Salivary Gland Development in Culture.

Salivary glands are branching organs which develop by bud and cleft formation to create an organ with a large surface area. The epithelium and mesenchyme signal back and forth to control this branching process, with additional cues provided by the parasympathetic nerves and blood vessels that surround the developing branches. This branching morphogenesis can be recapitulated successfully in organ culture , allowing access to the tissue to follow development and manipulate the tissue interactions, and signals. To culture glands, the filter-grid method has been widely used, allowing the development of salivary glands cultured as a whole organ, or the gland epithelium in isolation, or with the surrounding craniofacial tissue in a cranial slice. Here, we describe the methods for each approach and show the applicability of culturing glands from a wide variety of species: mouse , snake, and human. The resulting samples and data from these cultures can be employed for morphological and molecular analysis, with some examples described in this chapter, bringing valuable knowledge to our understanding of branching morphogenesis.

Depression and Antidepressants During Pregnancy: Craniofacial Defects Due to Stem/Progenitor Cell Deregulation Mediated by Serotonin.

Depression is a common and debilitating mood disorder that increases in prevalence during pregnancy. Worldwide, 7 to 12% of pregnant women experience depression, in which the associated risk factors include socio-demographic, psychological, and socioeconomic variables. Maternal depression could have psychological, anatomical, and physiological consequences in the newborn. Depression has been related to a downregulation in serotonin levels in the brain. Accordingly, the most commonly prescribed pharmacotherapy is based on selective serotonin reuptake inhibitors (SSRIs), which increase local serotonin concentration. Even though the use of SSRIs has few adverse effects compared with other antidepressants, altering serotonin levels has been associated with the advent of anatomical and physiological changes in utero, leading to defects in craniofacial development, including craniosynostosis, cleft palate, and dental defects. Migration and proliferation of neural crest cells, which contribute to the formation of bone, cartilage, palate, teeth, and salivary glands in the craniofacial region, are regulated by serotonin. Specifically, craniofacial progenitor cells are affected by serotonin levels, producing a misbalance between their proliferation and differentiation. Thus, it is possible to hypothesize that craniofacial development will be affected by the changes in serotonin levels, happening during maternal depression or after the use of SSRIs, which cross the placental barrier, increasing the risk of craniofacial defects. In this review, we provide a synthesis of the current research on depression and the use of SSRI during pregnancy, and how this could be related to craniofacial defects using an interdisciplinary perspective integrating psychological, clinical, and developmental biology perspectives. We discuss the mechanisms by which serotonin could influence craniofacial development and stem/progenitor cells, proposing some transcription factors as mediators of serotonin signaling, and craniofacial stem/progenitor cell biology. We finally highlight the importance of non-pharmacological therapies for depression on fertile and pregnant women, and provide an individual analysis of the risk-benefit balance for the use of antidepressants during pregnancy.

Comparing development and regeneration in the submandibular gland highlights distinct mechanisms.

A common question in organ regeneration is the extent to which regeneration recapitulates embryonic development. To investigate this concept, we compared the expression of two highly interlinked and essential genes for salivary gland development, Sox9 and Fgf10, during submandibular gland development, homeostasis and regeneration. Salivary gland duct ligation/deligation model was used as a regenerative model. Fgf10 and Sox9 expression changed during regeneration compared to homeostasis, suggesting that these key developmental genes play important roles during regeneration, however, significantly both displayed different patterns of expression in the regenerating gland compared to the developing gland. Regenerating glands, which during homeostasis had very few weakly expressing Sox9-positive cells in the striated/granular ducts, displayed elevated expression of Sox9 within these ducts. This pattern is in contrast to embryonic development, where Sox9 expression was absent in the proximally developing ducts. However, similar to the elevated expression at the distal tip of the epithelium in developing salivary glands, regenerating glands displayed elevated expression in a subpopulation of acinar cells, which during homeostasis expressed Sox9 at lower levels. A shift in expression of Fgf10 was observed from a widespread mesenchymal pattern during organogenesis to a more limited and predominantly epithelial pattern during homeostasis in the adult. This restricted expression in epithelial cells was maintained during regeneration, with no clear upregulation in the surrounding mesenchyme, as might be expected if regeneration recapitulated development. As both Fgf10 and Sox9 were upregulated in proximal ducts during regeneration, this suggests that the positive regulation of Sox9 by Fgf10, essential during development, is partially reawakened during regeneration using this model. Together these data suggest that developmentally important genes play a key role in salivary gland regeneration but do not precisely mimic the roles observed during development.

