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Nadine Dobrovolska�a-Zavadska�a and the dawn of developmental genetics
Abstract
In one of the first genetic screens aimed at identifying induced developmental mutants, Nadine Dobrovolskaïa-Zavadskaïa, working at the Pasteur Laboratory in the 1920s, isolated and characterized a mutation affecting Brachyury, a gene that regulates tail and axial development in the mouse. Dobrovolskaïa-Zavadskaïa's analysis of Brachyury and other mutations affecting tail development were among the earliest attempts to link gene action with a tissue-specific developmental process in a vertebrate. Her analyses of genes that interacted with Brachyury led to the discovery of the t-haplotype chromosome of mouse. After 70 years, Brachyury and the multiple genes with which it interacts continue to occupy a prominent focus in developmental biology research. A goal of this review is to identify the contributions that Dobrovolskaïa-Zavadskaïa made to our current thinking about Brachyury and how she helped to shape the dawn of the field of developmental genetics. BioEssays 23:365-371, 2001.
... Hence the name of the mutation: Brachyury (from Greek, "short tail"; Dobrovolskaïa-Zavadskaïa, 1927). The history of this discovery has been described in detail Korzh and Grunwald, 2001). When summing up her studies of Brachyury mutants, Dobrovolskaya-Zavadskaya wrote in 1934 that the analysis of the mutant allowed her to formulate the question of the genetic mechanism responsible for the development of the tail. ...
Atavisms have attracted people’s attention for a long time. First, atavisms excited their imagination and created fertile ground for myths and superstitions. With the development of science, atavisms became the subject of investigation, which soon provided evidence to support evolutionary theory. However, at the molecular level, the formation of atavisms remained insufficiently understood. Recent progress in comparative genomics and molecular developmental biology has helped in understanding the processes underlying the formation of one of the human atavisms: the vestigial tail.
... Continuing with the example of the T-box family, tbxta performs in zebrafish most functions of T, the gene whose discovery laid at the foundation of developmental biology and gave rise to the name of the whole family of T-box genes. [10,11] Zebrafish tbxta was renamed 12 times, whereas its more divergent ohnolog, tbxtb, was renamed six times. The latest renaming of the T gene occurred simply for the convenience of bioinformaticians to avoid its historical one-letter name. ...
We developed an ex silico evolutionary‐based systematic synteny approach to define and name the duplicated genes in vertebrates. The first convention for the naming of genes relied on historical precedent, the order in the human genome, and mutant phenotypes in model systems. However, total‐genome duplication that resulted in teleost genomes required the naming of duplicated orthologous genes (ohnologs) in a specific manner. Unfortunately, as we review here, such naming has no defined criteria, and some ohnologs and their orthologs have suffered from incorrect nomenclature, thus creating confusion in comparative genetics and disease modeling. We sought to overcome this barrier by establishing an ex silico evolutionary‐based systematic approach to naming ohnologs in teleosts. We developed software and compared gene synteny in zebrafish using the spotted gar genome as a reference, representing the unduplicated ancestral state. Using new criteria, we identified several hundred potentially misnamed ohnologs and validated the principle manually.
... The emergence of developmental genetics in the 1980s and 1990s has shifted the focus of researches on embryonic development from the cell-and tissuelevel morphological events to the dynamics and interactions of gene activities. Developmental genetics has first become most successful in the fly Drosophila melanogaster and the mouse Mus musculus, each of which already has a long and distinctive history in genetic research dating back to the beginning of the twentieth century (Kohler 1993;Korzh and Grunwald 2001). Due to its relative simplicity and the availability of highly powerful genetic tools, Drosophila has made the most groundbreaking contributions to our current understanding of animal development (Lawrence 1992 (Gurdon et al. 1958). ...
In the nineteenth century and the first half of the twentieth century, comparative embryology has been indispensable for reconstructing the evolutionary history of Metazoa. The rise of molecular phylogeny and developmental genetics in the last decade of the twentieth century, however, has radically changed the role of comparative embryology in the study of animal evolution. Now, comparative embryology is no longer directly used in building phylogenetic trees, and the role of development in evolution has been recast as the mediator of morphological changes. The new technological developments have enabled investigators to study gene expression patterns and gene functions in embryonic development of many different animal species. By comparing developmental data from different species and reconstructing how developmental mechanisms evolved along the phylogenetic tree, it is now possible to imagine how animal body plans originated and evolved. Therefore, although the role of comparative embryology in evolution research has changed a lot in the past 50 years, it continues to be the forefront of Metazoan evolution research in the twenty-first century.
... The Brachyury protein, which has a critical role in notochord formation, is encoded by the T gene and has recently gained much attention due to its association with familial chordoma [3] and its role in epithelial-mesenchymal transition (EMT). The brachyury mutation was first described in mice by Nadine Dobrovolskaïa-Zavadskaïa as a mutation that affected tail length [4]. Mouse embryos lacking the T gene failed to form notochords, posterior regions, and the allantois; these embryos died at appro-ximately 10 days of gestation. ...
Being a rare malignant bone tumor on the axial skeleton, chordoma is locally invasive and has a high rate of recurrence. Despite extensive studies, the mechanisms of chordoma recurrence after surgical intervention, as well as resistance to radiation and chemotherapy, remain elusive. In this study, primary chordoma cell lines PCH1 and PCH2 were established and characterized by chordoma specific markers. We found that the embryonic transcription factor Brachyury inhibits Paclitaxel induced apoptosis in different cells, including PCH1 and U2OS cells. T gene regulated genes were identified in PCH1 and U2OS using microarray. After comparing gene regulated by Brachyury in different cells and the chromatin immunoprecipitation assay, we identified carbonic anhydrase IX (CA9) as a common target gene of Brachyury. Besides, immunohistochemical staining of CA9 and Brachyury in chordoma tissues revealed that their expression levels were positively correlated. We further showed that CA9 is responsible for Paclitaxel resistance in PCH1 cell. Our data suggest that CA9 plays a role in Brachyury mediated Paclitaxel resistance and serves as a potential target for chordoma treatment.
