The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs1, 2. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell cycle inhibitors p16ink4a and p19Arf3. However, deletion of Ink4a/Arf only partially rescues Bmi1 null phenotypes4, indicating that other important targets of BMI1 exist. Here, using the continuously-growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression, and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated1, 2, 5–7, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.
The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal.
Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling, which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs, suggesting that they may influence self-renewal. To test this hypothesis, we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation, whereas SPRY4 KD enhanced survival. In both cases, after KD the cells were able to differentiate into cells of the three germ layers, although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage, explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs.
Tissue engineering; Regenerative medicine; Dental tissues; Scaffold
Stem cells are essential for the regeneration and homeostasis of many organs, such as tooth, hair, skin, and intestine. Although human tooth regeneration is limited, a number of animals have evolved continuously growing teeth that provide models of stem cell-based organ renewal. A well-studied model is the mouse incisor, which contains dental epithelial stem cells in structures known as cervical loops. These stem cells produce progeny that proliferate and migrate along the proximo-distal axis of the incisor and differentiate into enamel-forming ameloblasts. Here, we studied the role of E-cadherin in behavior of the stem cells and their progeny. Levels of E-cadherin are highly dynamic in the incisor, such that E-cadherin is expressed in the stem cells, downregulated in the transit-amplifying cells, re-expressed in the pre-ameloblasts and then downregulated again in the ameloblasts. Conditional inactivation of E-cadherin in the cervical loop led to decreased numbers of label-retaining stem cells, increased proliferation, and decreased cell migration in the mouse incisor. Using both genetic and pharmacological approaches, we showed that Fibroblast Growth Factors regulate E-cadherin expression, cell proliferation and migration in the incisor. Together, our data indicate that E-cadherin is an important regulator of stem cells and their progeny during growth of the mouse incisor.
E-cadherin; Epithelial stem cells; Cell migration; Cell proliferation; Incisor; Ameloblasts; Fibroblast Growth factors (FGFs); Sprouty genes; Mouse
Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, hair follicle and also in kidney nephrons. Here we demonstrate the outstanding regenerative potential of a rare population of Lgr5-expressing (Lgr5+) mammary epithelial cells (MECs). We found that Lgr5+ cells reside within the basal population, are superior to other basal cells in regenerating functional mammary glands (MGs), are exceptionally efficient in reconstituting MGs from single cells and exhibit regenerative capacity in serial transplantations. Loss-of-function, depletion experiments of Lgr5+ cells from transplanted MECs or from pubertal MGs revealed that these cells are not only sufficient but also necessary for postnatal mammary organogenesis.
Lgr5; stem cell; mammary gland; regenerative potential
The mouse incisor is a valuable but under-utilized model organ for studying the behavior of adult stem cells. This remarkable tooth grows continuously throughout the animal's lifetime and houses two distinct epithelial stem cell niches called the labial and lingual cervical loop (laCL and liCL, respectively). These stem cells produce progeny that undergo a series of well-defined differentiation events en route to becoming enamel-producing ameloblasts. During this differentiation process, the progeny move out of the stem cell niche and migrate toward the distal tip of the tooth. Although the molecular pathways involved in tooth development are well documented, little is known about the roles of miRNAs in this process. We used microarray technology to compare the expression of miRNAs in three regions of the adult mouse incisor: the laCL, liCL, and ameloblasts. We identified 26 and 35 differentially expressed miRNAs from laCL/liCL and laCL/ameloblast comparisons, respectively. Out of 10 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. In situ hybridization and target prediction analyses further supported the reliability of our microarray results. These studies point to miRNAs that likely play a role in the renewal and differentiation of adult stem cells during stem cell-fueled incisor growth.
The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry) genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK) signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1−/−;Spry2−/− embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.
