Tcof1兾Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities Jill Dixon*†, Natalie C. Jones†‡, Lisa L. Sandell‡, Sachintha M. Jayasinghe‡, Jennifer Crane‡, Jean-Philippe Rey‡, Michael J. Dixon*§¶, and Paul A. Trainor‡¶ *School of Dentistry and §Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom; and ‡Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110

Neural crest cells are a migratory cell population that give rise to the majority of the cartilage, bone, connective tissue, and sensory ganglia in the head. Abnormalities in the formation, proliferation, migration, and differentiation phases of the neural crest cell life cycle can lead to craniofacial malformations, which constitute one-third of all congenital birth defects. Treacher Collins syndrome (TCS) is characterized by hypoplasia of the facial bones, cleft palate, and middle and external ear defects. Although TCS results from autosomal dominant mutations of the gene TCOF1, the mechanistic origins of the abnormalities observed in this condition are unknown, and the function of Treacle, the protein encoded by TCOF1, remains poorly understood. To investigate the developmental basis of TCS we generated a mouse model through germ-line mutation of Tcof1. Haploinsufficiency of Tcof1 leads to a deficiency in migrating neural crest cells, which results in severe craniofacial malformations. We demonstrate that Tcof1兾Treacle is required cell-autonomously for the formation and proliferation of neural crest cells. Tcof1兾Treacle regulates proliferation by controlling the production of mature ribosomes. Therefore, Tcof1兾Treacle is a unique spatiotemporal regulator of ribosome biogenesis, a deficiency that disrupts neural crest cell formation and proliferation, causing the hypoplasia characteristic of TCS craniofacial anomalies. craniofacial development 兩 embryo 兩 mouse 兩 Treacher Collins syndrome

N

eural crest cells are a multipotent, migratory cell population that generate a diverse array of cell types during vertebrate development. These include bones, tendons, neurons, glia, melanocytes, and connective, endocrine, and adipose tissue. With a limited capacity for self-renewal and a wide range of differentiation fates, neural crest cells bear many of the hallmarks of stem cells (1). Neural crest cells are born at the interface between the surface ectoderm and the dorsal margin of the neural plate, a region termed the neural plate border (2, 3). This induction process requires a precise threshold concentration gradient of bone morphogenetic protein (BMP) signaling in the neural plate border, which is determined by contact-mediated interactions across the neural plate–surface ectoderm interface (4–7). The delamination of neural crest cells involves an epithelial-to-mesenchymal transformation, which is driven by the repression of cell adhesion molecules such as E-cadherin by Snail1 (6). In the head, cranial neural crest cells exhibit a unique ability to differentiate into cartilage and bone, underpinning their fundamental importance to vertebrate craniofacial evolution (7). Defects in neural crest cell formation, proliferation, migration, and兾or differentiation are considered responsible for craniofacial abnormalities, which constitute up to one-third of all congenital birth defects (8). Therefore, it is critical to understand the mechanisms regulating each phase of neural crest cell development to comprehend the precise etiology of specific craniofacial anomalies, and currently there is a dearth of knowledge describing the regulation of neural crest cell proliferation.

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0603730103

Treacher Collins syndrome (TCS) (Online Mendelian Inheritance in Man database accession no. 154500) is one example of a congenital craniofacial disorder, this condition being characterized by numerous anomalies that are restricted primarily to the head and neck. The phenotype of TCS includes hypoplasia of the facial bones, particularly the zygomatic complex and mandible, cleft palate, and middle and external ear defects that result in conductive deafness. TCS is caused by autosomal dominant mutations in the TCOF1 gene; however, the in vivo functions of TCOF1 and the nucleolar protein Treacle that it encodes (9) remain poorly understood. A number of hypotheses have been proposed to account for the origins of TCS craniofacial malformations. These include abnormal neural crest cell migration (10), improper cellular differentiation (11), and extracellular matrix abnormalities (12). However, to date there have been no molecular or cellular data to support any of these hypotheses. Consequently, we investigated the function of Tcof1兾Treacle in neural crest cell development and craniofacial morphogenesis. Through germ-line mutation of Tcof1 we generated a mouse model of TCS, and we demonstrate that Treacle is a novel spatiotemporal regulator of ribosome biogenesis that is cell-autonomously required for neural crest cell generation and proliferation. Results Tcof1 Is Expressed in a Dynamic Spatiotemporal Pattern. Individuals

affected by TCS exhibit distinctive craniofacial anomalies, and we hypothesized that these abnormalities result from defects in neural crest cell patterning. As a first step to determining the mechanistic basis of TCS and any role for Tcof1 in neural crest cells, we characterized in detail the spatiotemporal pattern of Tcof1 expression during early embryogenesis (Fig. 6, which is published as supporting information on the PNAS web site). In whole embryos we observed strong, spatiotemporally specific expression of Tcof1 in the neuroepithelium at embryonic day (E) 8.5 (Fig. 7A) and in the frontonasal and branchial arch mesenchyme at E9.5 (Fig. 7D). Section in situ hybridization for Tcof1 demonstrated the specificity of staining throughout the neuroepithelium at E8.5, which corresponds with the generation of neural crest cells. Interestingly, staining was most intense at the lateral edge of the neuroepithelium, which correlates with cells in the G1兾S phases of the cell cycle. Strong Tcof1 expression was also observed in migrating neural crest cells in the craniofacial mesenchyme but was noticeably absent in Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office. Freely available online through the PNAS open access option. Abbreviations: En, embryonic day n; BMP, bone morphogenic protein; TCS, Treacher Collins syndrome. †J.D.

and N.C.J. contributed equally to this work.