Balance Between Tooth Size and Tooth Number Is Controlled by Hyaluronan.

While the function of proteins and genes has been widely studied during vertebrate development, relatively little work has addressed the role of carbohydrates. Hyaluronan (HA), also known as hyaluronic acid, is an abundant carbohydrate in embryonic tissues and is the main structural component of the extracellular matrix of epithelial and mesenchymal cells. HA is able to absorb large quantities of water and can signal by binding to cell-surface receptors. During organ development and regeneration, HA has been shown to regulate cell proliferation, cell shape, and migration. Here, we have investigated the function of HA during molar tooth development in mice, in which, similar to humans, new molars sequentially bud off from a pre-existing molar. Using an ex vivo approach, we found that inhibiting HA synthesis in culture leads to a significant increase in proliferation and subsequent size of the developing molar, while the formation of sequential molars was inhibited. By cell shape analysis, we observed that inhibition of HA synthesis caused an elongation and reorientation of the major cell axes, indicating that disruption to cellular orientation and shape may underlie the observed phenotype. Lineage tracing demonstrated the retention of cells in the developing first molar (M1) at the expense of the generation of a second molar (M2). Our results highlight a novel role for HA in controlling proliferation, cell orientation, and migration in the developing tooth, impacting cellular decisions regarding tooth size and number.

Getting out of an egg: Merging of tooth germs to create an egg tooth in the snake.

BACKGROUND: The egg tooth is a vital structure allowing hatchlings to escape from the egg. In squamates (snakes and lizards), the egg tooth is a real tooth that develops within the oral cavity at the top of the upper jaw. Most squamates have a single large midline egg tooth at hatching, but a few families, such as Gekkonidae, have two egg teeth. In snakes the egg tooth is significantly larger than the rest of the dentition and is one of the first teeth to develop. RESULTS: We follow the development of the egg tooth in four snake species and show that the single egg tooth is formed by two tooth germs. These two tooth germs are united at the midline and grow together to produce a single tooth. In culture, this merging can be perturbed to give rise to separate smaller teeth, confirming the potential of the developing egg tooth to form two teeth. CONCLUSIONS: Our data agrees with previous hypotheses that during evolution one potential mechanism to generate a large tooth is through congrescence of multiple tooth germs and suggests that the ancestors of snakes could have had two egg teeth.

Tracking morphological complexities of organ development in culture.

Organogenesis is one of the most striking process during development. During this period, organ primordia pass throughout several stages in which the level of organisation increases in complexity to achieve the final organ architecture. Organ culture, a method in which an isolated organ is explanted and maintained ex-vivo, is an excellent tool for following the morphological dynamics during development. While most of the work has been made in early stages of development, culturing organs in mid-late stages is needed to understand the achievement of the final organ anatomy in the new-born. Here, we investigated the possibility of following morphological changes of the mice heart, lung, kidney and intestine using a filter-grid culture method for 7 days starting at E14.5. We observed that the anatomy, histology and survival of the cultured organs were indicative of a continuity of the developmental processes: they survived and morphodifferentiated during 5-7 days in culture. The exception was the heart, which started to die after 4 days. Using a second approach, we demonstrated that heart tissue can be easily cultured in body slices, together with other tissues such as the lung, with a healthier differentiation and longer survival. The culture method used here, permits a high-resolution imaging to identify the dynamic of organ architecture ex-vivo using morphovideos. We also confirmed the suitability of this system to perform lineage tracing using a vital dye in branching organs. In summary, this work tested the feasibility of monitoring and recording the anatomical changes that establish the final organ structure of the heart, lung, kidney and intestine. Additionally, this strategy allows the morphological study of organ development including fate maps with a relative long-term survival up to the onset of differentiation. This work contributes to elucidating how organs are formed, promoting the understanding of congenital malformations and to design organ replacement therapies.

