Vertebrate genome evolution and the zebrafish gene map.

In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.

Dental tissue regeneration - a mini-review.

BACKGROUND: with today's 21st century technological advancements, it is expected that individuals will either retain their natural teeth or obtain functional tooth replacements throughout their entire life. Modern dental therapies for the replacement of missing teeth largely utilize partial or complete dentures and titanium implants capped with prosthetic crowns. Although these prostheses serve a purpose, they are not equivalent, neither in function nor aesthetics, to natural teeth. Recent progress in dental tissue engineering has lent significant credibility to the concept that biological replacement teeth therapies may soon be available to replace missing teeth. OBJECTIVE: in this review, we summarize the emerging concepts of whole-tooth replacement strategies, using postnatal dental stem cells (DSCs) and dental tissue engineering approaches. METHODS: we provide a thorough and extensive review of the literature. RESULTS: current approaches to achieve clinically relevant biological replacement tooth therapies rely on the cultivation of DSCs capable of relaying odontogenic induction signals, through dental epithelial-mesenchymal cell interactions. DSC expansion and differentiation can be achieved by programming progenitor stem cells to adopt dental lineages, using instructive, bioengineered scaffold materials. Periodontal ligament regeneration in particular has demonstrated significant progress recently, despite the somewhat unpredictable clinical outcomes, with regard to its capacity to augment conventional metallic dental implants and as an important component for whole-tooth tissue engineering. Following recent advances made in DSC and tissue engineering research, various research groups are in the midst of performing 'proof of principle' experiments for whole-tooth regeneration, with associated functional periodontal tissues. This mini-review focuses on recent and promising developments in the fields of pulp and periodontal tissue DSCs that are of particular relevance for dental tissue and whole-tooth regeneration. CONCLUSION: continued advances in the derivation of useable DSC populations and optimally designed scaffold materials unequivocally support the feasibility of dental tissue and whole-tooth tissue engineering.

Localization of protamine 1 mRNA in different stages of the cycle of the rat seminiferous epithelium.

A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.

Tooth Bioengineering and Regenerative Dentistry.

Over the past 100 y, tremendous progress has been made in the fields of dental tissue engineering and regenerative dental medicine, collectively known as translational dentistry. Translational dentistry has benefited from the more mature field of tissue engineering and regenerative medicine (TERM), established on the belief that biocompatible scaffolds, cells, and growth factors could be used to create functional, living replacement tissues and organs. TERM, created and pioneered by an interdisciplinary group of clinicians, biomedical engineers, and basic research scientists, works to create bioengineered replacement tissues that provide at least enough function for patients to survive until donor organs are available and, at best, fully functional replacement organs. Ultimately, the goal of both TERM and regenerative dentistry is to bring new and more effective therapies to the clinic to treat those in need. Very recently, the National Institutes of Health/National Institute of Dental and Craniofacial Research invested $24 million over a 3-y period to create dental oral and craniofacial translational resource centers to facilitate the development of more effective therapies to treat edentulism and other dental-related diseases over the next decade. This exciting era in regenerative dentistry, particularly for whole-tooth tissue engineering, builds on many key successes over the past 100 y that have contributed toward our current knowledge and understanding of signaling pathways directing natural tooth and dental tissue development-the foundation for current strategies to engineer functional, living replacement dental tissues and whole teeth. Here we use a historical perspective to present key findings and pivotal advances made in the field of translational dentistry over the past 100 y. We will first describe how this process has evolved over the past 100 y and then hypothesize on what to expect over the next century.

Bioengineered dental tissues grown in the rat jaw.

Our long-term objective is to develop methods to form, in the jaw, bioengineered replacement teeth that exhibit physical properties and functions similar to those of natural teeth. Our results show that cultured rat tooth bud cells, seeded onto biodegradable scaffolds, implanted into the jaws of adult rat hosts and grown for 12 weeks, formed small, organized, bioengineered tooth crowns, containing dentin, enamel, pulp, and periodontal ligament tissues, similar to identical cell-seeded scaffolds implanted and grown in the omentum. Radiographic, histological, and immunohistochemical analyses showed that bioengineered teeth consisted of organized dentin, enamel, and pulp tissues. This study advances practical applications for dental tissue engineering by demonstrating that bioengineered tooth tissues can be regenerated at the site of previously lost teeth, and supports the use of tissue engineering strategies in humans, to regenerate previously lost and/or missing teeth. The results presented in this report support the feasibility of bioengineered replacement tooth formation in the jaw.

Bioengineered Tooth Buds Exhibit Features of Natural Tooth Buds.

Tooth loss is a significant health issue currently affecting millions of people worldwide. Artificial dental implants, the current gold standard tooth replacement therapy, do not exhibit many properties of natural teeth and can be associated with complications leading to implant failure. Here we propose bioengineered tooth buds as a superior alternative tooth replacement therapy. We describe improved methods to create highly cellularized bioengineered tooth bud constructs that formed hallmark features that resemble natural tooth buds such as the dental epithelial stem cell niche, enamel knot signaling centers, transient amplifying cells, and mineralized dental tissue formation. These constructs were composed of postnatal dental cells encapsulated within a hydrogel material that were implanted subcutaneously into immunocompromised rats. To our knowledge, this is the first report describing the use of postnatal dental cells to create bioengineered tooth buds that exhibit evidence of these features of natural tooth development. We propose future bioengineered tooth buds as a promising, clinically relevant tooth replacement therapy.

Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain.

Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

Bioengineered post-natal recombinant tooth bud models.

The long-term goal of this study is to devise reliable methods to regenerate full-sized and fully functional biological teeth in humans. In this study, three-dimensional (3D) tissue engineering methods were used to characterize intact postnatal dental tissue recombinant constructs, and dental cell suspension recombinant constructs, as models for bioengineered tooth development. In contrast to studies using mouse embryonic dental tissues and cells, here the odontogenic potential of intact dental tissues and dental cell suspensions harvested from post natal porcine teeth and human third molar wisdom tooth dental pulp were examined. The recombinant 3D tooth constructs were cultured in osteogenic media in vitro for 1 week before subcutaneous transplantation in athymic nude rat hosts for 1 month or 3 months. Subsequent analyses using X-ray, histological and immunohistochemical methods showed that the majority of the recombinant tooth structures formed calcified tissues, including osteodentin, dentin cementum, enamel and morphologically typical tooth crowns composed of dentin and enamel. The demonstrated formation of mineralized dental tissues and tooth crown structures from easily obtained post-natal dental tissues is an important step toward reaching the long-term goal of establishing robust and reliable models for human tooth regeneration. Copyright © 2014 John Wiley & Sons, Ltd.

Decellularized Tooth Bud Scaffolds for Tooth Regeneration.

Whole tooth regeneration approaches currently are limited by our inability to bioengineer full-sized, living replacement teeth. Recently, decellularized organ scaffolds have shown promise for applications in regenerative medicine by providing a natural extracellular matrix environment that promotes cell attachment and tissue-specific differentiation leading to full-sized organ regeneration. We hypothesize that decellularized tooth buds (dTBs) created from unerupted porcine tooth buds (TBs) can be used to guide reseeded dental cell differentiation to form whole bioengineered teeth, thereby providing a potential off-the-shelf scaffold for whole tooth regeneration. Porcine TBs were harvested from discarded 6-mo-old pig jaws, and decellularized by successive sodium dodecyl sulfate/Triton-X cycles. Four types of replicate implants were used in this study: 1) acellular dTBs; 2) recellularized dTBs seeded with porcine dental epithelial cells, human dental pulp cells, and human umbilical vein endothelial cells (recell-dTBs); 3) dTBs seeded with bone morphogenetic protein (BMP)-2 (dTB-BMPs); and 4) freshly isolated nondecellularized natural TBs (nTBs). Replicate samples were implanted into the mandibles of host Yucatan mini-pigs and grown for 3 or 6 mo. Harvested mandibles with implanted TB constructs were fixed in formalin, decalcified, embedded in paraffin, sectioned, and analyzed via histological methods. Micro-computed tomography (CT) analysis was performed on harvested 6-mo samples prior to decalcification. All harvested constructs exhibited a high degree of cellularity. Significant production of organized dentin and enamel-like tissues was observed in dTB-recell and nTB implants, but not in dTB or dTB-BMP implants. Micro-CT analyses of 6-mo implants showed the formation of organized, bioengineered teeth of comparable size to natural teeth. To our knowledge, these results are the first to describe the potential use of dTBs for functional whole tooth regeneration.

GelMA-Encapsulated hDPSCs and HUVECs for Dental Pulp Regeneration.

Pulpal revascularization is commonly used in the dental clinic to obtain apical closure of immature permanent teeth with thin dentinal walls. Although sometimes successful, stimulating bleeding from the periapical area of the tooth can be challenging and in turn may deleteriously affect tooth root maturation. Our objective here was to define reliable methods to regenerate pulp-like tissues in tooth root segments (RSs). G1 RSs were injected with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) encapsulated in 5% gelatin methacrylate (GelMA) hydrogel. G2 RSs injected with acellular GelMA alone, and G3 empty RSs were used as controls. White mineral trioxide aggregate was used to seal one end of the tooth root segment, while the other was left open. Samples were cultured in vitro in osteogenic media (OM) for 13 d and then implanted subcutaneously in nude rats for 4 and 8 wk. At least 5 sample replicates were used for each experimental group. Analyses of harvested samples found that robust pulp-like tissues formed in G1, GelMA encapsulated hDPSC/HUVEC-filled RSs, and less cellularized host cell-derived pulp-like tissue was observed in the G2 acellular GelMA and G3 empty RS groups. Of importance, only the G1, hDPSC/HUVEC-encapsulated GelMA constructs formed pulp cells that attached to the inner dentin surface of the RS and infiltrated into the dentin tubules. Immunofluorescent (IF) histochemical analysis showed that GelMA supported hDPSC/HUVEC cell attachment and proliferation and also provided attachment for infiltrating host cells. Human cell-seeded GelMA hydrogels promoted the establishment of well-organized neovasculature formation. In contrast, acellular GelMA and empty RS constructs supported the formation of less organized host-derived vasculature formation. Together, these results identify GelMA hydrogel combined with hDPSC/HUVECs as a promising new clinically relevant pulpal revascularization treatment to regenerate human dental pulp tissues.