... Mesodermal precursor cell markers, T and Mixl1, were suppressed by high glucose during early GR-E14 cardiogenesis. T deficiency in mice resulted in early embryonic lethality due to defects in mesoderm formation [47]. Mixl1 is required for axial mesendoderm morphogenesis in differentiating ES cells and murine embryos [48,49]. ...
Background
Babies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. ESC maintenance and differentiation are currently performed under high glucose conditions, whose adverse effects have never been clarified. Method
We investigated the effect of high glucose on cardiomyocyte differentiation from a well-characterized ESC line, E14, derived from mouse blastocysts. E14 cells maintained under high glucose (25 mM) failed to generate any beating cardiomyocytes using the hanging-drop embryonic body method. We created a glucose-responsive E14 cell line (GR-E14) through a graduated low glucose adaptation. The expression of stem cell markers was similar in the parent E14 cells and the GR-E14 cells. ResultsGlucose transporter 2 gene was increased in GR-E14 cells. When GR-E14 cells were differentiated into cardiomyocytes under low (5 mM) or high (25 mM) glucose conditions, high glucose significantly delayed the appearance and reduced the number of TNNT2 (Troponin T Type 2)-positive contracting cardiomyocytes. High glucose suppressed the expression of precardiac mesoderm markers, cardiac transcription factors, mature cardiomyocyte markers, and potassium channel proteins. High glucose impaired the functionality of ESC-derived cardiomyocytes by suppressing the frequencies of Ca2+ wave and contraction. Conclusions
Our findings suggest that high glucose inhibits ESC cardiogenesis by suppressing key developmental genes essential for the cardiac program.
... Mutations in the Brachyury (also known as T for tail) gene cause short, blunt tails in heterozygous mice (T/+), whereas T/T homozygotes die in utero (Dobrovolskaia-Zavadskaia 1927; Korzh and Grunwald 2001). Subsequent embryological investigations showed that mutant embryos had severe mesoderm abnormalities and failed to form a notochord (Chesley 1935; Gluecksohn-Schoenheimer 1944). ...
Mutations in the mouse Brachyury (T) gene are characterized by a dominant reduction of tail length and recessive lethality. Two quantitative trait loci, Brachyury-modifier 1 and 2 (Brm1 and Brm2) are defined by alleles that enhance the short-tail Brachyury phenotype. Here we report on a genetic analysis of a visible dominant mutation Abnormal feet and tail (Aft) located in the vicinity of Brm1. Affected animals display kinky tails and syndactyly in the hindlimbs, both likely resulting from a defect in apoptosis. We observed an unusual genetic incompatibility between Aft and certain genetic backgrounds. We show that Aft and T are likely to interact genetically, since some double heterozygotes are tailless. In addition to the tail and hindlimb phenotypes, Aft-bearing mutants display characteristic late-onset skin lesions. We therefore tested for allelism between Aft and a closely linked recessive mutation rough coat (rc) and found that these two mutations are likely nonallelic. Our results provide a valuable resource for the study of mammalian skin development and contribute to the genetic analysis of Brachyury function.
... The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis (for a review, see Korzh and Grunwald, 2001). The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development (Chelsey, 1935). ...
T-box transcription factors are important players in the molecular circuitry that generates lineage diversity and form in the developing embryo. At least seven family members are expressed in the developing mammalian heart, and the human T-box genes TBX1 and TBX5 are mutated in cardiac congenital anomaly syndromes. Here, we review T-box gene function during mammalian heart development in the light of new insights into heart morphogenesis. We see for the first time how hierarchies of transcriptional activation and repression involving multiple T-box factors play out in three-dimensional space to establish the cardiac progenitors fields, to define their subservient lineages, and to generate heart form and function.
... Homozygous mutations in the Brachyury gene, also referred to as T for tail, can have profound effects on the development of the notochord. Mice heterozygous for mutation in this gene exhibit blunt tails [35,36]. This T gene encodes a transcription factor [37], which is modulated by a number of Brachyurymodifier genes (Brm1 and Brm2) or Brachyury-interacting genes [38]. ...
CXCR2 plays an important role during cutaneous wound healing. Transgenic mice were generated using the keratin-14 promoter/enhancer to direct expression of wild-type human CXCR2 (K14hCXCR2 WT) or mutant CXCR2, in which the carboxyl-terminal domain (CTD) was truncated at Ser 331 and the dileucine AP-2 binding motif was mutated to alanine (K14hCXCR2 331T/LL/AA/IL/AA). Our results indicate that K14hCXCR2WT transgenic mice exhibited a normal phenotype, while K14hCXCR2 331T/LL/AA/IL/AA transgenic mice were born with tails of normal length, but three to eight days after birth their tails degenerated, leaving only a short tail stub. The tissue degeneration in the tail started between caudal somites with degeneration of bone and connective tissue distal to the constriction, which was replaced with stromal tissue heavily infiltrated with inflammatory cells. The tail lesion site revealed coagulation in enlarged vessels and marked edema that eventually led to loss of the distal tail. Moreover, 66% of the mice exhibited focal skin blemishes and inflammation that exhibited an increase in the number of sebaceous glands and blood vessels, enlargement of the hair follicles due to increased number of keratinocytes, reduction in the connective tissue content, and a thickening of the epidermis. Furthermore, immunohistochemical staining of the epidermis from tail tissue in the transgenic mice indicated a loss of the cell adhesion markers E-cadherin and desmoplakin. These data suggest that keratinocyte expression of a CTD mutant of CXCR2 has effects on homeostasis of the connective tissue in the tail, as well as the maintenance of the epidermis and its appendages.
The T / t locus was a major focus of study by mouse geneticists during the 20th century. In the 70s, as the study of cell surface antigens controlling transplantation antigens was taking off, several laboratories hypothesized that alleles of this locus would control cell surface antigens important for embryonic development. One such antigen, the embryonal carcinoma F9 antigen was said to be an example. Other antigens were described on sperm and embryos that were said to be controlled by alleles at the T / t complex. These findings were later found to be false. The history of the findings and their refutation is described.
