Gene expression in Plasmodium berghei ookinetes and early oocysts in a co-culture system with mosquito cells

Using an in vitro development system for Plasmodium berghei sporogonic stages and microarray technology we examined parasite gene expression during ookinete invasion of Aedes cells and the ensuing oocyst development. A number of genes were found to be differentially expressed. The most prominent class of up-regulated elements corresponded to products involved in protein synthesis and metabolism. Furthermore, several previously studied genes with a known in vivo developmental profile matched published data. A large number of genes with a hitherto unknown function during the life cycle stages studied also show a differential pattern of expression, indicating the involvement of their products in control and execution of active developmental processes.     

Differential gene expression in the ookinete stage of the malaria parasite Plasmodium berghei

Plasmodium, the malaria parasite, undergoes a complex developmental program in its mosquito vector. The ookinete is the parasite form which invades the mosquito midgut and is an important stage for genetic mixing. To identify genes expressed during ookinete development and mosquito midgut invasion, purified zygotes and ookinetes of the rodent parasite Plasmodium berghei were used to construct a suppression subtractive hybridization cDNA library, enriched in sequences expressed in the ookinete stage. In addition to four genes coding for previously described major ookinete-secreted proteins, we isolated ookinete-expressed sequences representing 18 predicted genes. Their gene products include proteins involved in signal transduction and regulatory processes. For six of these genes our analysis provides the first evidence for expression in the ookinete stage. A majority of the genes are not expressed in the zygote, the preceding developmental stage. Furthermore, four of the genes are also transcribed in sporozoites, and one of these in merozoites, suggesting that they code for proteins with a function common to Plasmodium invasive stages.     

Comparative spatiotemporal analysis of Hox gene expression in early stages of intermediate mesoderm formation

Background : Hox genes are key players in AP patterning of the vertebrate body plan and are necessary for organogenesis. Several studies provide evidence for the role Hox genes play during kidney development and especially regarding metanephros initiation and formation. However, the role Hox genes play during early stages of kidney development is largely unknown. A recent study in our lab revealed the role Hoxb4 plays in conferring the competence of intermediate mesodermal cells to respond to kidney inductive signals and express early kidney regulators. Results : As a first step in understanding the role Hox genes play in setting the formation of the pronephros morphogenetic field and the expression of early regulators of kidney development, we studied in detail the expression pattern of 10 Hox genes in relation to the 6th somite axial level, the anterior sharp border of the kidney field. Despite the idea of spatial co‐linearity as exemplified in the Hox gene expression pattern in late developmental stages, a very dynamic spatio‐temporal expression of these genes was found in early stages. Conclusions : Since mesodermal patterning occurs at gastrula stages, the relevance of a “Hox code” at early stages is questioned in this study. Developmental Dynamics 241:1637–1649, 2012. © 2012 Wiley Periodicals, Inc.     

Modeling stem cell development by retrospective analysis of gene expression profiles in single progenitor-derived colonies

The process of development of various cell types is often based on a linear or deterministic paradigm. This is true, for example, for osteoblast development, a process that occurs through the differentiation of a subset of primitive fibroblast progenitors called colony-forming unit-osteoblasts (CFU-Os). CFU-O differentiation has been subdivided into three stages: proliferation, extracellular matrix development and maturation, and mineralization, with characteristic changes in gene expression at each stage. Few analyses have asked whether CFU-O differentiation, or indeed stem cell differentiation in general, may follow more complex and nondeterministic paths, a possibility that may underlie the substantial number of discrepancies in published reports of progenitor cell developmental sequences. We analyzed 99 single colonies of osteoblast stem/primitive progenitor cells cultured under identical conditions. The colonies were analyzed by global amplification poly(A) polymerase chain reaction to determine which of nine genes had been expressed. We used the expression profiles to develop a statistically rigorous map of the cell fate decisions that occur during osteoprogenitor differentiation and show that different developmental routes can be taken to achieve the same end point phenotype. These routes appear to involve both developmental "dead ends" (leading to the expression of genes not correlated with osteoblast-associated genes or the mature osteoblast phenotype) and developmental flexibility (the existence of multiple gene expression routes to the same developmental end point). Our results provide new insight into the biology of primitive progenitor cell differentiation and introduce a powerful new quantitative method for stem cell lineage analysis that should be applicable to a wide variety of stem cell systems.     

