Role Of Genes In Development Research Articles

Mouse Cre drivers: tools for studying disorders of the human female neuroendocrine-reproductive axis†.

Benign disorders of the human female reproductive system, such primary ovarian insufficiency and polycystic ovary syndrome are associated with infertility and recurrent miscarriage, as well as increased risk of adverse health outcomes, including cardiovascular disease and type 2 diabetes. For many of these conditions, the contributing molecular and cellular processes are poorly understood. The overarching similarities between mice and humans have rendered mouse models irreplaceable in understanding normal physiology and elucidating pathological processes that underlie disorders of the female reproductive system. The utilization of Cre-LoxP recombination technology, which allows for spatial and temporal control of gene expression, has identified the role of numerous genes in development of the female reproductive system and in processes, such as ovulation and endometrial decidualization, that are required for the establishment and maintenance of pregnancy in mammals. In this comprehensive review, we provide a detailed overview of Cre drivers with activity in the neuroendocrine-reproductive axis that have been used to study disruptions in key intracellular signaling pathways. We first summarize normal development of the hypothalamus, pituitary, ovary, and uterus, highlighting similarities and differences between mice and humans. We then describe human conditions resulting from abnormal development and/or function of the organ. Finally, we describe loss-of-function models for each Cre driver that elegantly recapitulate some key features of the human condition and are associated with impaired fertility. The examples we provide illustrate use of each Cre driver as a tool for elucidating genetic and molecular underpinnings of reproductive dysfunction.

Identifying the role of ASPN and COMP genes in knee osteoarthritis development

BackgroundOsteoarthritis (OA) is a common cause of musculoskeletal disability among elders and is characterized by late-onset degeneration of articular cartilage. OA affects various joints, commonly hand, knee, and hip, with clinical features that are unique to each joint. This study was initiated to identify and evaluate the role of the ASPN and COMP genes in the development of knee OA.MethodsA case–control study was carried out involving 500 cases with knee OA (diagnosed by the American College of Rheumatology) and an equal number of healthy controls. Blood was drawn for genomic DNA isolation. PCR-RFLP and TaqMan assay methods were used to identify the SNPs. mRNA and protein expression of genes were carried out in peripheral blood lymphocytes (PBLs) by RT-PCR and Western immunoblotting. The data obtained were analyzed for the statistical significance between control and case groups.ResultsThe variant genotype of ASPN and COMP genes was found to be present at a relatively higher frequency in cases than controls. RT-PCR and immunochemical studies revealed increased mRNA and protein expression of such gene in PBLs isolated from cases of knee OA as compared to healthy control.ConclusionThe allelic alteration in ASPN and COMP genes in knee OA cases points to the role of these genes in the development of knee OA. Further, increased mRNA and protein expression of ASPN and COMP in peripheral blood samples of patients with the disease suggest that expression profile of candidate gene could be used as a biomarker for predicting the development and progression of knee OA.