¶To

whom correspondence may be addressed. E-mail: [email protected] or mike. [email protected].

© 2006 by The National Academy of Sciences of the USA

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Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved July 10, 2006 (received for review May 16, 2006)

Fig. 1. Analysis of DBA ⫻ C57BL兾6 Tcof1⫹兾⫺ embryos. Comparison of wildtype (A) and mutant (B) newborn pups. Tcof1⫹/⫺ mutant mice exhibit shortened and domed-shaped heads with frontonasal dysplasia (arrow). Skeletal staining of E17.5 wild-type (C, E, G, and I) and Tcof1⫹/⫺ (D, F, H, and J) embryos reveals hypoplasia of numerous craniofacial bones including the nasal (n), frontal (f), premaxillary (pmx), maxillary (mx), mandibular (md), and temporal bones (t) together with cleft palate (arrow) in mutant embryos. In comparisons of wild-type (K) and mutant (L) embryo skeletal preparations, cranioskeletal hypoplasia is evident at E15.5 in the frontal, premaxillary, and maxillary elements. Transverse histological sections of E14.5 wild-type (M) and mutant (N) embryos highlight the absence of complete midline fusion (arrowhead) and the lack of a nasal septum (ns) and conchae (c) in Tcof1⫹/⫺ embryos.

the more medially located cranial mesoderm (Fig. 7 B and C). At E9.5 Tcof1 was strongly expressed throughout the neural tube and could also be observed in craniofacial tissues heavily populated with neural crest cells such as the frontonasal, maxillary, and mandibular mesenchyme and the sensory ganglia (Fig. 7 E and F). By E10.5 the levels of Tcof1 expression had significantly weakened, becoming very diffuse, and by E11.5–E12.5 Tcof1 expression was largely undetectable. This demonstration of the dynamically regulated spatiotemporal pattern of expression of Tcof1 during neural crest cell formation and migration provided evidence of a tangible link between neural crest cells and TCS craniofacial abnormalities. Tcof1 Haploinsufficient Embryos Display Severe Cranioskeletal Defects. To investigate the role of Tcof1 in neural crest cell patterning,

our laboratory generated chimeric mice carrying a germ-line mutation in one allele of the murine orthologue of TCOF1. Breeding of male chimeras with female C57BL兾6 mice generated heterozygous offspring that exhibited a number of features reminiscent of the human disorder, including abnormalities of the maxilla and mandible. However, additional anomalies not observed in TCS patients, including severe developmental delay, anophthalmia, and exencephaly, were also found (13). The extreme nature of this phenotype, which resulted in premature neonatal death, precluded breeding and expansion of the mutant line and furthermore prevented a detailed analysis of neural crest cell patterning defects in these animals. To circumvent this lethality we crossed the Tcof1 mutation onto several different backgrounds and determined that, on the DBA genetic background, Tcof1 heterozygosity was compatible with postnatal life, enabling congenic lines to be generated (14). Although we maintained the Tcof1⫹/⫺ line on a pure DBA background, we outcrossed the Tcof1⫹/⫺ DBA line to C57BL兾6 to generate embryos with the characteristic features of TCS. Importantly, although the embryos analyzed were of mixed DBA ⫻ C57BL兾6 background, the characteristic mutant phenotype was consistently reproducible with minimal interembryo variability at each developmental stage. This permitted a detailed investigation of the function of Tcof1 and its relationship to neural crest cell patterning. Offspring of congenic DBA Tcof1 heterozygous mice intercrossed with wild-type C57BL兾6 mice exhibit a distinctive pheno13404 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0603730103

type that mimics TCS. Confirmed Tcof1⫹/⫺ neonates were characterized by a reduction in the size of the head, which was domed in appearance and shortened in the anteroposterior direction with obvious severe frontonasal dysplasia (Fig. 1 A and B). Neonates displayed gasping behavior and abdominal distension and died within 24 h of birth. Skeletal analyses at E17.5 highlighted the severity of the cranioskeletal abnormalities associated with heterozygosity of Tcof1. The cranial vault was domed and the nasal and frontal bones were dysmorphic and hypoplastic in Tcof1⫹/⫺ mutants (Fig. 1 C–F). Ventral views of the viscero- and chondrocranium revealed that the premaxilla, maxillary, and palatine bones were also abnormally shaped and truncated (Fig. 1 E and F). A midline cleft was visible between the displaced palatal shelves of the maxilla and palatine bones, allowing partial visualization of the presphenoid skull bone elements (arrow in Fig. 1F). Mandibles isolated from the same embryos were also shorter in mutant as compared with wild-type embryos (Fig. 1 G and H). In addition, the temporal bone was hypoplastic and dysmorphic in mutant embryos (Fig. 1 I and J). These cranioskeletal abnormalities in Tcof1⫹/⫺ mutants were evident as early as E15.5 in the form of frontonasal, premaxillary, and maxillary hypoplasia (Fig. 1 K and L). Transverse histological sections through the midfacial region of E14.5 Tcof1⫹/⫺ embryos confirmed the presence of cleft palate by demonstrating that the palatal shelves had failed to fuse completely (Fig. 1 M and N). Furthermore, the nasal passages were poorly formed, with no evidence of a nasal septum or conchae. Collectively, these data indicated that Tcof1⫹/⫺ neonates died from respiratory arrest due to malformations of the nasal, premaxilla, maxilla, and palatine skeletal elements. These defects are characteristic of the midfacial abnormalities associated with severe cases of TCS in humans. Interestingly, the cranioskeletal structures that are malformed in Tcof1⫹/⫺ embryos are derived primarily from neural crest cells. These combined observations strongly suggested that the craniofacial anomalies associated with TCS arise specifically as the result of defects in neural crest cell patterning. Tcof1 Haploinsufficient Embryos Exhibit Neural Crest Cell Hypoplasia.

