Innovation in dental education: The "On-the-Fly" approach to simultaneous development, implementation and evidence collection.

INTRODUCTION: This study outlines an approach for education innovation and addresses the ambivalence between evidence-based and non-evidence-based conditions. The "on-the-fly" approach was described as involving implementation during the development of an innovation for dental education. MATERIALS AND METHODS: The process of designing and implementing cutting-edge technology of the MOOG Simodont Dental Trainer (DT) whilst systematically collecting evidence illustrates the "on-the-fly" approach. RESULTS: Using the "on-the-fly" approach for developing, implementing and collecting evidence simultaneously in an academic environment appears feasible in serving both the professionals, users and developers and system designers. During the implementation of the new technology, growing evidence stepwise strengthened its position; therefore, showing stakeholders that evidence was used to improve the technology seemed to support and increase acceptance of the new technology. CONCLUSIONS: When pioneering an innovative technology in a specialty field, the development stage often precedes evidence for its effectiveness. Consciously choosing the "on-the-fly" approach clarifies to stakeholders in advance about the lack of evidence in an innovation and the need of their support to collect such evidence for improvement and in order to facilitate implementation.

Precious cargo: regulation of sex-specific germ cell development in mice.

Although mammalian sex determination is normally specified genetically by an XX or XY chromosome complement, germ cells develop as sperm or oocytes in response to molecular cues provided by the gonadal somatic cells. In an ovary, germ cells enter meiosis during fetal life, thereby committing to oogenesis. In a testis, germ cells do not enter meiosis until after birth, at puberty. Recent findings indicate that, in mice, the sex-specific timing of entry into meiosis is governed by the balance between 2 secreted signalling molecules, retinoic acid (RA), which promotes entry into meiosis, and fibroblast growth factor 9 (FGF9), which counteracts RA. The combined action of these 2 molecular regulators provides a safety mechanism to guard against germ cell dysregulation that can lead to infertility or germ cell cancers.

[Innovations in education for the digital student]. / Onderwijsinnovaties voor de digitale student.

A significant percentage of today's teaching staff received their professional training before the revolution in information and communication technology took place. Students, by contrast, are so-called 'digital natives': they grew up surrounded by digital technology. Present day students are used to multi-tasking and expect to be facilitated in using educationalfacilities regardless of time and place. Adapting higher education to present day students' study behaviour and expectations requires reconsideration of educationalform and methods. Several types of staff can be distinguished in their attitude towards technological innovation in education. Among them are staff who are reluctant in accepting innovations. Dental schools face the challenge of finding supportfor innovations with all their teaching staff and to better adapt to the twenty-first century student. In order to introduce technological innovations successfully, students need to become involved and sufficient attention must be paid to qualifying instructors.

Redd1 is a novel marker of testis development but is not required for normal male reproduction.

In an effort to identify novel candidate genes involved in testis determination, we previously used suppression subtraction hybridisation PCR on male and female whole embryonic (12.0-12.5 days post coitum) mouse gonads. One gene to emerge from our screen was Redd1. In the current study, we demonstrate by whole-mount in situ hybridisation that Redd1 is differentially expressed in the developing mouse gonad at the time of sex determination, with higher expression in testis than ovary. Furthermore, Redd1 expression was first detected as Sry expression peaks, immediately prior to morphological sex determination, suggesting a potential role for Redd1 during testis development. To determine the functional importance of this gene during testis development, we generated Redd1-deficient mice. Morphologically, Redd1-deficient mice were indistinguishable from control littermates and showed normal fertility. Our results show that Redd1 alone is not required for testis development or fertility in mice. The lack of a male reproductive phenotype in Redd1 mice may be due to functional compensation by the related gene Redd2.

Expression and functional analysis of Dkk1 during early gonadal development.

WNT signalling plays a central role in mammalian sex determination by promoting ovarian development and repressing aspects of testis development in the early gonad. Dickkopf homolog 1 (DKK1) is a WNT signalling antagonist that plays critical roles in multiple developmental systems by modulating WNT activity. Here, we examined the role of DKK1 in mouse sex determination and early gonadal development. Dkk1 mRNA was upregulated sex-specifically during testis differentiation, suggesting that DKK1 could repress WNT signalling in the developing testis. However, we observed overtly normal testis development in Dkk1-null XY gonads, and found no significant upregulation of Axin2 or Sp5 that would indicate increased canonical WNT signalling. Nor did we find significant differences in expression of key markers of testis and ovarian development. We propose that DKK1 may play a protective role that is not unmasked by loss-of-function in the absence of other stressors.

Analysis of gene function in cultured embryonic mouse gonads using nucleofection.