Editorial

Editorial

Salome Gluecksohn-Waelsch. 6 October 1907—7 November 2007

Salome Gluecksohn-Waelsch was a pioneer in establishing the field of mammalian developmental genetics, bringing together experimental embryology and genetics at a time when the role of genes in development was far from accepted. She studied in Germany in the 1930s with the renowned experimental embryologist Hans Spemann and then moved to New York City where she spent her entire professional career at Columbia University and the Albert Einstein College of Medicine of Yeshiva University. Her career was remarkable not only for its longevity—she continued experiments well into her 90s—but also for ushering in new ways of approaching developmental biology in mammals. In her studies of theT-complex in mice, she made use of naturally occurring mutations as nature's own experiments that allowed the investigation of the normal role of the genes in the events of morphogenesis. In her later work with the albino chromosomal deletions, she extended her studies to the genetics of physiological traits. Throughout the decades that saw a blossoming of the entire field of genetics, Salome Gluecksohn-Waelsch's work tackling some of the most perplexing problems in mammalian genetics firmly established the mouse as model organism, not only for studying development, but also for the eventual application of molecular biology techniques to development. Her published work is a beautifully coherent and rigorous opus, for which she received many honours. Her influence on a generation of geneticists, developmental biologists and the field of developmental genetics was profound.The life of Salome Gluecksohn–Waelsch spanned a century that suffered the destructive upheaval of two world wars but also saw phenomenal progress in the sciences, including embryology and genetics. At the start of Salome's career, these two fields were far apart and developmental genetics was barely a concept. Along with a few other pioneers, Salome was instrumental in establishing that genes actually had roles in development and in founding the field of mammalian developmental genetics. Her career laid the ground work for the eventual integration of genetic and developmental studies through molecular biology.Salome Gluecksohn–Waelsch published under four different names at different stages of her life and career: Salome Glücksohn, Salome Gluecksohn–Schoenheimer, Salome Gluecksohn–Waelsch, and Salome G. Waelsch. Among her colleagues and friends, she was almost universally known as Salome and so for the purpose of this biographical memoir, I have chosen to refer to her by her first name, out of friendship and respect.

Evolution and development of handedness: An Evo-Devo approach.

Hand preference is a sensorimotor skill whose development both reflects and promotes the development of hemispheric lateralization for manual and cognitive functions. Extensive comparative, crosscultural, and paleoanthropological evidence demonstrates the prevalence of limb lateralized preferences across vertebrate species and the prevalence of right-handedness within hominid evolution. Many reviews of the evolution and development of human handedness have proposed adaptive explanations for its evolution. However, during the last 3 decades a new approach to understanding evolution (the Extended Evolutionary Synthesis-EES) provided a persuasive alternative to the conventional (Neo-Darwinian Synthetic Theory-ST) evolutionary and developmental accounts. EES combines modern evolutionary and developmental research (Evo-Devo) in ways that alter understanding of natural selection, adaptation, and the role of genes in development and evolution. These changes make obsolete all past accounts of the evolution and development of lateralization and handedness because EES/Evo-Devo requires new study designs. The developmental trajectories of any structural or functional trait must be specified so that it may be related to variations in the developmental trajectories of other traits. First, we describe how the EES/Evo-Devo differs from the conventional ST, particularly for understanding of how traits develop. Then, we apply Evo-Devo to the study of handedness development in infancy and its relation to the development of other cognitive functions. Finally, we argue that identifying the development of atypical traits would benefit from knowledge of the range of individual differences in typical developmental trajectories of hand-use preference and their relation to variations in the developmental trajectories of cognitive functions.

Functional Genomics and Seed Development in Medicago truncatula: An Overview.

The study of seed development in the model species Medicago truncatula has made a significant contribution to our understanding of this process in crop legumes. Thanks to the availability of comprehensive proteomics and transcriptomics databases, coupled with exhaustive mutant collections, the roles of several regulatory genes in development and maturation are beginning to be deciphered and functionally validated. Advances in next-generation sequencing and the availability of a genomic sequence have made feasible high-density SNP genotyping, allowing the identification of markers tightly linked to traits of agronomic interest. A further major advance is to be expected from the integration of omics resources in functional network construction, which has been used recently to identify "hub" genes central to important traits.

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

Towards a comprehensive picture of the genetic landscape of complex traits.

The formation of phenotypic traits, such as biomass production, tumor volume and viral abundance, undergoes a complex process in which interactions between genes and developmental stimuli take place at each level of biological organization from cells to organisms. Traditional studies emphasize the impact of genes by directly linking DNA-based markers with static phenotypic values. Functional mapping, derived to detect genes that control developmental processes using growth equations, has proven powerful for addressing questions about the roles of genes in development. By treating phenotypic formation as a cohesive system using differential equations, a different approach-systems mapping-dissects the system into interconnected elements and then map genes that determine a web of interactions among these elements, facilitating our understanding of the genetic machineries for phenotypic development. Here, we argue that genetic mapping can play a more important role in studying the genotype-phenotype relationship by filling the gaps in the biochemical and regulatory process from DNA to end-point phenotype. We describe a new framework, named network mapping, to study the genetic architecture of complex traits by integrating the regulatory networks that cause a high-order phenotype. Network mapping makes use of a system of differential equations to quantify the rule by which transcriptional, proteomic and metabolomic components interact with each other to organize into a functional whole. The synthesis of functional mapping, systems mapping and network mapping provides a novel avenue to decipher a comprehensive picture of the genetic landscape of complex phenotypes that underlie economically and biomedically important traits.