The house mouse is the source of almost all genetic variation in laboratory mice; its genome was sequenced alongside that of humans, and it has become the model for mammalian speciation. Featuring contributions from leaders in the field, this volume provides the evolutionary context necessary to interpret these patterns and processes in the age of genomics. The topics reviewed include mouse phylogeny, phylogeography, origins of commensalism, adaptation, and dynamics of secondary contacts between subspecies. Explorations of mouse behaviour cover the nature of chemical and ultrasonic signalling, recognition, and social environment. The importance of the mouse as an evolutionary model is highlighted in reviews of the first described example of meiotic drive (t-haplotype) and the first identified mammalian speciation gene (Prdm9). This detailed overview of house mouse evolution is a valuable resource for researchers of mouse biology as well as those interested in mouse genetics, evolutionary biology, behaviour, parasitology, and archaeozoology.
Introduction. Skull base chordomas are rare slow-growing neoplasms that arise from notochord. Their morbidity is mainly related to highly aggressive local invasion and resistance to treatments. Due to its heterogeneous appearance and not fully understood clinical and molecular behaviors, the main goal of the present work is to identify clinical and bio-molecular markers as specific prognostic factors that could be used for the management of skull base chordoma patients. Achieving a detailed prognostic signature of skull base chordomas is of paramount importance to personalize the treatment to each specific patient. Moreover, sphingolipids analysis is emerging as a new approach in many cancers and it has never been applied in chordomas. Our aim is to investigate chordoma biological behavior and the role of ceramides production in this context of proliferation and invasion.
Patients and Methods. A retrospective review of all the patients diagnosed and treated for a skull base chordoma at the Fondazione IRCCS Istituto Neurologico “Carlo Besta” between January 1992 and December 2017 has been performed. Clinical, radiological, surgical and pathological data have been collected. A prospective collection of frozen surgical specimens has been performed to analyze ceramides species in chordomas. Sphingolipids were extracted from frozen tissues and total ceramides and dihydroceramides were evaluated by liquid chromatography and mass spectrometry. Survival analysis was performed according to Kaplan-Meier method. Univariate comparisons were conducted using Mann-Whitney, Chi-square and exact Fisher test. Simple linear regression and correlation with computation of Pearson coefficients analyses were conducted. Using a logistic regression model, statistically significant predictors were rated based on their odds ratios in order to build a personalized grading scale – the Peri-Operative Chordoma Scale (POCS).
Results. Eighty-seven consecutive patients were surgically treated for a skull base chordoma during the period of recruitment. Seventy-eight patients were eligible for the retrospective review. There were 38 males (48.7%) and 40 females (51.3%). The mean follow-up was 69 months (range, 3–233). One-hundred-fourteen surgical operations were performed in the initial recruitment or recurrent setting. The presence of motor deficits in skull base chordoma revealed to be a significant prognostic factor correlating with a worse PFS (p=0.0480). Calcification on KM analysis showed a correlation with better outcomes (OS) compared with tumor lacking any calcification on CT scan (p value=0.0402). The degree of MR contrast enhancement revealed to be a significant and strong prognostic factor in terms of OS and PFS (p≤0.0001 and 0.0010, respectively). Jugular foramen involvement represented a significant prognostic factor with a worse PFS in the cohort of primary skull base chordomas (p=0.0130). The presence of chordoma in the pre-brainstem cistern revealed to be a significant prognostic factor with a worse PFS in the cohort of recurrent skull base chordomas (p=0.0210). Brainstem dislocation represented a significant prognostic factor correlating with a both worse outcome in terms of OS and PFS in the cohort of recurrent skull base chordomas (p=0.0060 and 0.0030). Extent of resection represents a strong prognostic factor according to PFS in the cohort of primary skull base chordomas (p=0.0200). Patients operated by an experienced chordoma surgeon did better in terms of prolonged PFS in the cohort of primary patients (p=0.0340). Development of post-operative complications in primary skull base chordoma patients represented an important prognostic factor related to both OS and PFS (p≤0.0001 and 0.0360, respectively). In the cohort of recurrent chordomas, ∆KPS correlated to both OS and PFS (p=0.0010 and 0.0180, respectively). Moreover, post-operative radiation treatment correlated with prolonged OS (p=0.0020) and PFS (p=0.0100). The following factors were found to be statistically significant predictors of both PFS and OS in the logistic regression model: MR contrast enhancement (intense vs mild/no), preoperative motor deficit (yes vs no) and the development of any post-operative complications (yes vs no). A grading scale was obtained with scores ranging between 0 and 17 (Nagelkerke’s pseudo R2=0.656). The mean total ceramides and dihydroceramides species in primary chordomas were 808.4±451.4 pmol/mg (522.5-1760.2) and 30.7±16.4 pmol/mg (17.6-62.4), respectively. The mean total ceramides and dihydroceramides species in recurrent chordomas were 1488.1±763.8 pmol/mg (540.7-2787.5) and 67.2±45.5 pmol/mg (9.0-145.6), respectively. Total ceramides species were significantly higher in recurrent chordomas that underwent previous surgical resection and radiation therapy in comparison to the primary chordomas (p=0.0496). The mean total ceramides and dihydroceramides species in “intense enhancement” group were 1597.6±737.8 pmol/mg (592.7-2787.5) and 69.1±45.0 pmol/mg (17.8-145.6), respectively. The mean total ceramides and dihydroceramides species in “no or mild enhancement” group were 664.7±120.4 pmol/mg (522.5-826.0) and 31.5±13.6 pmol/mg (17.6-53.6), respectively. Total ceramides and dihydroceramides were significantly higher in “intense enhancement” chordomas in comparison to the “no/mild enhancement” chordomas (p=0.0290 and p=0.0186, respectively). Analyzing the association between ceramides level and MIB-1 within each skull base chordoma patient, total ceramides level showed a strong association (r=0.7257, r2=0.5267) with MIB-1 staining (p=0.0033). Analyzing the association between DHCer level and MIB-1 within each skull base chordoma patient, total DHCer level showed also strong association (r=0.6733, r2= 0.4533) with MIB-1 staining (p= 0.0083). Among the single ceramides species Cer C24:1 (r=0.8814, r2=0.7769, p≤0.0001), DHCer C24:1 (r=0.8429, r2=0.7104, p=0.0002) and DHCer C18:0 (r=0.9426, r2=0.8885, p≤0.0001) levels showed a significant correlation with MIB-1 staining. Final candidate predictive factors that well fitted the regression model were: cer24:1 (r=0.824, p≤0.001), and DHCer C18:0 (r=0.748, p=0.002).