To establish whether a migration defect was responsible for the craniofacial defects characteristic of TCS as originally proposed Dixon et al.

(10), we performed neural crest cell lineage tracing in combination with whole-embryo culture of wild-type and Tcof1⫹/⫺ mouse embryos (Fig. 2). Regions of the anterior hindbrain, midbrain, and forebrain were focally labeled with the lipophilic dye DiI at E8.25 to coincide with the emergence of the earliest waves of neural crest cells. Fluorescence analysis indicated that numerous DiI-labeled rhombomere 2- and rhombomere 4-derived neural crest cells migrated ventrolaterally in discrete streams into the first and second branchial arches, respectively, in wild-type embryos (Fig. 2 A, B, E, and F). An identical pattern of migration from the hindbrain to the branchial arches was observed in Tcof1⫹/⫺ mutant embryos (Fig. 2 C, D, G, and H). Similarly, no migratory or path-finding differences were observed in midbrain and forebrain neural crest cell migration to the maxillary and frontonasal regions of the face in comparisons between wild-type and Tcof1⫹/⫺ embryos (Fig. 3 A–D). Hence, the segmental migration of cranial neural crest cells is normal and unaffected in Tcof1⫹/⫺ embryos, which clearly demonstrates that aberrant neural crest cell migration is not the underlying cause of TCS craniofacial abnormalities. Therefore, Tcof1 does not play a role in neural crest cell migration. Interestingly, despite the absence of a migration defect, we did reproducibly observe fewer migrating neural crest cells in the Tcof1⫹/⫺ embryos compared with their wild-type littermates at each axial level examined in our lineage-tracing experiments (Fig. 2, compare B and F with D and H). The reduction in the number of migrating cranial neural crest cells was further supported by in situ hybridization staining for the neural crest marker Sox10, in which the staining was visibly more sparse in E8–E9 Tcof1⫹/⫺ embryos compared with wild-type littermates (Fig. 3 A–D). Consequently, by E10.5 defects in cranial neural crest cell-derived structures were clearly evident. Although the cranial sensory ganglia formed in the correct axial locations, they were significantly smaller and more disorganized in Tcof1⫹/⫺ embryos compared with wild-type littermates (Fig. 3 E and F). This, however, was a very subjective view, and it was critical in establishing the mechanistic basis of TCS to be able to consistently and reproducibly quantify the degree of neural crest cell reduction. This quantification was achieved by flow cytometric analyses of GFP-labeled neural crest cells. E9 embryos with GFP-labeled neural crest cells were obtained through intercrossing DBA Tcof1⫹/⫺ and homozygous C57BL兾6 Pax3GFP mice (Fig. 3G). Reproducible flow cytometry enumeration of GFP-positive neural crest cells demonstrated that, as a proportion of the total number of cells in the craniofacial mesenDixon et al.

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Fig. 2. Neural crest cells at the level of rhombomere 2 (r2) and rhombomere 4 (r4) in wild-type (A, B, E, and F) and Tcof1⫹/⫺ (C, D, G, and H) embryos were labeled with DiI (red) at E8.25 and cultured for 24 h. Fewer migrating neural crest cells from r2 and r4 populating the first branchial arch (ba1) (B and D) and second branchial arch (ba2) (F and H), respectively, in E9.5 Tcof1⫹/⫺ (D and H) embryos were observed.

Fig. 3. Neural crest cell quantification in Tcof1⫹兾⫺ embryos. Sox10 in situ hybridization in E8.5 (A and B), E9.5 (C and D), and E10.5 (E and F) wild-type (A, C, and E) and Tcof1⫹/⫺ (B, D, and F) embryos reveals the deficiency in cranial neural crest cell contribution to the frontonasal prominence (fnp), first and second branchial arches (ba1 and ba2; arrows in A and B), cranial ganglia (cg; arrows in C and D) such as the trigeminal ganglion (t in E), and to the hypoglossopharyngeal and vagal ganglia posterior to the otic vesicle (double arrows in E and F). (G) E9.0 wild-type embryo obtained by intercrossing C57BL兾6 Pax3GFP and DBA Tcof1⫹/⫺ mice to quantify the number of migrating cranial neural crest cells in flow cytometry analyses. Neural crest cells (GFP-labeled) contribute to the frontonasal process (fnp), the mesenchyme surrounding the optic placode (op), and the maxillary (mx) and mandibular (md) prominences of the first branchial arch (ba1), as well as the more caudal branchial arches. fb, forebrain; mb, midbrain; hb, hindbrain. (H) Histogram demonstrating a 22.3% reduction in the relative proportion of GFP-labeled migrating neural crest cells to cranial mesenchyme cells in Tcof1⫹/⫺ mutant (green bar) as compared with wild-type littermate (yellow bar) and positive control (red bar) embryos. The blue bar represents a negative control for GFP in wild-type DBA ⫻ C57BL兾6 embryos.