The Contributions of the Ribosome Biogenesis Protein Utp5/WDR43 to Craniofacial Development.

Fairly recently, it was recognized that human ribosomopathies-developmental defects caused by mutations in ribosome biogenesis proteins-can exhibit tissue-specific defects rather than the expected global defects. This apparent anomaly-that seemingly ubiquitously expressed and required ribosomal proteins can have distinct functions in cell and tissue differentiation-has spurred new areas of research focused on better understanding translational mechanisms, biogenesis, and function in diverse cell types. This renewed appreciation for, and need to better understand, roles for ribosomal proteins in human development and disease has identified surprising similarities and differences in a variety of human ribosomopathies. Here, we discuss ribosomal protein functions in health and disease, focusing on the ribosome biogenesis protein Utp5/WDR43. New and exciting research in this field is anticipated to provide insight into a variety of previously understudied craniofacial dysostoses and result in significantly improved knowledge and understanding of roles for translational machinery in human craniofacial development and disease.

Sequence homologies in the mouse protamine 1 and 2 genes.

To identify candidates for cis-acting sequences that regulate the stage and cell-specific expression of the two coordinately regulated protamine genes in the mouse, genomic clones were isolated and the nucleotide sequences of the 5' flanking regions and coding regions were compared. Unlike most histone genes and the multigene family of trout protamine genes which are intronless, each mouse protamine gene has a single, short intervening sequence. Although the coding regions do not share significant nucleotide homology, the 5' flanking regions contain several short homologous sequences that may be involved in gene regulation. An additional shared sequence is present in the 3' untranslated region surrounding the poly(A) addition signal in both genes.

Nucleotide sequence of a cDNA clone encoding mouse transition protein 1.

We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455-464; Hecht et al. (1986) Exp. Cell Res. 164, 183-190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.

Functional characterization and genetic mapping of alk8.

The novel type I TGFbeta family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.

Developmental analysis and computer modelling of bioengineered teeth.

Here we present the developmental progression of bioengineered pig teeth from 1 to 25 weeks of development. We demonstrate that 2-25 week implants contained embryonic tooth bud- and cap-stage tooth structures consisting of dental epithelium expressing the sonic hedgehog gene and condensed dental mesenchyme. Implants harvested at 18-25 weeks also contained tooth bud-like structures, as well as mature tooth structures containing enamel, dentin and pulp tissues. Immunohistochemical analyses confirmed the expression of dentin- and enamel-specific proteins in differentiated bioengineered tooth tissues. Three-dimensional computer modelling further demonstrated a spatial organization of enamel, dentin and pulp tissues resembling that of natural teeth. We conclude that bioengineered teeth commonly exhibit morphological stages characteristic of naturally forming teeth. Furthermore, the presence of immature tooth buds at all times assayed and increased numbers of bioengineered tooth structures over time suggests that porcine dental progenitor cells maintain the ability to form teeth for at least 25 weeks.

Cloning and complete coding sequence of a novel human cathepsin expressed in giant cells of osteoclastomas.

A gene encoding a possible novel human cathepsin, a cysteine proteinase that is distinct from previously characterized enzymes, has been identified by differential screening of a human osteoclastoma cDNA library. This molecule, termed cathepsin X, appears to represent the human homolog of the osteoclast-expressed rabbit cathepsin OC-2. Cathepsin X (GenBank accession number U20280) is 93.9% identical to OC-2 at the amino acid level, and is 92% identical at the nucleotide level within the coding region. Cathepsin X is 52.2 and 46.9% identical to cathepsins S and L, respectively, and is therefore clearly distinct from these enzymes. Cathepsin X mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization. These data strongly support the hypothesis that cathepsin X represents a novel cysteine proteinase which is expressed at high levels in osteoclasts.

Regulation of tooth development by the novel type I TGFbeta family member receptor Alk8.

We have recently identified, in zebrafish, a novel type I receptor of the TGFbeta family, alk8, that participates in Bmp signaling pathways to mediate early dorsoventral patterning of neurectodermal and mesendodermal tissues. Since Bmps play significant roles in tooth specification, initiation, and differentiation, we hypothesized that alk8 may play a role in directing the Bmp-mediated epithelial mesenchymal cell interactions regulating tooth development. Immunohistochemical analysis demonstrates that Alk8 is expressed in developing zebrafish and mouse teeth. Examination of tooth development in zebrafish with disrupted alk8 signaling revealed specific defects in tooth development. Ectopic expression of constitutively active Alk8 results in the formation of elongated tooth structures, while expression of dominant-negative Alk8 results in arrested tooth development at the bud stage. These results are consistent with the established requirements for Bmp signaling in tooth development and demonstrate that Alk8 is a key regulator of tooth development.

Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig's root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Bioengineered teeth from cultured rat tooth bud cells.

The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.