Localized all-cell knock-out (LACKO) strategy is needed for studying adult stage diseases

Knock-out (KO) mouse models have been increasingly used to dissect the roles of genes in development, diseases, and injuries. The conventional KO approach allows study of the role of the targeted genes in all cells, but it sometimes results in embryonic lethality. Using the classical conditional KO approach, reseachers can avoid embryonic lethality, but they cannot modulate genes in a temporally controllable way. The inducible KO technique, which has been used to study the role of a gene in life processes at the adult stage, avoids the potential interfering role of changed structures and functions of the tissues/organs resulting from the early KO of the gene in the non-inducible conditional knock-out approach. However, it is difficult to develop clinically applicable therapies for some diseases or injuries based on the results obtained from inducible KO studies since the total summed role of the genes of interest in those diseases or injuries cannot be determined and, therefore, the potential therapeutic effects of the applied modulators of the activity of the targeted genes cannot be predicted. To solve this problem of the classical conditional and inducible KO approaches, researchers need to simultaneously knock out a gene in all cells locally-a process called the localized all-cell KO (LACKO) strategy. We describe the concept of this new strategy in detail in this article.

Deregulation of Developmental Genes in Pancreatic Malignancies

The concept that neoplastic diseases are characterized by a deregulation of the genes that control, during embryogenesis, the correct development it has now been fully accepted by the scientific community. In fact, the common mechanisms between the two processes are very numerous, and for this reason at the end of eighties, the role of developmental genes in neoplastic transformation and progression has been investigated. A pivotal role, in this context, is played by the homeoboxcontaining genes. These genes represent a wide family, subdivided in several classes, of transcription factors mostly involved with the determination of the developmental identity of animal body plan [1]. Their alteration in cancer has been widely described in the last twenty years, particularly for tumors of the gastrointestinal tract [2]. The homeobox genes control embryonic development of the pancreas, especially some genes of the subfamily PAX, such as PAX 4, involved in the differentiation of pancreatic beta cells [3]. But, the gene involved mainly in morphogenesis and functionality of the pancreas, is PDX-1, whose altered expression has been described abundantly in pancreas tumor evolution and progression [4,5]. Another homeobox gene described as a prognostic marker in this tumor is CDX-2, able to down regulate cyclin D1 by inhibiting pancreatic cancer cell proliferation [6]. Furthermore, in several studies it has been shown the pivotal role played by homeobox genes of class 1 (HOX genes in humans) in the pancreas development and carcinogenesis [7]. HOX B2 is expressed aberrantly in pancreatic cancer cells and is associated with a poor prognosis [8]. Moreover, downregulation of HOXD13 is strongly related to clinic outcome of patients with pancreatic tumors [9]. Finally, more recently, it was shown that HOXB7 is able to promote invasion and predict survival in pancreatic adenocarcinoma [10]. In future, the use of ever more advanced and reliable techniques, as gene arrays and tissue microarrays, will allow us to evaluate the simultaneous expression of these markers on large casuistries of pancreatic malignancies, permitting not only to establish their prognostic value, but also to stratify patients to establish new therapeutic strategies. For many homeobox genes this possibility has already been made in other human cancers, while only one study was performed for targeting of PDX1 in pancreatic tumor cells [11]. However, more accurate functional studies are require for investigating the aberrant activity of these developmental-related genes, and for establishing personalized therapies for pancreatic tumors, as well as has happened for many other known biomarkers.