chyme, there were ⬇22% fewer migrating cranial neural crest cells in Tcof1⫹/⫺ embryos compared with wild-type littermates (Fig. 3H and Table 1, which is published as supporting information on the PNAS web site). Taken together with the dynamic expression pattern exhibited by Tcof1, this finding suggested that Treacle was required for neural crest cell generation, proliferation, and兾or viability. Loss of Tcof1 therefore results in a neural crest cell deficit, which causes hypoplasia of the cranial sensory ganglia and skeletal elements. Neural Crest Cell Hypoplasia Is a Direct Consequence of Neuroepithelial-Specific Apoptosis. Collectively, our analyses indicated that there

was a significant deficiency in the number of migrating cranial neural crest cells in Tcof1⫹/⫺ mutant embryos, which underlies the PNAS 兩 September 5, 2006 兩 vol. 103 兩 no. 36 兩 13405

Fig. 4. Neuroepithelial-specific apoptosis in Tcof1⫹兾⫺ embryos. TUNEL staining for apoptosis (green) in E8.25 wild-type (A) and Tcof1⫹/⫺ (B) embryos and in a cultured Tcof1⫹/⫺ embryo (C) in which forebrain-derived (fb), midbrain-derived (mb), and hindbrain-derived (hb) neural crest cells were labeled with DiI (red). Double-stained cells (yellow) in the neural plate (np) and not in migrating neural crest cells (red) highlight the specificity of apoptosis in the neuroepithelium of Tcof1⫹/⫺ embryos. Caspase3 immunohistochemistry of parasagittal cranial sections from E8.75 wild-type (D) and Tcof1⫹/⫺ (E) embryos revealed enhanced caspase3 activity in the neuroepithelium (arrows) of Tcof1⫹/⫺ mutants.

neurogenic and cranioskeletal hypoplasia observed in TCS individuals. This deficiency could arise through compromised viability of migrating neural crest cells or anomalies in neural crest cell formation. To discriminate between these mechanisms we initially assayed for apoptosis using TUNEL staining in E8–E9.5 embryos, which corresponded with the formation and migration phases of neural crest cell development. We detected a surprisingly high level of apoptosis in Tcof1⫹/⫺ embryos relative to their wild-type littermates, which was largely confined to the neural plate (Fig. 4 A and B). These observations suggested that Treacle was critically required for neuroepithelial survival and neural crest cell formation but was not essential for neural crest cell viability. To confirm the viability of migrating cranial neural crest cells, we broadly labeled the midbrain and anterior hindbrain of E8–E8.5 mutant embryos with DiI (red fluorescence) and cultured the embryos for at least 6 h. This time period was sufficient for the newly formed cranial neural crest cells to migrate to the periphery of the head. These labeled embryos were then assayed for apoptosis by TUNEL (green fluorescence). The absence of double-labeled (yellow fluorescence) migrating neural crest cells conclusively demonstrated that migrating neural crest cells in Tcof1⫹/⫺ embryos were viable (Fig. 4C). In contrast, colabeled DiI- and TUNEL-positive cells highlighted the spatiotemporal restriction of cell death to the neuroepithelium. The elevated levels of apoptosis were mediated by caspase3 as evidenced by enhanced activated capsase3 immunostaining in the neuroepithelium of mutant embryos (Fig. 4 D and E and data not shown). Consequently, during neural crest cell induction the neural plate in Tcof1⫹/⫺ embryos is significantly thinner than in wild-type littermates (Fig. 4 D and E). Thus, haploinsufficiency of Tcof1 leads to the apoptotic depletion of neural stem cells and migrating neural crest cells, which strongly argues that Treacle is critically required for neural crest cell formation. To further refine the mechanistic origins of TCS craniofacial abnormalities and the role of Tcof1, it was important to investigate where Tcof1 functions in the neural crest cell formation process. Because much of the information for neural crest cell patterning is derived from the neural tube and the hindbrain in particular, we first assayed for the presence of any anteroposterior patterning or segmentation defects in the neural tube during early embryogenesis. Otx2, which is strongly expressed in the forebrain and midbrain neuroepithelium of wild-type embryos, was unaltered in Tcof1⫹/⫺ embryos (Fig. 7 A and B, which is published as supporting information on the PNAS web site). Similarly, Msx2 strongly labeled the dorsal folds of the neural plate in both wild-type and Tcof1⫹/⫺ 13406 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0603730103