The sense of taste is important for an animal's ability to survive and thrive, because it enables discrimination between nutritious substances and toxins. Taste buds are housed largely on the tongue in structures called papillae; of the three types of gustatory papillae, the circumvallate papilla (CVP) is the largest. In rodents, a single CVP is located in the posterior midline of the tongue housing hundreds of taste buds, whereas in other mammals up to dozens of CVPs can be found. However, despite the great variation in the number of CVPs in mammals, its status as the largest of the taste papillae, and its importance in taste function, very little is known about its development. We identified members of the FGF signaling pathway as determinants of CVP number. We propose that perturbations to the FGF signaling pathway may have been involved in the dramatic differences in CVP number that arose during mammalian evolution.
Wnt signaling is essential for tooth formation. Dact proteins modulate Wnt signaling by binding to the intracellular protein Dishevelled (Dvl). Comparison of all known mouse Dact genes, Dact1-3, from the morphological initiation of mandibular first molar development after the onset of the root formation using sectional in situ hybridization showed distinct, complementary and overlapping expression patterns for the studied genes. While Dact2 expression was restricted to the dental epithelium including the enamel knot signaling centers and tooth specific preameloblasts, Dact1 and Dact3 showed developmentally regulated expression in the dental mesenchyme. Both mRNAs were first detected in the presumptive dental mesenchyme. After being downregulated from the condensed dental mesenchyme of the bud stage tooth germ, Dact1 was upregulated in the dental follicle masenchyme at the cap stage and subsequently also in the dental papilla at the bell stage where the expression persisted to the postnatal stages. In contrast, Dact3 transcripts persisted throughout the dental mesenchymal tissue components including the tooth-specific cells, preodontoblasts before transcripts were largely downregulated from the tooth germ postnatally. Collectively these results suggest that Dact1 and -3 may contribute to early tooth formation by modulation of Wnt signaling pathways in the mesenchyme, including preodontoblasts, whereas Dact2 may play important signal-modulating roles in the adjacent epithelial cells including the enamel knot signaling centers and preameloblasts. Future loss-of-function studies will help elucidate whether any of these functions are redundant, particularly for Dact1 and Dact3.
Mammalian lungs are branched networks containing thousands to millions of airways arrayed in intricate patterns that are crucial for respiration. How such trees are generated during development, and how the developmental patterning information is encoded, have long fascinated biologists and mathematicians. However, models have been limited by a lack of information on the normal sequence and pattern of branching events. Here we present the complete three-dimensional branching pattern and lineage of the mouse bronchial tree, reconstructed from an analysis of hundreds of developmental intermediates. The branching process is remarkably stereotyped and elegant: the tree is generated by three geometrically simple local modes of branching used in three different orders throughout the lung. We propose that each mode of branching is controlled by a genetically-encoded subroutine, a series of local patterning and morphogenesis operations, which are themselves controlled by a more global master routine. We show that this hierarchical and modular program is genetically tractable, and it is ideally suited to encoding and evolving the complex networks of the lung and other branched organs.
An understanding of the factors that promote or inhibit tooth development is essential for designing biological tooth replacements. The embryonic mouse dentition provides an ideal system for studying such factors because it consists of two types of tooth primordia. One type of primordium will go on to form a functional tooth, whereas the other initiates development but arrests at or before the bud stage. This developmental arrest contributes to the formation of the toothless mouse diastema. It is accompanied by the apoptosis of the rudimentary diastemal buds, which presumably results from the insufficient activity of anti-apoptotic signals such as fibroblast growth factors (FGFs). We have previously shown that the arrest of a rudimentary tooth bud can be rescued by inactivating Spry2, an antagonist of FGF signaling. Here, we studied the role of the epithelial cell death and proliferation in this process by comparing the development of a rudimentary diastemal tooth bud (R2) and the first molar in the mandibles of Spry2−/− and wild-type (WT) embryos using histological sections, image analysis and 3D reconstructions. In the WT R2 at embryonic day 13.5, significantly increased apoptosis and decreased proliferation were found compared with the first molar. In contrast, increased levels of FGF signaling in Spry2−/− embryos led to significantly decreased apoptosis and increased proliferation in the R2 bud. Consequently, the R2 was involved in the formation of a supernumerary tooth primordium. Studies of the revitalization of rudimentary tooth primordia in mutant mice can help to lay the foundation for tooth regeneration by enhancing our knowledge of mechanisms that regulate tooth formation.
