expression of transcriptional targets of the pathway, Ptch1 and Gli1. We found that the expression of both Ptch1 and Gli1 is decreased in the Prickle1^Bj/Bj mutants at E12.5 (Fig. 7g,h).

Osteoblast migration is reduced in the Prickle1 ^Bj/Bj frontal bones

The frontal bone develops in two parts: the body of the frontal bone differentiates in situ and mineralizes anterior to the eye, followed by apical extension towards the dorsal part of the skull by growth of the osteogenic front and insertion of migratory osteoblasts^3,4,34. Prickle1 protein is a core component in the Wnt/PCP pathway. One of the functions of the Wnt/PCP pathway is required for cell movement and intercalation. We wanted to test whether the Prickle1^Bj/Bj frontal bones were smaller because the Prickle1 mutation decreases the migration potential of these cells. We performed whole mount in situ hybridization to markers of migratory populations of frontal bone cells Twist1, Msx1, Msx2, and Engrailed1 (En1). We found decreased expression of Twist1, Msx1, Msx2, and En1 in the frontal bone region by wholemount in situ staining (Supp Fig. 2a–d). We further confirmed this finding in coronal sections of E12.5 animals (Fig. 8). Indeed, we find that the expression of Twist1, Msx1, Msx2 is decreased in the Prickle1^Bj/Bj frontal bone (Fig. 8e–g). The expression of En1 is similar between genotypes in the frontal bone condensation, but is reduced in the dermis between the condensation and the surface ectoderm (Fig. 8h). These data support the hypothesis that the early apical migration of cranial mesenchyme is reduced in the mutant. During later stages, when the osteoblasts of the frontal bone are migrating to insert into the osteogenic front⁴, the expression of these three markers were examined in sagittal section of E15.5 wild type and mutant littermates. Our results show that the expression level of En1, Twist1, Msx1 and Msx2 in both the frontal bone and osteogenic front are reduced in the mutant (Supp Fig. 2i–l). These data suggest that Prickle1 protein function is necessary for mediating cell migration of osteoblasts precursors during all stages of skull vault development.

Discussion

We found that the Prickle1^Bj mutation results in defects in two processes in frontal bone development: delayed osteoblast differentiation and reduced migration in the frontal bone. The frontal bone defects that we observe are within the phenotypic spectrum for Cleidocranial Dysplasia (CCD). In humans, 70 percent of CCD cases result from mutations in RUNX2 and have a triad of phenotypes including enlarged fontanelles, hypoplastic clavicles and supernumerary teeth. In toto, our Prickle1^Bj/Bj mutants look similar to the published allelic series that identified dose dependant and threshold requirements of Runx2 in the development of the phenotypic spectrum of CCD^7,8. Across the five different genotypes that express variable amounts of Runx2, the most consistent phenotype is the enlarged fontanelle and develops in concert with decreased Osterix expression^7,8. In the Prickle1^Bj/Bj mice, we found both decreased Runx2 and Osx expression in the frontal bone primordia, leading to smaller frontal bones and an enlarged fontanelle. These data suggest that Wnt/PCP signaling may be responsible for aspects of transcriptional control of Runx2 expression. The link between mutations in Prickle1, Wnt/PCP signaling and the development of phenotypic variants in CCD patients warrants further exploration.

Bones develop through two different processes – intramembranous and endochondral ossification^35,36,37. In the neural crest skeleton, the skull vault undergoes intramembranous ossification^1,38,39. In mice, frontal bone development starts with mesenchymal condensations cranial to the eye at E12.5 and osteoblast differentiation begins at E13.5³⁶. In Beetlejuice mutants, the frontal bone is hypoplastic. Runx2, Osterix and ALP expression domains are decreased at E12.5 and E15.5 with no observed changes in apoptosis or proliferation. We hypothesize that the domains are smaller because either fewer neural crest cells are recruited into the primordia or because the signaling environment surrounding the frontal bone primordia (including the dermis, dura and brain) inhibits the recruitment or maturation of the osteoblasts into the primordia. We are currently determining if the smaller frontal bones result from autonomous or non-autonomous functions of Prickle1.