Fig. 5. Cellular analysis of Tcof1⫹兾⫺ embryos. (A–D) BrdU incorporation (pink in A and B) and Y10B immunostaining (red in C and D) of transverse sections of E8.75 wild-type (A and C) and Tcof1⫹/⫺ (B and D) embryos. A reduction in cell proliferation (arrows) in the neuroepithelium (ne) and cranial mesenchyme (cm) corresponds with diminished production of mature 28S rRNA (arrows in D) in Tcof1⫹/⫺ embryos. No proliferation anomalies were observed in the endoderm (arrowheads in A and B) or surface ectoderm (se). (E–H) TUNEL-stained embryos after homotopic hindbrain neural plate (np) transplantations of DiI-labeled (red) wildtype neuroepithelium in cultured E8.25 wild-type (E) and Tcof1⫹/⫺ (F) embryos. Transplanted cells exhibit minimal apoptosis and generate migrating neural crest cells. (G and H) E8.75–E9.0 TUNEL-stained embryos after homotopic transplantations of DiI-labeled Tcof1⫹/⫺ hindbrain neuroepithelium in cultured E8.25 wildtype (G) and Tcof1⫹/⫺ (H) embryos. Transplanted cells exhibit significant apoptosis (arrows in G and H).

embryos (Fig. 7 C and D). Furthermore, using genetic markers such as Krox20, which distinguishes rhombomeres 3 and 5 in the hindbrain, we could discern no segmental patterning defects in the neural tubes of Tcof1⫹/⫺ embryos (Fig. 7 E and F). Therefore, there were no obvious anteroposterior or segmentation defects underlying the neuroepithelial apoptosis and corresponding reduction in the number of cranial neural crest cells. Hence, we focused our analyses on the morphogenetic process of neural crest cell induction and assayed for BMP and WNT signaling owing to their well characterized roles in neural crest cell development (15, 16). Bmp2 is normally strongly expressed in the surface ectoderm immediately adjacent to the neuroepithelium, and no differences in spatiotemporal expression or intensity were observed in comparisons between wild-type and mutant embryos (Fig. 7 G and H). Similarly, no differences were observed between wild-type and mutant embryos for Wnt1 activity, which is strongly expressed throughout the dorsal neuroepithelium during the induction phase of neural crest cell development (Fig. 7 I and J). Therefore, the conserved morphogenetic signals involved in vertebrate neural crest cell induction remain unaltered in Tcof1⫹/⫺ embryos, suggesting that the defect in neural crest cell formation lies downstream of BMP兾WNT signaling. In contrast, we observed a significant reduction in Snail1 expression in mutant compared with wild-type embryos, which was consistent with diminished epithelial-to-mesenchymal transformation and generation of neural crest cells (Fig. 7 K and L). These data indicate that the function of Tcof1 lies upstream of Snail1 in the generation of neural crest cells. Therefore, although the morphogenetic machinery for neural crest stem cell induction is spatiotemporally intact in Tcof1⫹/⫺ embryos, haploinsufficiency of Tcof1 results in elevated neuroepithelial apoptosis and a diminished capacity to generate neural crest stem cells, leading directly to hypoplasia of migrating cranial neural crest cells and also of their derived craniofacial structures. Deficient Production of Mature Ribosomes Causes Apoptosis and Reduced Proliferation. As a corollary to the elevated levels of

apoptosis observed specifically in the neuroepithelium of Tcof1⫹/⫺ embryos, it was important to assay for alterations to cell proliferation in the neuroepithelium and neural crest. We therefore Dixon et al.

Treacle Functions Cell-Autonomously. Our data suggest a model in

which haploinsufficiency of Tcof1 leads to insufficient mature ribosome biogenesis specifically in neuroepithelial cells and neural crest cells. As a direct result, the proliferative capacity of neuroepithelial and neural crest cells is compromised. The spatiotemporal correlation between these defects and the domain of activity of Tcof1 predicts that Treacle functions in a cell-autonomous manner. Therefore, to test our prediction we homotopically transplanted small focal regions of DiI-labeled midbrain and hindbrain reciprocally between E8.5 wild-type and Tcof1⫹/⫺ mutant embryos (Fig. 5 E and F). The viability of the grafted cells was then assayed by TUNEL staining after 6- to 8-h periods of in vitro whole-embryo culture. Wild-type DiI-labeled midbrain or rhombomere 2 cells transplanted homotopically into wild-type isochronic host embryos incorporated into the neural plate and generated neural crest cells while exhibiting minimal apoptosis as evidenced by the absence of TUNEL staining (Fig. 5E). Similarly, wild-type midbrain or rhombomere 2 cells were also viable when grafted homotopically into Tcof1⫹/⫺ isochronic host embryos (Fig. 5F). Transplanted wild-type cells incorporated into the neural plate and generated neural crest cells despite being surrounded by a sea of endogenous apoptosis within the host Tcof1⫹/⫺ neural plate. These observations imply that Treacle functions in a cell-autonomous manner. We confirmed the cell-autonomous function of Treacle by transplanting Tcof1⫹/⫺ midbrain or rhombomere 2 cells homotopically into wild-type and Tcof1⫹/⫺ isochronic host embryos (Fig. 5). Tcof1⫹/⫺ neuroepithelial cells incorporated into the neural plate of wild-type and Tcof1⫹/⫺ host embryos, and in both cases a significant proportion of the transplanted cells exhibited elevated levels of apoptosis. In the wild-type host environment a focal region of apoptosis was observed coinciding with the DiI-labeled transplanted Tcof1⫹/⫺ cells (Fig. 5G). In the Tcof1⫹/⫺ mutant host environment, despite high levels of endogenous apoptosis throughDixon et al.