Conclusion. Our clinical analysis showed that pre-operative clinical symptoms (motor and cranial nerve deficits), anatomical location (jugular foramen, pre-brainstem cisterns and brainstem dislocation), surgical features (extent of tumor resection and surgeon’s experience), development of post-operative complications and KPS decline represent significant prognostic factors. The degree of MR contrast enhancement significantly correlated to both OS and PFS. We also preliminarily developed the Peri-Operative Chordoma Scale (POCS) to aid the practitioner in the personalized management of patients undergoing potential adjuvant therapies. Our lipid analysis showed ceramides as promising tumoral bio-markers in skull base chordomas. Long and very long chain ceramides, such as Cer C24:1 and DHCer C24:1, may be related to a prolonged tumor survival, aggressiveness and the understanding of their effective biological role will hopefully shed lights on the mechanisms of chordoma radio-resistance, tendency to recur and use of agents targeting ceramide metabolism. Such results should be validated in future larger clinical, in-vitro and in-vivo studies to confirm such intricate link between ceramides and chordoma aggressive behavior.
Attempts to understand the inheritance of coat color and unusual behaviors such as waltzing in mice long preceded the re-discovery of Mendel’s laws in 1900. Thus, it may not be surprising that they were confirmed in 1901 by the Frenchman Lucien Cuenot in mice. However, the further development of mouse genetics was mostly to be an Anglophone event. Geneticists in England, and to a much larger extent, the United States were to establish mice as a major organism for genetic studies. Another major topic for mouse genetics, the T/t complex was also discovered in France but its continued study was elsewhere.
High osmolarity, bound water, and hydrostatic pressure contribute to notochord mechanics and its morphogenesis into the nucleus pulposus (NP) compartment of the intervertebral disc. Indeed, the osmoadaptive transcription factor, nuclear factor of activated T-cells 5 (NFAT5 aka TonEBP), is robustly expressed by resident cells of the notochord and NP. Nevertheless, the molecular mechanisms that drive notochord osmoregulation and the functions of NFAT5 in disc embryogenesis remain largely unexplored. In this study, we show that deletion of NFAT5 in mice results in delayed vertebral column development and a reduced NP aspect ratio in the caudal spine. This phenotype is associated with lower levels of the T-box transcription factor, Brachyury, delayed expression of notochord phenotypic markers, and decreased collagen II deposition in the perinotochordal sheath and condensing mesenchyme. In addition, NFAT5 mutants showed a stage-dependent dysregulation of sonic hedgehog (Shh) signaling with non-classical expression of Gli1. Generation of mice with notochord-specific deletion of IFT88 (ShhcreERT2;Ift88f/f) supported this mode of Gli1 regulation. Using isolated primary NP cells and bioinformatics approaches, we further show that Ptch1 and Smo expression is controlled by NFAT5 in a cell autonomous manner. Altogether, our results demonstrate that NFAT5 contributes to notochord and disc embryogenesis through its regulation of hallmark notochord phenotypic markers, extracellular matrix, and Shh signaling.
Diabetic nephropathy (DN) is a leading cause of end-stage renal disease in the developed world. Accordingly, an urgent need exists for new, curative treatments as well as for biomarkers to stratify risk of DN among individuals with diabetes mellitus. A barrier to progress in these areas has been a lack of animal models that faithfully replicate the main features of human DN. Such models could be used to define the pathogenesis, identify drug targets and test new therapies. Owing to their tractability for genetic manipulation, mice are widely used to model human diseases, including DN. Questions have been raised, however, about the general utility of mouse models in human drug discovery. Standard mouse models of diabetes typically manifest only modest kidney abnormalities, whereas accelerated models, induced by superimposing genetic stressors, recapitulate key features of human DN. Incorporation of systems biology approaches and emerging data from genomics and metabolomics studies should enable further model refinement. Here, we discuss the current status of mouse models for DN, their limitations and opportunities for improvement. We emphasize that future efforts should focus on generating robust models that reproduce the major clinical and molecular phenotypes of human DN.
The house mouse is the source of almost all genetic variation in laboratory mice; its genome was sequenced alongside that of humans, and it has become the model for mammalian speciation. Featuring contributions from leaders in the field, this volume provides the evolutionary context necessary to interpret these patterns and processes in the age of genomics. The topics reviewed include mouse phylogeny, phylogeography, origins of commensalism, adaptation, and dynamics of secondary contacts between subspecies. Explorations of mouse behaviour cover the nature of chemical and ultrasonic signalling, recognition, and social environment. The importance of the mouse as an evolutionary model is highlighted in reviews of the first described example of meiotic drive (t-haplotype) and the first identified mammalian speciation gene (Prdm9). This detailed overview of house mouse evolution is a valuable resource for researchers of mouse biology as well as those interested in mouse genetics, evolutionary biology, behaviour, parasitology, and archaeozoology.
The idea of a major developmental transition that includes the activation of the embryonic genome has a long history. In the 1950-1960s this concept was developed to a large extent due to the efforts of Alexander Neyfakh, who described a specific type of deleterious effect resulting from X-ray irradiation of fish eggs. He interpreted the radiation-sensitive target as the nucleus and established the onset of the function of the zygotic genome, naming it the morphogenetic function of nuclei, what we call now the midblastula transition. Most of his studies were performed using the loach (Misgurnus fossilis), a European teleost. Neyfakh's efforts paved the way to understanding the whole phenomenon of the maternal-zygotic transition.