Mammalian sex determination is a dynamic process involving balanced gene expression leading to the development of either a testis or an ovary. Candidate sex-determining genes have been identified through microarray-based studies of gonadal gene expression; however, few methods exist for validation. This study describes a new technique for transfecting gonads using nucleofection. Fifteen micrograms of expression plasmid DNA was transfected into E11.5 gonads, cultured for 3 days and gene expression analyzed. Following optimization, we consistently achieved cell transfection efficiencies of 11% of cells using pMax-GFP plasmid. To test the applicability of nucleofection to studies of gene function, a testis-determining gene was transfected into gonads and its ability to sex-reverse was examined. When Sry was transfected into female (XX) gonads, upregulation of its target gene Sox9 was observed, as well as a downregulation of the ovarian gene Foxl2. Conversely, when shSox9 was introduced into male (XY) gonads, reduction of Sox9 and its target gene, Amh was observed, with a concomitant upregulation of Foxl2. Nucleofection-based gene delivery can recapitulate in vivo events of gonadal development that demonstrates 'proof-of-principle' of the method as a screening tool to evaluate the cellular function of potential sex-determining and gonadal differentiation genes.

The delicate balance between male and female sex determining pathways: potential for disruption of early steps in sexual development.

Testes and ovaries develop from the same primordial structures, the genital ridges, in the mammalian foetus. Male development depends critically on the correct functioning of the Y-linked testis-determining gene, Sry. However, Sry is highly vulnerable to mutation, and so does not provide a very robust sex-determining mechanism. Both in testes and in ovaries, proper gonadal development involves co-ordinated regulation of the bipotential fates of a number of different cell lineages, and is dependent on intercellular signalling mechanisms. If either the testicular or ovarian pathway stalls in the early stages, mechanisms operate to engage the alternative pathway. For these reasons, the early steps in mammalian sexual development are vulnerable to genetic and environmental perturbation, and represent possible points of action of endocrine disrupting compounds.

Profiles of gonadal gene expression in the developing bovine embryo.

Our current understanding of the molecular basis of sex determination and gonadal development in humans is mostly an extrapolation of knowledge gained from studies in the mouse. However, the timing of gene expression in the mouse is unusual among mammals, and it is therefore important that data from other models are also available to help elucidate this pivotal process in human development. Here we describe the sequence of molecular and morphological events marking testis differentiation in bovine embryos. The genital ridges first appeared at CRL 12 (day 32). SRY expression began at CRL 18 (day 37) and peaked at CRL 20 (day 39), leading to a cascade of regulatory, signaling, and steroidogenic gene expression at later stages, detected by quantitative real-time RT-PCR and immunohistochemistry. Testis cords were distinguishable at CRL 27 (day 42). We conclude that the timing of gene expression observed in developing human embryos is much more similar to bovine development than it is to the mouse. Therefore, Bos taurus may represent a useful model in which to study gene expression during sex determination, relevant to human development.

Identification of suitable normalizing genes for quantitative real-time RT-PCR analysis of gene expression in fetal mouse gonads.

In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.

[Is there a future for administrative psychiatry?]. / Heeft beleidspsychiatrie toekomst?

The role of the psychiatrist as administrator was first defined in the psychiatrist profile of the Dutch Psychiatric Association (1996). According to that profile the psychiatrist was the 'playing captain' of a multidisciplinary team. However, this phraseology was no longer used in the revised profile (2005); there the psychiatrist had become primarily a medical specialist. As a result of the broad acceptance of the Canmeds competence-based training, the management of psychiatry has now become one of the seven core skills of the psychiatric profession. Competence-based training in the future will put more emphasis on management skills, the psychiatrist will once more become a 'playing captain' and there will again be a future for administrative psychiatry.

Involvement of homeobox genes in mammalian sexual development.

In eutherian mammals, sexual development hinges on the differentiation of the gonadal primordia into either testes or ovaries, in turn determined by the activity of the Y-linked gene Sry. Organogenesis of the gonads requires input from a complex network of morphogenetic signals, many of which are still uncharacterised. Homeobox genes are known to play important roles in embryo morphogenesis and organogenesis in a number of systems. Here, we review current data relating to possible roles of the extended homeobox gene family on gonad development, sex-specific morphogenesis and gametogenesis, and speculate that these genes may play a broader role in these processes than is currently suspected.

Characterisation of urogenital ridge gene expression in the human embryonal carcinoma cell line NT2/D1.

The study of the mammalian sex-determining pathway has been hampered by the lack of cell culture systems to investigate the underlying molecular relationships between sex-determining genes. Recent approaches using high-throughput genome-wide studies have revealed a number of sexually dimorphic genes expressed in the developing mouse gonad. Here, we investigated a human testicular cell line in terms of its expression of known sex-determining genes and newly identified candidates. The human embryonal carcinoma cell line NT2/D1 was screened for the expression of 46 genes with known or potential roles in the sex-determining and differentiation pathway. Forty genes tested were expressed in NT2/D1 cells including the testis-determining genes SRY, SOX9, SF-1, DHH and FGF9. Genes not expressed included WT1, DAX1 and the ovary-specific genes FOXL2 and WNT4. Cell-specific markers demonstrate that NT2/D1 cells reflect a number of cell types in the gonad including Sertoli, Leydig and germ cells. Our results suggest that male pathways initiated by SRY, SOX9 and SF-1 remain intact in these cells. Lack of expression of ovary-specific genes is consistent with a commitment of these cells to the male lineage. Manipulation of gene expression in this cell line could be an important new in vitro tool for the discovery of new human sex-determining genes.