The Prickle1^Bj/Bj skulls develop smaller but normally shaped frontal bones without affecting the parietal bones. We found that most of the proximal-distal shortening is found in the nasal bone, while the parietal bone is unaffected. Intriguingly, the Prickle1^Bj/Bj frontal bone contributes to a greater proportion of the proximal-distal length of the neurocranium. Molecularly, we observe decreased Wnt/β-catenin in the Bj mutant frontal bone primordium at E12.5. In agreement with previous reports regarding differences between the frontal and parietal bones^27,40, we observe a defect only in the Prickle1^Bj/Bj frontal bone. Our data provides more support to the hypothesis that the parietal bone is not sensitive to changes in Wnt/β-catenin signaling and therefore develops normally in Prickle1^Bj/Bj. We also observe decreased HH signaling in the Bj mutant frontal bone primordium at E12.5. The Prickle1^Bj/Bj frontal bone phenotype is consistent with the frontal bone phenotype observed in the Ihh^-/- mice^31,33. Together the published phenotypes and our data suggest that both Wnt/β-catenin and HH signaling contribute to the migratory behavior of the frontal bones osteoblasts. We currently favor the model where the cells within the Prickle1^Bj/Bj mutant frontal bone primordia have prematurely sensed the completion of migration through Prickle1 function. As a result, the osteoblasts decrease both Wnt and HH signaling, thus preventing additional migration.

En1, Twist1 and Msx1, and Msx2 are expressed during embryonic craniofacial bone development and are required for early migration and survival of cranial neural crest mesenchyme^{6,41,42,43,44,45}. We found that Twist1, Msx1, and Msx2 expression was reduced in the frontal bone primordia at E12.5 and E15.5 (Fig. 8, Supp Fig. 2). These results show that Prickle1 is upstream of Twist1 and Msx2 to control apical expansion and migration of frontal bone osteoblasts. This suggests that there are subpopulations of neural crest cells that respond differently to the same signals–one population that is contributing to the suture and a second population that drives apical expansion of the frontal bone. However, from our analysis, we are unable to distinguish between whether the Prickle1^Bj/Bj osteoblasts have decreased migratory ability or whether there is a reduced population of apical migratory osteoblasts in the Prickle1^Bj/Bj frontal bone. Clarifying the identity, specific molecular markers and spatiotemporal location of these populations will be critical for understanding calvarial patterning and growth.

In this work, we have shown that the Prickle1^Bj mice are a new model for understanding the etiology of microcephaly²⁰. The genetic, molecular and physical mechanisms underlying the development of microcephaly involves a combination of decreased growth of the craniofacial region and reduced expansion of the brain. Currently, few animal models exist to determine how growth patterns of the face and skull contribute to the development of microcephaly. We are currently undertaking work to determine how the migration of cells and expansion of each compartment (the brain, skull vault and cranial base) contributes to the development of microcephaly. We further hypothesize that decreased growth in all regions combined together contributes to the development of the microcephaly.

Methods

Mutant lines and animal husbandry

We obtained the founder animals from Dr. Cecilia W. Y. Lo (University of Pittsburgh) and maintained the Beetlejuice mutant line outbred on C57/BL6J background²⁰. We genotyped the Beetlejuice line using a custom SNP assay (Invitrogen), Taqman Genotyping MasterMix, and read the results on a StepOnePlus machine. For embryo collection, we performed timed matings, and the day of the plug was designated as E0.5. For BrdU labeling, we intraperitoneally injected 10 mg/kg BrdU in sterile PBS one-hour prior to collection. The embryos were collected via C-section after CO2 euthanasia of the pregnant dam on the appropriate day and embryo staging was confirmed by morphology. Animal care and use described here was approved and complies with the guidelines of the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Skeletal preparation and histological analyses

After collection, embryos, and P0 fetuses were fixed in 4% paraformaldehyde at 4 °C overnight and embedded in paraffin using standard protocols. For histological analysis, 8-μm-thick sagittal and coronal sections were cut and placed on TESPA-coated Superfrost Plus slides. hematoxylin and eosin, Safranin O and Von Kossa staining were conducted using standard protocols. For wholemount staining of the skeleton, alcian blue and alizarin red staining, embryos of different stage as well as postnatal day (P0) mice were first fixed at 95% ethanol at room temperature overnight, and stained according to standard protocols²⁰.