Several factors contributed to the discovery of Wnt genes. On the one hand, the development of research tools in mammalian genetics such as mouse strains with high incidence of cancer in 1920-1930s led to discovery of the mouse mammary tumor virus in 1949. This virus in turn was used in 1982 to identify int-1 (Wnt1) in mammals. On the other hand, a discovery of the wingless mutant in 1973 resulted in cloning of the wingless (wg) gene in 1987. A comparative analysis of int-1 and wg led to the realization that these genes are evolutionarily related. This brought about a harmonization of names for a whole family of these genes now known as the Wnt family of genes. This review mainly focuses on what stood at the cradle of research on Wnt genes, and in particular on those elements that were nearly lost in history.
The BTBR mouse strain harbors alleles promoting insulin resistance. When made genetically obese (ob/ob), these mice develop severe type 2 diabetes (fasting glucose >400 mg/dL). By contrast, C57BL/6 ob/ob mice are able to compensate for the obesity-induced insulin resistance by increasing pancreatic insulin secretion and thus maintain only slightly elevated plasma glucose levels (<250 mg/dL). Islet insulin secretory responses to glucose are undiminished in the remaining islets of BTBR ob/ob mice. A genome-wide linkage analysis identified 3 major loci influencing plasma glucose and/or insulin levels in an F2ob/ob sample derived from the 2 strains. A locus on chromosome 2 affects insulin sensitivity and is independent of obesity. Loci on chromosomes 16 and 19 affect fasting glucose and insulin levels and likely affect beta-cell mass or function. Analysis of mRNA expression patterns revealed a reduction in lipogenic gene expression in adipose tissue associated with obesity. Conversely, hepatic lipogenic gene expression increases in obese mice, but to a much greater extent in the diabetes-resistant C57BL/6 strain. We propose that hepatic lipogenic capacity affects susceptibility to obesity-induced diabetes.
Inbred mouse strains provide genetic diversity comparable to that of the human population. Like humans, mice have a wide range of diabetes-related phenotypes. The inbred mouse strains differ in the response of their critical physiological functions, such as insulin sensitivity, insulin secretion, beta-cell proliferation and survival, and fuel partitioning, to diet and obesity. Most of the critical genes underlying these differences have not been identified, although many loci have been mapped. The dramatic improvements in genomic and bioinformatics resources are accelerating the pace of gene discovery. This review describes how mouse genetics can be used to discover diabetes-related genes, summarizes how the mouse strains differ in their diabetes-related phenotypes, and describes several examples of how loci identified in the mouse may directly relate to human diabetes.
See related article, pages 1433–1442
Cardiogenesis is a complex phenomenon: its success—and ultimately life births—depends on factors acting in a combinatorial or hierarchical fashion and turning on and off gene transcription. Actually, the incidence of cardiac defects at birth is relatively high (1% to 2%), and our comprehension of these phenomena very limited. Hence, the role of transcription factors in cardiac specification and maturation has claimed increasing attention in recent years, providing a complex and evolving picture of the molecular and cellular processes involved but leaving many questions unanswered. In this issue of Circulation Research , Postma et al1 add to a growing list of regulatory factors/functions a step forward in our understanding of cardiac morphogenesis and disclosing novel features of Tbx5.
Tbx5 belongs to the T-box gene family; the first member was identified in 1927 by a genetist who selected a mouse strain with truncated tail carrying a heterozygous mutation in a locus called T (reviewed in2). More than 60 years later the gene was cloned and named Brachyury (short-tail in Greek),3 but its functional role remained obscure because the T -gene product lacked homology to any previously characterized protein. Between 1993 and 1997, it was described as a novel DNA-binding protein and the crystallographic structure of the domain, termed the T-box , revealed a new feature of protein-DNA interaction.4 In recent years, studies in transgenic mice lines and in patients carrying spontaneous mutations have demonstrated that T-box genes act as crucial regulators for the morphogenesis of a wide range of tissues and organs (skeletal or genital apparatus, cardiovascular or endocrine system) and, when defective, as major contributors to several human syndromes.
To set T-box genes in their functional scenario, it is worthwhile to recall that they have been detected from the oocyte to …
Principles of Development
To analyse the roles of FGF activity and brachyury during gastrulation we have directly compared the consequences of inhibition of FGF-receptor signalling with the phenotype of the zebrafish brachyury mutant, no tail (ntl). We show that expression of ntl is regulated by FGF and that inhibition of FGF receptor-signalling leads to complete loss of the trunk and tail. Since the ntl mutant lacks the tail and notochord but has an otherwise normal trunk, this demonstrates that trunk development is dependent upon an unidentified gene, or set of genes, referred to as no trunk (ntk) which is regulated by FGF. We propose a model to explain the FGF-dependent regulation of ntl and ntk that accounts for the above phenotypes. Consistent with this model, over-expression of eFGF led to suppression of anterior fates and development of trunk and tail derivatives only. In addition, widespread activation of convergence and extension movements resulted in the formation of multiple axis-like structures. Expression of eve1 and cad1 was also regulated by FGF activity, suggesting that during gastrulation FGF activity is normally restricted to the germ ring where these genes, and ntl, are expressed. Taken together these data suggest that the germ ring acts as a posteriorising centre during AP patterning, mediated by FGF activity in this tissue.
The mouse T (Brachyury) gene is required for normal mesoderm development and the extension of the body axis. Recently, two mutant alleles of a zebrafish gene, no tail (ntl), have been isolated (Halpern, M. E., Ho., R. K., Walker, C. and Kimmel, C. B. (1993) Cell 75, 99-111). ntl mutant embryos resemble mouse T/T mutant embryos in that they lack a differentiated notochord and the caudal region of their bodies. We report here that this phenotype is caused by mutation of the zebrafish homologue of the T gene. While ntl embryos express mutant mRNA, they show no nuclear protein product. Later, expression of mRNA in mutants, but not in wild types, is greatly reduced along the dorsal midline where the notochord normally forms. This suggests that the protein is required for maintaining transcription of its own gene.
What is it that defines an animal? The definition provided here, made on the basis of developmental biology, suggests methods for resolving phylogenetic problems.