Unlike humans, who have a continuous row of teeth, mice have only molars and incisors separated by a toothless region called a diastema. Although tooth buds form in the embryonic diastema, they regress and do not develop into teeth. Here, we identify members of the Sprouty (Spry) family, which encode negative feedback regulators of fibroblast growth factor (FGF) and other receptor tyrosine kinase signaling, as genes that repress diastema tooth development. We show that different Sprouty genes are deployed in different tissue compartments—Spry2 in epithelium and Spry4 in mesenchyme—to prevent diastema tooth formation. We provide genetic evidence that they function to ensure that diastema tooth buds are refractory to signaling via FGF ligands that are present in the region and thus prevent these buds from engaging in the FGF-mediated bidirectional signaling between epithelium and mesenchyme that normally sustains tooth development.
The mouse incisor has two unusual features: it grows continuously and it is covered by enamel exclusively on the labial side. The continuous growth is driven in part by epithelial stem cells in the cervical loop region that can both self-renew and give rise to ameloblasts. We have previously reported that ectopic enamel is found on the lingual side of the incisor in mice with loss-of-function of sprouty (spry) genes. Spry2+/−; Spry4−/− mice, in which three sprouty alleles have been inactivated, have ectopic enamel as a result of upregulation of epithelial-mesenchymal FGF signaling in the lingual part of the cervical loop. Interestingly, lingual enamel is also present in the early postnatal period in Spry4−/− mice, in which only two sprouty alleles have been inactivated, but ectopic enamel is not found in adults of this genotype. To explore the mechanisms underlying the disappearance of lingual enamel in Spry4−/− adults, we studied the fate of the lingual enamel in Spry4−/− mice by comparing the morphology and growth of their lower incisors with wild type and Spry2+/−; Spry4−/− mice at several timepoints between the perinatal period and adulthood. Ameloblasts and enamel were detected on the lingual side in postnatal Spry2+/−; Spry4−/+ incisors. By contrast, new ectopic ameloblasts ceased to differentiate after postnatal day 3 in Spry4−/− incisors, which was followed by a progressive loss of lingual enamel. Both the posterior extent of lingual enamel and the time of its last deposition were variable early postnatally in Spry4−/− incisors, but in all Spry4−/− adult incisors the lingual enamel was ultimately lost through continuous growth and abrasion of the incisor.
Rodent incisors grow throughout adult life, but are prevented from becoming excessively long by constant abrasion, which is facilitated by the absence of enamel on one side of the incisor. Here we report that loss-of-function of Sprouty genes, which encode antagonists of receptor tyrosine kinase signaling, leads to bilateral enamel deposition, thus impeding incisor abrasion and resulting in unchecked tooth elongation. We demonstrate that Sprouty genes function to ensure that enamel-producing ameloblasts are generated on only one side of the tooth by inhibiting the formation of ectopic ameloblasts from self-renewing stem cells, and that they do so by preventing the establishment of an epithelial-mesenchymal FGF signaling loop. Interestingly, whilst inactivation of Spry4 alone initiates ectopic ameloblast formation in the embryo, the dosage of another Sprouty gene must also be reduced to sustain it after birth. These data reveal that the generation of differentiated progeny from a particular stem cell population can be differently regulated in the embryo and adult.
ameloblast; enamel; FGF signaling; Sprouty genes; stem cells
Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle defect. While hemizygous males typically present with hyperammonemic coma in infancy, reports of rare late-onset presentations exist, with poor outcomes in males up to 58 years old. Relatives with mutations identical to affected patients often remain asymptomatic, and it is likely that environmental and genetic factors influence disease penetrance and expression. Here, we present our investigation of a patient with late-onset presentation, and we emphasize the potential role of environmental and genetic factors on disease expression. The patient was a previously healthy 62-year-old man who developed mental slowing, refractory seizures, and coma over an 8-day period. Interestingly, the patient had recently used home gardening fertilizers and pesticides. Evaluations for drug and alcohol use, infections, and liver disease were negative. Despite aggressive therapy, blood NH3 concentration peaked at 2,050 μM and the patient died from cerebral edema and cerebellar herniation. Analysis of the OTC gene showed a Pro-225-Thr (P225T) change in exon 7, a mutation that has been previously implicated in OTC deficiency. This case illustrates that OTC deficiency can cause acute, severe hyperammonemia in a previously healthy adult and that the P225T mutation can be associated with late-onset OTC deficiency. We speculate that exposure to organic chemicals might have contributed to the onset of symptoms in this patient. This case also emphasizes that persistent hyperammonemia may cause irreversible neurologic damage and that after the diagnosis of hyperammonemia is established in an acutely ill patient, certain diagnostic tests should be performed to differentiate between urea cycle disorders and other causes of hyperammonemic encephalopathy.