Fgf10 and Sox9 are essential for the establishment of distal progenitor cells during mouse salivary gland development.

Salivary glands are formed by branching morphogenesis with epithelial progenitors forming a network of ducts and acini (secretory cells). During this process, epithelial progenitors specialise into distal (tips of the gland) and proximal (the stalk region) identities that produce the acini and higher order ducts, respectively. Little is known about the factors that regulate progenitor expansion and specialisation in the different parts of the gland. Here, we show that Sox9 is involved in establishing the identity of the distal compartment before the initiation of branching morphogenesis. Sox9 is expressed throughout the gland at the initiation stage before becoming restricted to the distal epithelium from the bud stage and throughout branching morphogenesis. Deletion of Sox9 in the epithelium results in loss of the distal epithelial progenitors, a reduction in proliferation and a subsequent failure in branching. We demonstrate that Sox9 is positively regulated by mesenchymal Fgf10, a process that requires active Erk signalling. These results provide new insights into the factors required for the expansion of salivary gland epithelial progenitors, which can be useful for organ regeneration therapy.

Epithelial topography for repetitive tooth formation.

During the formation of repetitive ectodermally derived organs such as mammary glands, lateral line and teeth, the tissue primordium iteratively initiates new structures. In the case of successional molar development, new teeth appear sequentially in the posterior region of the jaw from Sox2(+) cells in association with the posterior aspect of a pre-existing tooth. The sequence of molar development is well known, however, the epithelial topography involved in the formation of a new tooth is unclear. Here, we have examined the morphology of the molar dental epithelium and its development at different stages in the mouse in vivo and in molar explants. Using regional lineage tracing we show that within the posterior tail of the first molar the primordium for the second and third molar are organized in a row, with the tail remaining in connection with the surface, where a furrow is observed. The morphology and Sox2 expression of the tail retains characteristics reminiscent of the earlier stages of tooth development, such that position along the A-P axes of the tail correlates with different temporal stages. Sox9, a stem/progenitor cell marker in other organs, is expressed mainly in the suprabasal epithelium complementary with Sox2 expression. This Sox2 and Sox9 expressing molar tail contains actively proliferating cells with mitosis following an apico-basal direction. Snail2, a transcription factor implicated in cell migration, is expressed at high levels in the tip of the molar tail while E-cadherin and laminin are decreased. In conclusion, our studies propose a model in which the epithelium of the molar tail can grow by posterior movement of epithelial cells followed by infolding and stratification involving a population of Sox2(+)/Sox9(+) cells.

Genome-wide expression profile of the response to spinal cord injury in Xenopus laevis reveals extensive differences between regenerative and non-regenerative stages.

BACKGROUND: Xenopus laevis has regenerative and non-regenerative stages. As a tadpole, it is fully capable of functional recovery after a spinal cord injury, while its juvenile form (froglet) loses this capability during metamorphosis. We envision that comparative studies between regenerative and non-regenerative stages in Xenopus could aid in understanding why spinal cord regeneration fails in human beings. RESULTS: To identify the mechanisms that allow the tadpole to regenerate and inhibit regeneration in the froglet, we obtained a transcriptome-wide profile of the response to spinal cord injury in Xenopus regenerative and non-regenerative stages. We found extensive transcriptome changes in regenerative tadpoles at 1 day after injury, while this was only observed by 6 days after injury in non-regenerative froglets. In addition, when comparing both stages, we found that they deployed a very different repertoire of transcripts, with more than 80% of them regulated in only one stage, including previously unannotated transcripts. This was supported by gene ontology enrichment analysis and validated by RT-qPCR, which showed that transcripts involved in metabolism, response to stress, cell cycle, development, immune response and inflammation, neurogenesis, and axonal regeneration were regulated differentially between regenerative and non-regenerative stages. CONCLUSIONS: We identified differences in the timing of the transcriptional response and in the inventory of regulated transcripts and biological processes activated in response to spinal cord injury when comparing regenerative and non-regenerative stages. These genes and biological processes provide an entry point to understand why regeneration fails in mammals. Furthermore, our results introduce Xenopus laevis as a genetic model organism to study spinal cord regeneration.