Tetrapod fore-and hindlimbs have evolved from the pectoral and pelvic fins of an ancient vertebrate ancestor. In this ancestor, the pectoral fin appears to have arisen following the rostral homeotic recapitulation of an existing pelvic appendage (Tabin and Laufer (1993), Nature 361, 692-693). Thus the basic appendage outgrowth program is reiterated in both tetrapod fore- and hindlimbs and the pectoral and pelvic fins of extant teleost fishes (Sordino et al. (1995) Nature 375, 678-681). Recently a novel family of putative transcription factors, which includes the T (Brachyury) locus, has been identified and dubbed the "T-box' family. In mice, all of these genes have expression patterns indicative of involvement in embryonic induction (Chapman et al. (1996) Dev. Dyn., in press), and four (Tbx2-Tbx5) are represented as two cognate, linked gene pairs (Agulnik et al., (1996), Genetics, in press). We now report that, whereas Tbx2 and Tbx3 are expressed in similar spatiotemporal patterns in both limbs, Tbx5 and Tbx4 expression is primarily restricted to the developing fore- and hindlimb buds, respectively. These observations suggest that T-box genes have played a role in the evolution of fin and limb morphogenesis, and that Tbx5 and Tbx4 may have been divergently selected to play a role in the differential specification of fore- (pectoral) versus hind- (pelvic) limb (fin) identity.
1. The irradiation of the testicles in mice, with all kinds of doses till that giving complete sterilisation, did not produce in the majority of cases (about 3000 descendants) any hereditary effect.
2. Two mutations only (on 35 irradiated breeders) were obtained: first, a waltzing mouse, breeding true as mendelian recessive, and second, a short‐tailed mouse, so far only known in the hybrid state, but mutating continuously and giving different new forms of tail (anoure, filiforme tail, kinky brachyure, helicoid type, interruption of the skeleton, etc.).
3. These two mutations—“waltzing” and “short tail”—had already been observed in this locality by previous investigators, and besides, one male of our own stock presented a small tail abnormality, which passed unperceived before the irradiation and which proved afterwards to be hereditary. The mutations obtained must not, therefore, be considered as new ones produced by the rays. It was concluded that the rays only revealed a pre‐existing latent state which otherwise would have remained undetected.
4. New evidence supporting the last assumption is the non‐appearance of waltzing or of any tail mutation in a new stock of more than three thousand control mice—descendants from a few normal parents and reared by inbreeding.
5. In the light of these results, the living hereditary material, as represented by different species, may be considered to be quite stable and incapable of being changed in its genetic structure, by any kind of external agency. This accounts for the existence of very ancient species on the earth, for the extreme rarety of the natural mutants, and for the negative results of a great number of attempts to change hereditary behaviour of animals and plants by artificial means.
6. The variability of species and the recorded cases of positive results in attempts to produce a new form by means of different artificial procedures, may be accounted for by the assumption that there exists in different species a certain number of scattered single individuals—potential mutants—whose hereditary material conceals different degrees of instability.
7. This instability probably remains very often unperceived; sometimes it may manifest itself, as well in laboratory conditions as in the state of nature, through spontaneous mutations—source of variation. But the rate of these mutations is in general a very low one, even in Drosophila. This rate may be greatly increased, and the concealed mutability of some apparently normal individuals brought to light, by means of different external agents, especially by irradiating the gonads of the animals used for breeding.
8. The utilisation of penetrating rays seems to be a good method, as they appear to have an elective action on the potential mutants, as was shown by our own findings and by all the recent successful experiments on Drosophila. Yet the rays can hardly be looked upon as a real cause of mutations, and, therefore, the term of “producing” mutations were better abandoned as a misleading one.
9. Hybridisation, natural selection, etc., cannot create new forms. An independent act of nature, creating at least a predisposition to change, precedes all other agencies, though it may not always be at once evident.
10. The rôle of natural selection, as a survival in competitive conditions, is to fix a few of the new forms which are better adapted to a given environment, and to eliminate the majority of them. Hence natural selection cannot be considered as an agent favouring the variability of species on the earth, it is rather an agent limiting this variability.
11. Artificial selection made for purposes of domestication and under laboratory conditions tends, on the contrary, to conserve those mutations appearing spontaneously, or revealed by artificial means, which would perish under nature conditions.
12. This hypothesis of stable species with single changeable individuals amongst them, which are the source of new forms on the earth, would settle numerous experimental controversies. Moreover, it is in full accord with the established impossibility of the inheritance of somatic fluctuations resulting from the adaptation of the individuals to environmental conditions.
13. The present hypothesis conceives the building of organic life resulting from the process of evolution as based on three pillars: (1) stability of existing species as the expression of the conservative principle of life, (2) variability of single individuals as the manifestation of the creative power of nature, and (3) natural selection as the casting away of products which appear less fitted for the struggle for existence under given conditions, and thus determining that the species are adapted to the environment. The struggle for existence plays a special part in phenotypic perfection of individuals.
Industrial methods, and industrially produced instruments, reagents and living organisms are central to research activities today. They play a key role in the homogenization and the diffusion of laboratory practices, thus in their transformation into a stable and unproblematic knowledge about the natural world. This book displays the - frequently invisible - role of industry in the construction of fundamental scientific knowledge through the examination of case studies taken from the history of nineteenth and the twentieth century physics, chemistry and biomedical sciences.
A novel family of transcription factors that appears to play a critical role in the development of all animal species was recently uncovered on the basis of homology to the DNA binding domain of the Brachyury, or T locus, gene product. Phylogenetic studies have shown the ancient origin of this gene family, which has been named the T-box family, prior to the divergence of metazoa from a common ancestral type. T-box genes have now been identified in the genomes of C. elegans, Drosophila, sea urchin, ascidian, amphioxus, Xenopus, chick, zebrafish, mouse, and human and will probably be found in the genomes of all animals. Although functional analyses of T-box family members have just begun, the results show a wide range of roles in developmental processes that extend over time from the unfertilized egg through organogenesis. Only a few mutations in T-box genes are known, but all have drastic effects on development, including a targeted mutation in mice causing an embryonic lethal phenotype, and two human T-box gene mutations that results in developmental syndromes. This review presents a current overview of progress made in the analysis of T-box genes and their products in a variety of model systems. BioEssays 20:9–19, 1998. © 1998 John Wiley & Sons, Inc.