Hyperammonemia; OTC; Ornithine transcarbamylase; Urea cycle; Late onset
Dental epithelial stem cells (DESCs) drive continuous growth in the adult mouse incisors. To date, a robust system for the primary culture of these cells has not been reported, and little is known about the basic molecular architecture of these cells or the minimal extracellular scaffolding that is necessary to maintain the epithelial stem cell population in an undifferentiated state. We report a method of isolating DESCs from the cervical loop of the mouse mandibular incisor. Cells were viable in a two-dimensional culture system and did not demonstrate preferential proliferation when grown on top of various substrates. Characterization of these cells indicated that E-cadherin, integrin alpha-6, and integrin beta-4 mark the DESCs both in vivo and in vitro. We also grew these cells in a three-dimensional microenvironment and obtained spheres with an epithelial morphology and expression patterns. Insights into the mechanisms of stem cell maintenance in vitro will help lay the groundwork for the successful generation of bioengineered teeth from adult DESCs.
Sagittal craniosynostosis is the most common form of craniosynostosis, affecting approximately one of 5,000 newborns. We conducted the first genome-wide association study (GWAS) for non-syndromic sagittal craniosynostosis (sNSC) using 130 non-Hispanic white (NHW) case-parent trios. Robust associations were observed in a 120 kb region downstream of BMP2, flanked by rs1884302 (P = 1.13 × 10−14; odds ratio [OR] = 4.58) and rs6140226 (P = 3.40 × 10−11; OR = 0.24) and within a 167 kb region of BBS9 between rs10262453 (P = 1.61 × 10−10; OR=0.19) and rs17724206 (P = 1.50 × 10−8; OR = 0.22). We replicated the associations to both loci [rs1884302 (P = 4.39 × 10−31); rs10262453 (P = 3.50 × 10−14)] in an independent NHW population of 172 unrelated sNSC probands and 548 controls. Both BMP2 and BBS9 are genes with a role in skeletal development warranting functional studies to further understand the etiology of sNSC.
genome-wide association study; non-syndromic sagittal craniosynostosis; BMP2; BBS9; meta-analysis; nonsyndromic
Odontogenesis relies on the reciprocal signaling interactions between dental epithelium and neural crest-derived mesenchyme, which is regulated by several signaling pathways. Subtle changes in the activity of these major signaling pathways can have dramatic effects on tooth development. An important regulator of such subtle changes is the fine tuning function of microRNAs (miRNAs). However, the underlying mechanism by which miRNAs regulate tooth development remains elusive. This study determined the expression of miRNAs during cytodifferentiation in the human tooth germ and studied miR-34a as a regulator of dental papilla cell differentiation. Using microarrays, miRNA expression profiles were established at selected times during development (early bell stage or late bell stage) of the human fetal tooth germ. We identified 29 differentially expressed miRNAs from early bell stage/late bell stage comparisons. Out of 6 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. miR-34a was selected for further investigation because it has been previously reported to regulate organogenesis. miR-34a mimics and inhibitors were transfected into human fetal dental papilla cells, mRNA levels of predicted target genes were detected by quantitative real-time PCR, and levels of putative target proteins were examined by western blotting. ALP and DSPP expression were also tested by qPCR, western blotting, and immunofluorescence. Findings from these studies suggested that miR-34a may play important roles in dental papilla cell differentiation during human tooth development by targeting NOTCH and TGF-beta signaling.