Regulation of gene expression dynamics during developmental transitions by the Ikaros transcription factor

The DNA-binding protein Ikaros is a potent tumor suppressor and hematopoietic regulator. However, the mechanisms by which Ikaros functions remain poorly understood, due in part to its atypical DNA-binding properties and partnership with the poorly understood Mi-2/NuRD complex. In this study, we analyzed five sequential stages of thymocyte development in a mouse strain containing a targeted deletion of Ikaros zinc finger 4, which exhibits a select subset of abnormalities observed in Ikaros-null mice. By examining thymopoiesis in vivo and in vitro, diverse abnormalities were observed at each developmental stage. RNA sequencing revealed that each stage is characterized by the misregulation of a limited number of genes, with a strong preference for stage-specific rather than lineage-specific genes. Strikingly, individual genes rarely exhibited Ikaros dependence at all stages. Instead, a consistent feature of the aberrantly expressed genes was a reduced magnitude of expression level change during developmental transitions. These results, combined with analyses of the interplay between Ikaros loss of function and Notch signaling, suggest that Ikaros may not be a conventional activator or repressor of defined sets of genes. Instead, a primary function may be to sharpen the dynamic range of gene expression changes during developmental transitions via atypical molecular mechanisms that remain undefined.     

Developmental expression profiles of Xenopus laevis reference genes.

Cell differentiation depends mainly on specific mRNA expression. To quantify the expression of a particular gene, the normalisation with respect to the expression of a reference gene is carried out. This is based on the assumption that the expression of the reference gene is constant during development, in different cells or tissues or after treatment. Xenopus laevis studies have frequently used eEF-1 alpha, GAPDH, ODC, L8, and H4 as reference genes. The aim of this work was to examine, by real-time RT-PCR, the expression profiles of the above-mentioned five reference genes during early development of X. laevis. It is shown that their expression profiles vary greatly during X. laevis development. The developmental changes of mRNA expression can thus significantly compromise the relative mRNA quantification based on these reference genes, when different developmental stages are to be compared. The normalisation against total RNA is recommended instead.     

Expression profiling of the developing mouse lung: insights into the establishment of the extracellular matrix

We have undertaken a comprehensive gene expression profiling of the entire process of murine lung development using oligonucleotide-based microarrays. Our data reveals the expression pattern of approximately 11,000 genes throughout the morphologic stages of lung development. This includes known genes with unappreciated pulmonary expression and novel genes with undefined functions. Traditional gene expression analysis techniques verify a high degree of confidence in the microarray data. Examination of the data confirms previously known patterns of expression for extracellular matrix genes and provides new information regarding relationships in temporal expression among groups of these genes. Large-scale cluster analysis reveals associations in the expression profile of specific genes with defined developmental processes. For instance, we identify groups of genes, which are coordinately expressed with extracellular matrix genes during lung development. These data should serve as a resource for the pulmonary research community and assist in deciphering the molecular mechanisms governing normal lung development as well as those involved in aberrant developmental pathology.     

Developmental markers of B cells are superior to those of T cells for identification of stages with distinct gene expression profiles

B and T lymphocytes develop through a series of cellular stages, which are defined by recombination status of the immunoglobulin and T cell receptor loci and can be separated by analysis of cell-surface markers. We evaluated how well 26 and 41 samples from five and eight developmental stages of B and T cell development, respectively, could be correctly assigned to their lineage of origin and developmental stage by analysis of the expression of 13,026 genes and expressed sequence tags (ESTs). The RNA expression patterns of eight genes correctly classified all 67 samples as belonging to the B cell or to the T cell lineage. Ninety-two to 100% of B-lineage samples could be correctly assigned to the protein-defined developmental stage by the RNA expression pattern of 29 genes. By contrast, RNA expression patterns of 39 genes were necessary to correctly assign 85-100% of T-lineage samples to the correct developmental stage. The sets of genes used for these classifications contain ESTs as well as known genes that have not previously been associated with lymphocyte development. Graphical display of the classifications shows that B-lineage samples are well separated from T-lineage samples, and samples from the five stages of B cell development are well separated from each other. By contrast, samples from the eight stages of T cell development cannot be separated precisely. We conclude that the protein markers currently widely used for separating stages of B cell development better identify molecularly distinct stages than those used for separating stages of T cell development.     