There has been a revival of interest in the problems of differentiation and embryonic development that are considered to be among the frontiers of biology. The enthusiasm generated by the discoveries in the field of molecular biology seemed to justify the belief that when there is sufficiently extensive knowledge of microorganisms, it will find a solution to the problems of differentiation. The impact of molecular biology on the study of development has been essentially methodological and conceptual. One of the factors that have adversely affected research progress in developmental biology has been the divorce between genetics and embryology. The contribution of molecular biology that marked the turning point in the study of both cellular and developmental processes has been the concrete definition of the term “gene expression”; this has made it possible at last to design experiments aimed at investigating the nucleocytoplasmic interactions in the course of development. The study of RNA synthesis has allowed rapid advances in the interpretation of the processes of development. This has been largely due to the study of the synthesis of the ribosomal RNA.
A novel family of transcription factors that appears to play a critical role in the development of all animal species was recently uncovered on the basis of homology to the DNA binding domain of the Brachyury, or T locus, gene product. Phylogenetic studies have shown the ancient origin of this gene family, which has been named the T-box family, prior to the divergence of metazoa from a common ancestral type. T-box genes have now been identified in the genomes of C. elegans, Drosophila, sea urchin, ascidian, amphioxus, Xenopus, chick, zebrafish, mouse, and human and will probably be found in the genomes of all animals. Although functional analyses of T-box family members have just begun, the results show a wide range of roles in developmental processes that extend over time from the unfertilized egg through organogenesis. Only a few mutations in T-box genes are known, but all have drastic effects on development, including a targeted mutation in mice causing an embryonic lethal phenotype, and two human T-box gene mutations that results in developmental syndromes. This review presents a current overview of progress made in the analysis of T-box genes and their products in a variety of model systems. BioEssays 20:9–19, 1998.
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During embryonic development, initially similar fields can develop into distinct structures, such as the vertebrate fore- and hindlimbs. Although considerable progress has been made in our understanding of the genetic control underlying the establishment of the different limb axes, the molecular cues that specify the differential development of the fore- and hindlimbs are unknown. Possible candidates for genes determining limb identity are Pitx1, a gene whose transcripts are detected in the early hind- but not forelimb bud, and two members of the T-box (Tbx) gene family, Tbx4 and Tbx5, which are specifically expressed in the hindlimb and forelimb buds, respectively. Here we show that Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the activity of three signalling proteins that are required for limb outgrowth and patterning: fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and Wnt. In addition, we provide evidence that Tbx4 and Tbx5 are involved in controlling limb identity. Our findings provide insight into how similar developmental fields can evolve into homologous but distinct structures.
A novel family of transcription factors that appears to play a critical role in the development of all animal species was recently uncovered on the basis of homology of the DNA binding domain of the Brachyury, or T locus, gene product. Phylogenetic studies have shown the ancient origin of this gene family, which has been named the T-box family, prior to the divergence of metazoa from a common ancestral type. T-box genes have now been identified in the genomes of C. elegans, Drosophila, sea urchin, ascidian, amphioxus, Xenopus, chick, zebrafish, mouse, and human and will probably be found in the genomes of all animals. Although functional analyses of T-box family members have just begun, the results show a wide range of roles in developmental processes that extend over time from the unfertilized egg through organogenesis. Only a few mutations in T-box genes are known, but all have drastic effects on development, including a targeted mutation in mice causing an embryonic lethal phenotype, and two human T-box gene mutations that results in developmental syndromes. This review presents a current overview of progress made in the analysis of T-box genes and their products in a variety of model systems.
Genetic analysis of seven dominant short tailed mutations independently induced by radiation of male mice showed that six were allelic to T (Brachyury) but not identical to it. Homozygotes for each mutant die at least 2 days earlier than T/T homozygotes; two that were studied histologically are indistinguishable from one another. The development of these abnormal embryos is arrested by seven days of gestation, when cells of embryonic ectoderm cease proliferation and become pycnotic. Endoderm and extra-embryonic ectoderm do not seem to be primarily affected, and survive and grow for at least 2 days more. Serological studies of one of these mutations suggest that it is a deletion. A review is presented of these and other T-like mutations that have been described; from this it appears that five different categories of T-like mutants are discernible.
At the turn of the past century, the field of heredity included embryology, regeneration, and genetics. Discussions of genetics necessarily entailed a theory of development, and any theory of development had to show why eggs of different species developed in different ways. Thus, the theories of William Keith Brooks (1) or August Weismann (2) did not distinguish between genetics and embryology. The developmental mechanics of His, Roux, and Driesch likewise contained explicit genetic components whereby the hereditary determinants (thought to reside within either the cytoplasm or the nucleus) were seen to direct the processes of organ formation and cell differentiation.
During the first day of embryogenesis in the zebrafish, a precise and relatively simple network of neurons develops, pioneering axonal pathways and apparently functioning to mediate reflexive motor responses to touch stimuli. We have begun to use zygotic lethal mutations to analyze the assembly of this 'primary' embryonic nervous system. Here we focus on spinal primary motoneurons, their inputs from hindbrain Mauthner neurons, and their outputs to segmental body wall muscle. The mutation nic-1 blocks synaptic transmission between nerve and muscle, yet embryonic primary motoneurons appear normal, suggesting that functional interactions with their targets are not involved in regulating their development. The mutation spt-1 directly disrupts development of this muscle, and the mutation cyc-1 appears to directly block specification of the floor plate. Both spt-1 and cyc-1 affect aspects of primary neuronal development, and they probably do so indirectly. The nonautonomous actions of these mutations are local and they produce variable neuronal phenotypes. The observations can be interpreted to mean that some cellular interactions that specify the neurons and their axonal paths occur at close range and involve multiple, possibly combinatorial, transmitter-independent pathways.