Developmental aspects of cardiac arrhythmogenesis

The transcriptional regulation orchestrating the development of the heart is increasingly recognized to play an essential role in the regulation of ion channel and gap junction gene expression and consequently the proper generation and conduction of the cardiac electrical impulse. This has led to the realization that in some instances, abnormal cardiac electrical function and arrhythmias in the postnatal heart may stem from a developmental abnormality causing maintained (epigenetic) changes in gene regulation. The role of developmental genes in the regulation of cardiac electrical function is further underscored by recent genome-wide association studies that provide strong evidence that common genetic variation, at loci harbouring these genes, modulates electrocardiographic indices of conduction and repolarization and susceptibility to arrhythmia. Here we discuss recent findings and provide background insight into these complex mechanisms.

Use of fully modified 2′‐O‐methyl antisense oligos for loss‐of‐function studies in vertebrate embryos

Antisense oligonucleotides are commonly employed to study the roles of genes in development. Although morpholino phosphorodiamidate oligonucleotides (morpholinos) are widely used to block translation or splicing of target gene products' the usefulness of other modifications in mediating RNase-H independent inhibition of gene activity in embryos has not been investigated. In this study, we investigated the extent that fully modified 2'-O-methyl oligonucleotides (2'-OMe oligos) that can function as translation inhibiting reagents in vivo, using Xenopus and zebrafish embryos. We find that oligos against Xenopus β-catenin, wnt11, and bmp4 and against zebrafish chordin (chd), which can efficiently and specifically generate embryonic loss-of-function phenotypes comparable with morpholino injection and other methods. These results show that fully modified 2'-OMe oligos can function as RNase-H independent antisense reagents in vertebrate embryos and can thus serve as an alternative modification to morpholinos in some cases.

The innate and the acquired: Useful clusters or a residual distinction from folk biology?

The idea of the innate and the acquired is a part of folk-biology but is also used by biologists, psychologists and cognitive scientists in their disciplines. Are they right to do so? Innateness is often defined by appealing to the role of genes in development, to the role of Darwinian evolution in shaping developmental processes, to the non-involvement of learning during development, to developmental robustness, and to modularity. We argue that all such definitions are unsatisfactory. Some are unsatisfactory because they are based on simplistic and empirically outmoded views of development. Others are empirically defensible but are unsatisfactory because they do not capture the full breadth of the use of the term "innate" and, due to this restriction, they can easily lead to inferential mistakes. The definition of acquired behavior has been used with greater sophistication and is generally regarded as being heterogeneous. Nevertheless, in as much as the overall category has been seen in opposition to the innate, it has been an obstacle to a thorough investigation of how behavior develops. We suggest that a useful way forward is to examine whether or not the empirically well-established properties often associated with the concept of innate and the concept of acquired form theoretically useful clusters. This path leads to a much fuller appreciation of the view favored by Gilbert Gottlieb, according to which development involves the continuous interplay of the organism (and its genes) with its environment.

Prospero Mutants Induce Precocious Sexual Behavior in Drosophila Males

Brain maturation, a developmental process influenced by both endogenous and environmental factors, can affect sexual behavior. In vertebrates and invertebrates, sexual maturation is under the influence of hormones and neuromodulators, but the role of developmental genes in this process is still poorly understood. We report that prospero (pros), a gene crucial for nervous system development, can change the age of onset of sexual behavior in Drosophila melanogaster males: adult males carrying a single copy of several pros mutations court females and mate at a younger age than control males. However, these pros mutations had no effect on female sexual receptivity and did not alter other male phenotypes related to mating behavior. The Pros protein was detected in several brain and sensory structures of immature adult males, some of which are normally involved in the regulation of male specific behaviors. Our data suggest that the altered pros expression affects the age of onset of male mating behavior.