out the neural plate, extensive DiI and TUNEL costaining was observed at the axial level of transplantation (Fig. 5H). Therefore, Treacle plays key cell-autonomous roles in neural crest cell generation and proliferation through its critical role in regulating the production of mature ribosomes that are integral to these processes. Discussion Haploinsufficiency of Tcof1 in mice causes major craniofacial malformations, including hypoplasia of the frontonasal and maxillary regions, cleft palate, and mandibular hypoplasia. On a mixed DBA兾C57 background Tcof1⫹/⫺ embryos reproducibly recapitulate the midfacial anomalies characteristic of severe cases of TCS in humans, and using this mouse model we have uncovered its mechanistic etiology and pathogenesis. Our results clearly reveal that the cranioskeletal hypoplasia that is characteristic of TCS craniofacial abnormalities arises as a direct result of a deficiency in the number of cranial neural crest cells, which occurs because of defects in neural crest cell formation and proliferation. Tcof1兾 Treacle elicits its proliferation function cell-autonomously through dynamically regulating the spatiotemporal production of mature ribosomes in neuroepithelial and neural crest cells. In support of a link between Tcof1 and ribosome biogenesis, biochemical evidence has recently demonstrated that Treacle binds upstream binding factor, which, together with promoter selectivity factor SL1, forms a complex that is important for the activity of RNA polymerase I (RNA pol I in Fig. 8, which is published as supporting information on the PNAS web site) (21). Interestingly, we observed a reduction in Ubf activity in Tcof1⫹/⫺ embryos compared with wild-type littermates (data not shown), suggesting that Treacle may regulate proliferation directly through Ubf. Because this is a key component and rate-limiting step of ribosome biogenesis (22), it strongly supports a cell proliferation function for Tcof1兾Treacle. Furthermore, down-regulation of Treacle by using specific short interfering RNA in HeLa cell culture assays resulted in the inhibition of ribosomal DNA transcription and cell proliferation (21). These in vitro cell culture data correlate well with our in vivo observations in Tcof1⫹/⫺ embryos and validate a role for Treacle in driving the formation and proliferation of neural crest stem cells through the regulation of ribosome biogenesis. Interestingly, it has been estimated that in proliferating cells up to 95% of all transcription is dedicated to ribosome biogenesis (23). The revelation that Treacle is a critical regulator of neural crest cell formation and proliferation and that genetically it functions between BMP兾WNT and Snail1 is significant because BMP, WNT, and Snail have all been shown to regulate the cell cycle during neural crest cell induction. The formation and delamination of neural crest cells depend on the successful transition from G1 to S phase, and blocking BMP, WNT, or Snail1 signaling disrupts this transition, resulting in the inhibition of neural crest cell formation and delamination (17, 24) This finding argues in favor of a functional role for Treacle and the generation of mature ribosomes in the control of cell-cycle progression between BMP兾WNT and Snail1. In support of this idea, a recent study in frog oocytes demonstrated that Treacle methylates pre-RNA during G1 (25). Thus, in the absence of Treacle, insufficient ribosome biogenesis restricts cell-cycle progression, causing reduced proliferation, cellcycle arrest, and apoptosis. In summary, TCS is a rare congenital birth defect caused by mutations in TCOF1 and whose characteristic craniofacial abnormalities arise uniquely as a consequence of a specific spatiotemporal disruption of ribosome biogenesis in the neural plate and neural crest cells. The lack of mature ribosomes compromises the ability of neuroepithelial cells to proliferate and leads to neuroepithelial apoptosis. Furthermore, this anomaly results in a deficiency in the number of migrating cranial neural crest cells, the effect of which is compounded by their reduced proliferative capacity. These effects are caused cell-autonomously through haploinsufficiency of Tcof1, which highlights the essential novel roles played by Treacle PNAS 兩 September 5, 2006 兩 vol. 103 兩 no. 36 兩 13407

DEVELOPMENTAL BIOLOGY

analyzed proliferation in cultured E8.5–E9.0 embryos that were pulsed with BrdU. Analyses of BrdU-labeled embryos clearly demonstrated a significant reduction in neuroepithelial cell proliferation in Tcof1⫹/⫺ embryos compared with wild-type littermates (Fig. 5 A and B). Although this finding was not surprising considering the high level of apoptosis observed in the neural plate, we also discovered that there was reduced proliferation in the neural crest-derived cranial mesenchyme. It is important to note, however, that there may be decreased proliferation in the paraxial mesoderm, which also contributes to the cranial mesenchyme. In contrast, proliferation in the cranial endoderm was not affected, which suggests that the proliferation anomaly was specific to the neuroepithelium and neural crest in Tcof1⫹/⫺ embryos (Fig. 5 A and B). These observations demonstrate that Treacle is dynamically required for cell proliferation in the neuroepithelium and also in the neural crest, which correlates with the specific expression pattern of Tcof1 during embryogenesis. Consistent with the nucleolar localization of Treacle, the phosphoprotein encoded by Tcof1 (17), we hypothesized that Treacle may regulate neural crest cell proliferation by playing key roles in ribosome biogenesis. The final stage of ribosome maturation occurs as ribosomal subunits such as 28S rRNA are processed from rRNA precursors and transferred to the cytoplasm. Hence, we analyzed wild-type and mutant embryos by immunohistochemistry with the monoclonal anti-rRNA antibody Y10B (18, 19), which serves as a marker of mature ribosomal integrity by specifically labeling the 28S subunit of rRNA (18, 20) (Fig. 5C). We observed significantly reduced Y10B immunoreactivity in the neuroepithelium and neural crest-derived craniofacial mesenchyme of E8.75–E9 Tcof1⫹/⫺ embryos as compared with wild-type littermates (Fig. 5 C and D). In contrast, no changes were observed in the surface ectoderm or endoderm. These data suggest that Treacle plays a critical spatiotemporally specific role in regulating the production of mature ribosomes in the neuroepithelium and neural crest.