The murine developmental mutation T identifies an essential gene in mesoderm formation. Embryos lacking normal gene activity fail to form the notochord, the entire posterior region and the allantois, and die at about 10 days of gestation. We have isolated the T gene using a combination of molecular and genetic techniques, thus making molecular tools available to study processes underlying mesoderm formation in the mouse.
The discovery, more than 60 years ago, of a mutant mouse with a short tail led to the birth of the new field of developmental genetics. Over the years since, numerous investigators have probed the biology of the original short-tail mutation at the T locus, as well the naturally-occurring t haplotypes that were uncovered as a result of their interaction with this mutation. Although the T locus ranks among the best characterized developmental loci in the mouse, it was not among the first to be cloned. This situation has now been rectified with two recent reports from Herrmann, Lehrach and their colleagues. While the T locus is expressed uniquely in the embryonic tissues predicted from the mutant phenotype, the gene itself, as well as the predicted amino acid sequence of the T product, show no strong homology to any known sequence. For the moment, at least, the mystery behind the function of the T locus still awaits definitive resolution.
Sonic hedgehog, a secreted signalling molecule known to play a role in the patterning of the central nervous system and the limb in vertebrates, also controls differentiation of the somites.
Recent studies have uncovered new roles for the notochord and floor plate in patterning adjacent cells, elaborating their importance as essential organizers of neural and paraxial tissue. The identification of key molecules that mediate the ability of notochord and floor plate to induce cells to adopt distinct fates has provided a first step in elucidating the mechanisms underlying these events.
The T locus encodes a product with DNA binding activity that is likely to play a role in the development of all vertebrate organisms. We have identified and characterized a novel family of mouse genes that share a protein motif, the T-box, with the prototypical T locus. The T-box domain of the T locus co-localizes with its DNA binding activity. Each T-box gene is expressed in a unique temporal and spatial pattern during embryogenesis. Phylogenetic analysis suggests that at least three T-box genes were present in the common ancestor to vertebrates and invertebrates. Thus, members of the T-box family could have played a role in the evolution of all metazoan organisms.
Dorsal mesoderm is thought to provide important signals for axis formation and neural differentiation in vertebrate embryos. We have examined induction and patterning in a zebrafish mutant, no tail, that lacks a derivative of dorsal mesoderm, the notochord. Despite the absence of a differentiated notochord, development of the central nervous system including floor plate appears normal, likely owing to the presence of notochord precursor cells. In contrast, somites are misshapen, and muscle pioneer cells are absent. Wild-type cells transplanted into mutant hosts can autonomously differentiate into notochord and thereby rescue somitic defects, suggesting that interactions between notochord and paraxial mesoderm are necessary for proper somite patterning. Thus, cells derived from dorsal mesoderm may have multiple signaling functions during zebrafish embryogenesis.
The left-right body axis is coordinately aligned with the orthogonal dorsoventral and anterioposterior body axes. The developmental mechanisms that regulate axis coordination are unknown. Here it is shown that the cardiac left-right orientation in zebrafish (Danio rerio) is randomized in notochord-defective no tail and floating head mutants. no tail (Brachyury) and floating head (Xnot) encode putative transcription factors that are expressed in the organizer and notochord, structures which regulate dorsoventral and anterioposterior development in vertebrate embryos. Results from dorsal tissue extirpation and cardiac primordia explantation indicate that cardiac left-right orientation is dependent on dorsoanterior structures including the notochord and is specified during neural fold stages in Xenopus laevis. Thus, the notochord coordinates the development of all three body axes in the vertebrate body plan.
Classical embryology experiments have indicated the existence of dorsal-type and ventral-type mesoderms that arise as a consequence of mesoderm induction during vertebrate development. Here we report that the zebrafish tbx6 gene, a member of the Brachyury-related T-box family of genes, is exclusively expressed by ventral mesendoderm. Three observations link the expression of tbx6 to ventral mesoderm specification. First, the gene is initially expressed at the onset of gastrulation within a ventrolateral subpopulation of cells that express the pan-mesodermal gene, no tail (Brachyury). Second, the mesoderm-inducing factors activin and bFGF activate tbx6 expression in animal caps. Third, dorsalization of the mesendodermal precursor population following exposure of embryos to lithium ions causes down-regulation of tbx6 transcription. tbx6 is expressed transiently in the involuting derivatives of the ventral mesendoderm, which give rise to nonaxial mesodermal tissues; its expression is extinguished as tissue differentiation progresses. Transcription of tbx6 commences about an hour after initiation of expression of the pan-mesendodermal gene no tail and the organizer gene goosecoid. The dependence of tbx6 expression on no tail activity was examined in no tail mutant embryos. The activation of tbx6 transcription in ventral mesoderm does not depend on no tail gene activity. However, no tail appears to contribute to the maintenance of normal levels of tbx6 transcription and may be required for tbx6 transcription in the developing tail.
Mutational analyses have shown that the genes no tail (ntl, Brachyury homolog), floating head (flh, a Not homeobox gene), and cyclops (cyc) play direct and essential roles in the development of midline structures in the zebrafish. In both ntl and flh mutants a notochord does not develop, and in cyc mutants the floor plate is nearly entirely missing. We made double mutants to learn how these genes might interact. Midline development is disrupted to a greater extent in cyc;flh double mutants than in either cyc or flh single mutants; their effects appear additive. Both the notochord and floor plate are completely lacking, and other phenotypic disturbances suggest that midline signaling functions are severely reduced. On the other hand, trunk midline defects in flh;ntl double mutants are not additive, but are most often similar to those in ntl single mutants. This finding reveals that loss of ntl function can suppress phenotypic defects due to mutation at flh, and we interpret it to mean that the wild-type allele of ntl (ntl+) functions upstream to flh in a regulatory hierarchy. Loss of function of ntl also strongly suppresses the floor plate deficiency in cyc mutants, for we found trunk floor plate to be present in cyc;ntl double mutants. From these findings we propose that ntl+ plays an early role in cell fate choice at the dorsal midline, mediated by the Ntl protein acting to antagonize floor plate development as well as to promote notochord development.