Immunohistochemistry

Immunohistochemistry and immunofluorescence

Sections were blocked with 5% normal goat serum followed by primary antibodies incubation at 4 °C overnight. Antigen retrieval was conducted in sodium citrate buffer at 95 °C for 20 minutes. The primary antibodies used were as follows: Prickle1 (ab15577, Abcam) and active β-catenin (05–665, Millipore). We used the Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (A11001, Invitrogen). For immunohistochemistry with DAB detection, we used the primary antibodies above and the Vectastain ABC systems (Vector Labs) amplification system and ImmPACT DAB (Vector Labs) for detection.

Proliferation studies

BrdU samples underwent pre-treatment with Proteinase K, Exonuclease III and Dpn1. BrdU was detected using the RPN202 primary antibody (GE Healthcare Life Sciences) and Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody. The samples were mounted with Prolong-Gold with DAPI (Invitrogen). The BrdU-positive cells and all cells (DAPI-stained) were counted in the frontal bone at E12.5 by two observers. The phospho-histone H3 (PHH3; #9701, CST) positive cells were detected using DAB. We compared the number of PHH3 positive cells and the ratio of BrdU-positive cells per frontal bone using a paired t-test (p < 0.05). Cells were counted in at least one section from three individual littermate pairs of wild-type and Prickle1^Bj/Bj mice.

In situ hybridization

RNA in situ hybridization of both whole mount and section were performed according to standard protocols and detected with BM Purple²⁷. The probes used were as follows: Runx2, ALP, Osterix, En1, Twist1, Msx1, Msx2, Ptch1, Gli1²⁷. Each experiment was conducted in three different wild-type and mutant mice.

Real-time PCR

RNA was extracted from frontal bone as well as parietal bone using the Trizol reagent (Invitrogen) according to standard procedures. Real-time PCR was performed on StepOnePlus Real-Time PCR System (Applied Biosystems) using a SYBR Green Kit (Roche). We performed ΔΔ-CT calculations with GAPDH as the control gene. Error bars represent the range for the expression for the seven samples and we compared expression using an unpaired T-test. The primers for Prickle1 are Forward: 5-TGCTCAGGAGATCCAAGTCC-3, Reverse: 5-CTCTCTTCAAAGTGATACGC-3.

Microscopy, Imaging, and Measurement

Histological images were collected on a Zeiss Axioskop A1 microscope using a MrC3 camera and the Zen imaging software. Flourescent immunoflourescence images were captured on an Olympus Flou-View 100 confocal microscope. The images were compiled in Adobe Photoshop. Wholemount images were captured on a Leica M165FC dissecting microscope using a DFC450 camera with the Leica LAS software. We measured the proximal-distal length of the nasal, frontal and parietal bone as well as the total length of the skull vault using Leica Application Suite software (LAS v4.4). We used GraphPad Prism 5 to perform the Student’s t-test (p < 0.05).

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

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Acknowledgements

The authors are grateful for the support by P30-NIH-DE020740 and start-up funds from the University of Pittsburgh School of Dental Medicine (HLSR). We acknowledge the Center for Biological Imaging at the University of Pittsburgh for the use of the Olympus Flouview 100 Microscope.

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Authors and Affiliations

Center for Craniofacial Regeneration, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Yong Wan, Brandi Lantz, Brian J. Cusack & Heather L. Szabo-Rogers
Department of Developmental Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Heather L. Szabo-Rogers
McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Heather L. Szabo-Rogers

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Yong Wan
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Brandi Lantz
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Brian J. Cusack
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Heather L. Szabo-Rogers
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Contributions

Y.W. & B. L. performed the phenotype and histological analysis and wrote the paper. B.J.C., B.L. and Y.W. performed mouse husbandry. H.L.S.R. conceived and managed the project, analyzed data and wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Heather L. Szabo-Rogers.

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Wan, Y., Lantz, B., Cusack, B. et al. Prickle1 regulates differentiation of frontal bone osteoblasts. Sci Rep 8, 18021 (2018). https://doi.org/10.1038/s41598-018-36742-0

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Received: 27 October 2017
Accepted: 27 November 2018
Published: 21 December 2018
DOI: https://doi.org/10.1038/s41598-018-36742-0

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