in the formation and proliferation of neural crest cells through its involvement in generating mature ribosomes and possibly promoting cell-cycle progression. The early mechanistic onset of TCS abnormalities during the formation and proliferation phases of neural crest cell development highlights the difficulty in detecting and treating the problem in humans. However, our observations potentially open up avenues for rescuing TCS abnormalities by enhancing cell-cycle progression and inhibiting neuroepithelial apoptosis. Materials and Methods Genotyping of Mouse Embryos. Congenic DBA Tcof1 heterozygotes

and wild-type C57BL兾6 mice were bred to generate F1 progeny on a mixed DBA ⫻ C57BL兾6 background. Mutant embryos were obtained by timed matings, the morning of the vaginal plug being considered E0.5. Genotyping was performed by using previously described methods (13).

events before enumerating the percentage of cells that were expressing GFP. This percentage represents the number of migrating neural crest cells as a proportion of the total number of cells in the craniofacial mesenchyme in individual wild-type (n ⫽ 7) and Tcof1 heterozygous mutant (n ⫽ 4) embryos. Analyses of Cell Death. E7.5–E10.5 embryos were assessed for

neuroepithelial cell death by TUNEL staining by using the FITC Cell Death Detection Kit (Roche, Palo Alto, CA) according to the manufacturer’s instructions. E8–E9.5 embryos were also assessed for neuroepithelial cell death by wax section immunostaining with an anti-caspase3 antibody (R & D Systems, Minneapolis, MN). Briefly, embryos were fixed in 4% paraformaldehyde and paraffinprocessed routinely. Embryos were sectioned sagittally at 10–12 ␮m, deparaffinized, and incubated with a 1:50 dilution of caspase3. Sections were counterstained with hematoxylin. Analysis of Proliferation. E8.5 embryos were cultured in medium

Skeletal, Histological, and Expression Analyses. Skeletal staining,

histological analysis, and whole-mount and section in situ hybridization were performed as described previously (13, 26, 27). Isolation, Culture, Labeling, and Transplantation of Embryos. E8.25–

E8.75 embryos were dissected, cultured, and labeled, and tissue transplants were all performed as previously described (28, 29). A minimum of 10 wild-type and Tcof1 heterozygous embryos were used in each of the labeling and transplantation experiments described. Flow Cytometry and Neural Crest Cell Quantification. DBA Tcof1 heterozygotes were bred with C57BL兾6 Pax3GFP homozygous mice. Pax3GFP is a phenotypically normal targeted knockin that labels the neuroepithelium and migrating cranial neural crest cells with GFP. E9 embryos were harvested, and, after a brief incubation of the cranial region in 1 mg兾ml dispase at room temperature, tungsten needles were used to completely remove the cranial neuroepithelium. The remaining cranial tissue was incubated briefly in 0.25% trypsin with EDTA at 37°C and gently triturated to generate a single-cell suspension. Single-cell suspensions were resuspended in PBS with 2% FBS and incubated with 2 ␮g兾ml 7-aminoactinmycin D for 5 min on ice to discriminate live from dead cells. The 488-nm laser-excited fluorescence was measured by using a CyAn flow cytometer (DakoCytomation, Fort Collins, CO). The GFP log signal was detected by using FL1 (530兾40 nm) PMT, and the 7-aminoactinmycin D log signal was evaluated by using FL4 (680兾30 nm) PMT. The data were analyzed by using FlowJo software (Tree Star, Ashland, OR) with a gating strategy that removed nonnucleated and 7-aminoactinmycin D positively stained 1. Trainor, P., Bronner-Fraser, M. & Krumlauf, R. (2004) in Handbook of Stem Cells: Embryonic Stem Cells, eds. Lanza, G., Weissman, I., Thomson, J., Pedersen, R., Hogan, B., Gearhart, J., Blau, H., Melton, D., Moore, M., Verfaillie, C., et al. (Academic, Boston), pp. 219–232. 2. Selleck, M. A. & Bronner-Fraser, M. (1995) Development (Cambridge, U.K.) 121, 525–538. 3. Bronner-Fraser, M. & Fraser, S. (1989) Neuron 3, 755–766. 4. Moury, J. D. & Jacobson, A. G. (1990) Dev. Biol. 141, 243–253. 5. Rollhauser-ter Horst, J. (1977) Anat. Embryol. 151, 309–316. 6. Cano, A., Perez-Moreno, M. A., Rodrigo, I., Locascio, A., Blanco, M. J., del Barrio, M. G., Portillo, F. & Nieto, M. A. (2000) Nat. Cell Biol. 2, 76–83. 7. Gans, C. & Northcutt, R. (1983) Science 220, 268–274. 8. Jones, N. C. & Trainor, P. A. (2004) Expert Opin. Biol. Ther. 4, 645–657. 9. Treacher Collins Syndrome Collaborative Group (1996) Nat. Genet. 12, 130–136. 10. Poswillo, D. (1975) Br. J. Oral Surg. 13, 1–26. 11. Wiley, M. J., Cauwenbergs, P. & Taylor, I. M. (1983) Acta Anat. (Basel) 116, 180–192. 12. Herring, S. W., Rowlatt, U. F. & Pruzansky, S. (1979) Am. J. Med. Genet. 3, 225–229. 13. Dixon, J., Brakebusch, C., Fassler, R. & Dixon, M. J. (2000) Hum. Mol. Genet. 9, 1473–1480. 14. Dixon, J. & Dixon, M. J. (2004) Dev. Dyn. 229, 907–914. 15. Kanzler, B., Foreman, R. K., Labosky, P. A. & Mallo, M. (2000) Development (Cambridge, U.K.) 127, 1095–1104. 16. Ittner, L. M., Wurdak, H., Schwerdtfeger, K., Kunz, T., Ille, F., Leveen, P., Hjalt, T. A., Suter, U., Karlsson, S., Hafezi, F., et al. (2005) J. Biol. 4, 11.