Comparative analysis of anti-mouse SRY antibodies.

The Y chromosome gene SRY is the initiator of male sexual differentiation in mammals, but the molecular and cellular mechanisms operating downstream of SRY remain undefined. A deeper understanding of these issues relies on the ability to visualize SRY protein endogenously under a number of experimental conditions. Here we compare the specificity and effectiveness of several available antibodies to mouse SRY. Two antibodies cross-reacted with other SOX proteins in immunofluorescence analyses of transfected cells, and one of these two was unable to detect SRY on Western blots. A third antibody was both avid and specific, and was able to detect endogenous SRY in developing Sertoli cells in mouse genital ridges. Our findings underline the need to distinguish between useful and spurious reagents for biochemical and immunolocalization studies involving mouse SRY protein.

Regulation of Amh during sex determination in chickens: Sox gene expression in male and female gonads.

During mammalian sexual development, the SOX9 transcription factor up-regulates expression of the gene encoding anti-Müllerian hormone (AMH), but in chickens, Sox9 gene expression reportedly occurs after the onset of Amh expression. Here, we examined expression of the related gene Sox8 in chicken embryonic gonads during the sex-determining period. We found that cSox8 is expressed at similar levels in both sexes at embryonic day 6 and 7, and only at the anterior tip of the gonad, suggesting that SOX8 is not responsible for the sex-specifi c increase in cAmh gene expression at these stages. We also found that several other chicken Sox genes (cSox3, cSox4 and cSox11) are expressed in embryonic gonads, but at similar levels in both sexes. Our data suggest that the molecular mechanisms involved in the regulation of Amh genes of mouse and chicken are not conserved, despite similar patterns of Amh expression in both species.

Sox genes and cancer.

Sox genes encode transcription factors belonging to the HMG (High Mobility Group) superfamily. They are conserved across species and involved in a number of developmental processes. In vitro studies have shown at least one Sox gene to be capable of inducing oncogenic transformation of fibroblast cells. In addition, overexpression and/or amplification of Sox genes are associated with a large number of tumour types in vivo. We review here the available evidence linking Sox gene expression and cancer, and show that this link is supported by extensive EST database analysis. This work provides a basis for further studies aimed at investigating the possible role of Sox genes in the oncogenic process.

Molecular characterization of three gonad cell lines.

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce SOX9 transcription upon expression of SRY, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate SOX9 expression upon transfection of SRY-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of SOX9 expression during testis determination.

Dppa3 is a marker of pluripotency and has a human homologue that is expressed in germ cell tumours.

We identified a transcript named 11M2 on the basis of its strong male-specific expression pattern in the developing mouse gonad. 11M2 was found to be expressed by gonad primordial germ cells (PGCs) of both sexes and down-regulated in female PGCs as they enter prophase I of the first meiotic division, similar to the expression of OCT4. Mouse EST analysis revealed expression only in early-stage embryos, embryonic stem cells and pre-meiotic germ cells. 11M2 corresponds to a recently reported gene variously known as PGC7, STELLA or DPPA3. We have identified the human orthologue of DPPA3 and find by human EST analysis that it is expressed in human testicular germ cell tumours but not in normal human somatic tissues. The expression patterns of mouse and human DPPA3, in undifferentiated embryonic cells, embryonic germ cells and adult germ cell tumours, together suggest a role for this gene in maintaining cell pluripotentiality.

Origin and possible roles of the Sox8 transcription factor gene during sexual development.

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a critical gene involved in mammalian sex determination and differentiation. Both genes encode proteins with the ability to bind similar DNA target sequences, and to activate transcription in in vitro assays. Expression studies indicate that the two genes have largely overlapping patterns of activity during mammalian embryonic development. A knockout of Sox8 in mice has no obvious developmental phenotype, suggesting that the two genes are able to act redundantly in a variety of developmental contexts. In particular, both genes are expressed in the developing Sertoli cell lineage of the developing testes in mice, and both proteins are able to activate transcription of the gene encoding anti-Müllerian hormone (AMH), through synergistic action with steroidogenic factor 1 (SF1). We have hypothesized that Sox8 may substitute for Sox9 in species where Sox9 is expressed too late to be involved in sex determination or regulation of Amh expression. However, our studies involving the red-eared slider turtle indicate that Sox8 is expressed at similar levels in males and females throughout the sex-determining period, suggesting that Sox8 is neither a transcriptional regulator for Amh, nor responsible for sex determination or gonad differentiation in that species. Similarly, Sox8 is not expressed in a sexually dimorphic pattern during gonadogenesis in the chicken. Since a functional role(s) for Sox8 is implied by its conservation during evolution, the significance of Sox8 for sexual and other aspects of development will need to be uncovered through more directed lines of experimentation.