Dynamic relationship of the epithelium and mesenchyme during salivary gland initiation: the role of Fgf10.

Salivary glands provide an excellent model for the study of epithelial-mesenchymal interactions. We have looked at the interactions involved in the early initiation and development of murine salivary glands using classic recombination experiments and knockout mice. We show that salivary gland epithelium, at thickening and initial bud stages, is able to direct salivary gland development in non-gland pharyngeal arch mesenchyme at early stages. The early salivary gland epithelium is therefore able to induce gland development in non-gland tissue. This ability later shifts to the mesenchyme, with non-gland epithelium, such as from the limb bud, able to form a branching gland when combined with pseudoglandular stage gland mesenchyme. This shift appears to involve Fgf signalling, with signals from the epithelium inducing Fgf10 in the mesenchyme. Fgf10 then signals back to the epithelium to direct gland down-growth and bud development. These experiments highlight the importance of epithelial-mesenchymal signalling in gland initiation, controlling where, when and how many salivary glands form.

Organized emergence of multiple-generations of teeth in snakes is dysregulated by activation of Wnt/beta-catenin signalling.

In contrast to mammals, most reptiles constantly regenerate their teeth. In the snake, the epithelial dental lamina ends in a successional lamina, which proliferates and elongates forming multiple tooth generations, all linked by a permanent dental lamina. To investigate the mechanisms used to control the initiation of new tooth germs in an ordered sequential pattern we utilized the polyphodont (multiple-generation) corn snake (Pantherophis guttatus). We observed that the dental lamina expressed the transcription factor Sox2, a multipotent stem cell marker, whereas the successional lamina cells expressed the transcription factor Lef1, a Wnt/ß-catenin pathway target gene. Activation of the Wnt/ß-catenin pathway in culture increased the number of developing tooth germs, in comparison to control untreated cultures. These additional tooth germs budded off from ectopic positions along the dental lamina, rather than in an ordered sequence from the successional lamina. Wnt/ß-catenin activation enhanced cell proliferation, particularly in normally non-odontogenic regions of the dental lamina, which widely expressed Lef1, restricting the Sox2 domain. This suggests an expansion of the successional lamina at the expense of the dental lamina. Activation of the Wnt/ß-catenin pathway in cultured snake dental organs, therefore, led to changes in proliferation and to the molecular pattern of the dental lamina, resulting in loss of the organised emergence of tooth germs. These results suggest that epithelial compartments are critical for the arrangement of organs that develop in sequence, and highlight the role of Wnt/ß-catenin signalling in such processes.

Spinal cord regeneration in Xenopus tadpoles proceeds through activation of Sox2-positive cells.

BACKGROUND: In contrast to mammals, amphibians, such as adult urodeles (for example, newts) and anuran larvae (for example, Xenopus) can regenerate their spinal cord after injury. However, the cellular and molecular mechanisms involved in this process are still poorly understood. RESULTS: Here, we report that tail amputation results in a global increase of Sox2 levels and proliferation of Sox2(+) cells. Overexpression of a dominant negative form of Sox2 diminished proliferation of spinal cord resident cells affecting tail regeneration after amputation, suggesting that spinal cord regeneration is crucial for the whole process. After spinal cord transection, Sox2(+) cells are found in the ablation gap forming aggregates. Furthermore, Sox2 levels correlated with regenerative capabilities during metamorphosis, observing a decrease in Sox2 levels at non-regenerative stages. CONCLUSIONS: Sox2(+) cells contribute to the regeneration of spinal cord after tail amputation and transection. Sox2 levels decreases during metamorphosis concomitantly with the lost of regenerative capabilities. Our results lead to a working hypothesis in which spinal cord damage activates proliferation and/or migration of Sox2(+) cells, thus allowing regeneration of the spinal cord after tail amputation or reconstitution of the ependymal epithelium after spinal cord transection.