13408 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0603730103

containing 10 ␮g兾␮l BrdU (Roche) for ⬇2 h. For immunohistochemistry studies embryos were fixed in 4% paraformaldehyde, paraffin-processed, and sectioned at 10–12 ␮m in transverse planes. BrdU incorporation was revealed by using a standard antigenretrieval method with citrate buffer (pH 6.0), and the slides were subsequently incubated with a 1:50 dilution of mouse anti-BrdU (Amersham Pharmacia Biosciences, Piscataway, NJ). Detection of BrdU signal was accomplished by using Alexa Fluor-conjugated anti-mouse antibody (Molecular Probes, Carlsbad, CA) and then counterstained with DAPI by using established protocols. Analysis of rRNA Integrity. Deparaffinized transverse sections

(10–12 ␮m) prepared from E8.5 embryos were processed for immunohistochemical staining with Y10B, a mouse monoclonal antibody to rRNA (19). To determine the extent of rRNA degradation, the slides were incubated with a 1:500 dilution of supernatant from the Y10B hybridoma and subsequently a 1:200 dilution of biotinylated horse anti-mouse antibody (Vector Laboratories, Burlingame, CA) as described previously (19). We thank Dr. Robb Krumlauf and the M.J.D. and P.A.T. groups for comments on the manuscript. We greatly appreciate the technical assistance of Sharon Beckham, Teri Johnson, and Mike Morgan. We acknowledge Dr. Rubel (University of Washington, Seattle, WA) for generously providing the Y10B antibody. Research in the P.A.T. laboratory is supported by March of Dimes Research Grant 6FY05-82, National Institute of Dental and Craniofacial Research Grant R01 DE 016082-01, the Hudson Foundation, and the Stowers Institute for Medical Research. Research in the M.J.D. laboratory is supported by National Institutes of Health Grant P50 DE 016215 and Medical Research Council (U.K.) Grant G81兾535. 17. Marsh, K. L., Dixon, J. & Dixon, M. J. (1998) Hum. Mol. Genet. 7, 1795–1800. 18. Lerner, E. A., Lerner, M. R., Janeway, C. A., Jr., & Steitz, J. A. (1981) Proc. Natl. Acad. Sci. USA 78, 2737–2741. 19. Garden, G. A., Canady, K. S., Lurie, D. I., Bothwell, M. & Rubel, E. W. (1994) J. Neurosci. 14, 1994–2008. 20. Garden, G. A., Hartlage-Rubsamen, M., Rubel, E. W. & Bothwell, M. A. (1995) Mol. Cell. Neurosci. 6, 293–310. 21. Valdez, B. C., Henning, D., So, R. B., Dixon, J. & Dixon, M. J. (2004) Proc. Natl. Acad. Sci. USA 101, 10709–10714. 22. Poortinga, G., Hannan, K. M., Snelling, H., Walkley, C. R., Jenkins, A., Sharkey, K., Wall, M., Brandenburger, Y., Palatsides, M. & Pearson, R. B., et al. (2004) EMBO J. 23, 3325–3335. 23. Martin, D. E., Soulard, A. & Hall, M. N. (2004) Cell 119, 969–979. 24. Burstyn-Cohen, T., Stanleigh, J., Sela-Donenfeld, D. & Kalcheim, C. (2004) Development (Cambridge, U.K.) 131, 5327–5339. 25. Gonzales, B., Henning, D., So, R. B., Dixon, J., Dixon, M. J. & Valdez, B. C. (2005) Hum. Mol. Genet. 14, 2035–2043. 26. Kaufman, M. H. (1992) The Atlas of Mouse Development (Academic, London). 27. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. R. (2003) Manipulating the Mouse Embryo (Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY). 28. Trainor, P. A. & Tam, P. P. L. (1995) Development (Cambridge, U.K.) 121, 2569 –2582. 29. Trainor, P. & Krumlauf, R. (2000) Nat. Cell Biol. 2, 96–102.

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