The Role of Developmental Genes in Chordoma

Chordoma is a rare tumor arising from notochordal remnants, and most often located at either end of the spinal axis. Recently gene expression analysis has revealed that brachyury, a tissue-specific transcription factor expressed in the nucleus of notochord cells, is highly expressed in chordoma, but was not found to be expressed in a wide variety of non-neoplastic tissues or 42 other types of neoplasms. Since brachyury is essential for notochord development and maintenance, we hypothesized that brachyury plays a pathogenic role in chordoma development and/or survival. To test this hypothesis, we propose to knock-down expression of brachyury in chordoma cells in vitro using RNAi techniques and assess the effect of reduced brachyury expression on cellular phenotype, including cellular proliferation, growth in soft agar, wound healing, and apoptosis. If our work further implicates brachyury in chordoma, future work would utilize that information to guide development of targeted therapeutics for chordoma.

Preparation, Culture, and Immortalization of Mouse Embryonic Fibroblasts

Genetically manipulated transgenic and gene-targeted mouse models are indispensible tools used in defining the role of genes in development and organ function. Similarly, cells isolated from these mice bear the same genetic alterations and therefore can be extremely useful for studying the molecular and cellular mechanisms and regulatory gene networks involving the mutated gene under well defined culture conditions. In this unit, detailed protocols regarding isolation of mouse embryonic fibroblasts (MEFs) from mouse embryos, culture of MEFs, and immortalization of MEFs are described. These protocols should be particularly useful to those investigators who intend to establish primarily cultured MEFs and to immortalize primary MEFs into stable cell cultures with a permanent growth feature from wild-type and mutant mouse embryos.

Neural cells without functional N-Methyl- d-Aspartate (NMDA) receptors contribute extensively to normal postnatal brain development in efficiently generated chimaeric NMDA R1 −/− → +/+ mice

Embryonic stem (ES) cells have revolutionised our understanding of animal physiology. Analysis of chimaeric mice generated from these cells allows us to study the role of genes in development and function of the nervous system. The NMDA receptor, one of the two major ionotropic glutamate receptors, has been proposed to play fundamental roles in the survival, migration, differentiation, and activity-dependent maturation of neural cells. The NMDA receptor subunit 1 (NR1) gene is indispensable for receptor function, and knock-out mice die at birth, inhibiting the study of glutamate signalling in postnatal neurons. Homozygous NR1−/− ES cells were derived from matings of heterozygous mice under feeder-free conditions. Chimaeras were made by incorporating these ES cells into wild-type blastocysts and by the classical aggregation of morulae between wild-type and NR1−/− embryos. The resulting chimaeras survive and develop normally. NR1−/− neurons, identified by their lacZ label, were analysed and quantified in developing and adult brains with varying knock-out contributions in every single brain region. Specifically, postnatal ontogenesis of cerebellum and hippocampus was normal. Accordingly, in chimaeric mice, NMDA receptor-initiated signals are not required for the migration, differentiation, and survival of most types of neurons in the central nervous system, in a cell-autonomous way.

Can it be a "sin" to understand disease? On "genes" and "eugenics" and an "unconnected connection".

Particularly, but not exclusively, in Germany, concerns are uttered as to the consequences of modern biotechnological advances and their range of applications in the field of human genetics. Whereas the proponents of this research are mainly focussing on the possible knowledge that could be gained by understanding the causes of developmental processes and of disease on the molecular level, the critics fear the beginnings of a new eugenics movement. Without claiming a logical relationship between genetic sciences and eugenics movements, it is nevertheless suggested in this article that a connection between both can become established when the distinction between scientifically validated statements on one hand and guiding hypotheses and assumptions on the other hand is blurred, as is observed particularly when scientists report their results to the public. This claim is demonstrated in comparisons between the current state of scientific knowledge on the role of genes in development and causation of diseases, and the way this is presented to the public. It is required that a debate on biotechnology should include reflections on the validity of claims made by scientists.

Genetic initiation, progression and prognostic markers in transitional cell carcinoma of the bladder: a summary of the structural and transcriptional changes, and the role of developmental genes.

Genetic initiation, progression and prognostic markers in transitional cell carcinoma of the bladder: a summary of the structural and transcriptional changes, and the role of developmental genes.