Impaired cell cycle regulation of the osteoblast-related heterodimeric transcription factor Runx2-Cbfbeta in osteosarcoma cells.

Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfbeta heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G(1). Runx2 levels are modulated during the cell cycle, which are maximal in G(1) and minimal beyond the G(1)/S phase transition (S, G(2), and M phases). It is not known whether Cbfbeta gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfbeta are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfbeta gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G(1) (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfbeta protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfbeta levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G(1) and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma.

Early requirement of Hyaluronan for tail regeneration in Xenopus tadpoles.

Tail regeneration in Xenopus tadpoles is a favorable model system to understand the molecular and cellular basis of tissue regeneration. Although turnover of the extracellular matrix (ECM) is a key event during tissue injury and repair, no functional studies to evaluate its role in appendage regeneration have been performed. Studying the role of Hyaluronan (HA), an ECM component, is particularly attractive because it can activate intracellular signaling cascades after tissue injury. Here we studied the function of HA and components of the HA pathway in Xenopus tadpole tail regeneration. We found that transcripts for components of this pathway, including Hyaluronan synthase2 (HAS2), Hyaluronidase2 and its receptors CD44 and RHAMM, were transiently upregulated in the regenerative bud after tail amputation. Concomitantly, an increase in HA levels was observed. Functional experiments using 4-methylumbelliferone, a specific HAS inhibitor that blocked the increase in HA levels after tail amputation, and transgenesis demonstrated that the HA pathway is required during the early phases of tail regeneration. Proper levels of HA are required to sustain proliferation of mesenchymal cells in the regenerative bud. Pharmacological and genetic inhibition of GSK3beta was sufficient to rescue proliferation and tail regeneration when HA synthesis was blocked, suggesting that GSK3beta is downstream of the HA pathway. We have demonstrated that HA is an early component of the regenerative pathway and is required for cell proliferation during the early phases of Xenopus tail regeneration. In addition, a crosstalk between HA and GSK3beta signaling during tail regeneration was demonstrated.

Ameloblastin and amelogenin expression in posnatal developing mouse molars.

Ameloblastin and amelogenin are structural proteins present in the enamel matrix of developing teeth. Here we report the results of in situ hybridization analyses with DNA probes of ameloblastin and amelogenin expression in the mandibular first molars of ICR/Jcl mice from postnatal day 1 to day 15. Ameloblastin mRNA expression was observed in ameloblasts at day 2 while amelogenin mRNA was detected in secretory ameloblasts at day 3. Significant expression of both molecules was observed at days 4, 5 and 6, after which the levels decreased. Amelogenin expression ended on day 10, while ameloblastin mRNA was only weakly detected on day 12. Neither amelogenin nor ameloblastin expression was observed in day 15 mouse molars. Amelogenin and ameloblastin mRNAs were restricted to ameloblasts. We conclude that amelogenin and ameloblastin expression is enamel-specific, and suggest that these genes might be involved in the mineralization of enamel. It is possible that ameloblastin could participate in the attachment of ameloblasts to the enamel surface. In this case, the downregulation of expression may indicate the beginning of the maturation stage in which the ameloblasts tend to detach from the enamel layer.

Mouse tooth development time sequence determination for the ICR/Jcl strain.

To establish the normal dental development pattern of the ICR/Jcl strain of mouse, we analyzed a significant number of observations of the different developmental stages of the first mandibular molar, accurately recording the chronology of their daily embryonic development. Proliferation of the dental sheet began at day 12.5 in utero (E-12.5), the bud stage appeared at days E-13.5 and E-14.5, the cap stage was observed at days E-14.5, E-15.5 and E-16.5 and the early bell stage at day E-17.5. The presence of predentin was observed at day E-18.5 and dentin was observed 1 and 2 days after birth (D-1 and D-2). The late bell stage with presence of enamel was detected more than 3 days after birth. Embryonic and dental development in the ICR/Jcl strain of mouse is faster than in other well-known strains. The establishment of this developmental pattern will be useful for future investigations of transgenic mice.