Gene expression signatures identify novel regulatory pathways during murine lung development: implications for lung tumorigenesis

Oligonucleotide array based analysis was conducted to examine the temporal pattern of gene expression across the various stages of lung development to identify regulatory pathways at key developmental time points. Whole embryo total RNA or embryonic lung total RNA was harvested from A/J mice at seven developmental stages. To investigate changes in gene expression during lung development, four samples from each stage were examined using Affymetrix U74Av2 murine oligonucleotide microarrays. From the over 12 000 genes and ESTs represented on the array, 1346 genes and ESTs were identified as having a significant change in expression between at least one time point and the others (p<0.001, Kruskal-Wallis test). Within this group of ~1300 genes, four patterns of expression were seen: (1) upregulation during the embryonic period of development (up-down); (2) upregulation during the postnatal period of lung development (down-up) and (3) fluctuating expression, up initially, down for one or more time points, and then up again (up-down-up); and (4) vice versa (down-up-down). Expression patterns of genes previously reported to be involved in pulmonary development were also examined. Using the pathway visualisation tool, GenMapp, at least three regulatory pathways were found to contain clusters of differentially expressed genes: Wnt signalling, cell cycle, and apoptosis. Furthermore, we have shown that many of the genes involved in lung development are either known oncogenes or tumour suppressor genes altered in lung cancer, such as Cyr61, Rassf1a, and Dutt1/Robo1, or putative lung cancer genes. In addition, the genes identified pertinent to early development may also serve as candidate susceptibility genes for various inherited lung cancer disorders as well as for various heritable disorders of lung development. These results will contribute to our understanding of novel aspects of the regulatory machinery for embryonic lung development and of the genes involved in lung tumorigenesis.     

Immunohistochemical Localization of Barx2 in the Developing Fetal Mouse Submandibular Glands

The development of mouse submandibular gland (SMG) begins at embryonic day 11.5–12 (E11.5–12), during which successive rounds of epithelial clefting and branching create complex epithelial tree-like structures. Homeobox genes regulate place-dependent morphogenesis, including epithelial-mesenchymal interactions, and control the expression patterns of signaling molecules. The Barx2 containing Homeobox exerts several key roles in development. Some studies have shown that the Barx2 plays important roles in the epithelial-mesenchymal interactions of organogenesis. However, the mechanisms of Barx2 associated with the development of SMG are obscure. In this study, we demonstrated for the first time the exact spatial and temporal Barx2 expression pattern in SMG epithelial tissue during development using immunohistochemical staining and Real-Time quantitative PCR. Barx2 was expressed in the nucleus of the epithelial cells located in the proliferative and differentiative regions of the developing SMG during the early development stages (E11.5–E13.5). After the E14.5-time period, the expression gradually decreased, and at E16.5, expression mostly disappeared despite the fact that evidence of cytodifferentiation, such as the appearance of proacinar cells, distinct lumen formation, and secretory products, was beginning to be observed. Results of Real-Time PCR demonstrated that the amount of Barx2 mRNA expression in SMG was maximal on E14.5, and gradually decreased by E18.5. These results indicate that Barx2 is associated with early stage epithelial tissue development, and can be a useful epithelial marker of the SMG during early developmental stages.     

An automated in situ hybridization screen in the Medaka to identify unknown neural genes.

Despite the fact that a large body of factors that play important roles in development are known, there are still large gaps in understanding the genetic pathways that govern these processes. To find previously unknown genes that are expressed during embryonic development, we optimized and performed an automated whole-mount in situ hybridization screen on medaka embryos at the end of somitogenesis. Partial cDNA sequences were compared against public databases and identified according to similarities found to other genes and gene products. Among 321 isolated genes showing specific expression in the central nervous system in at least one of five stages of development, 55.14% represented genes whose functions are already documented (in fish or other model organisms). Additionally, 16.51% were identified as conserved unknown genes or genes with unknown function. We provide new data on eight of these genes that presented a restricted expression pattern that allowed for formulating testable hypotheses on their developmental roles, and that were homologous to mammalian molecules of unknown function. Thus, gene expression screening in medaka is an efficient tool for isolating new regulators of embryonic development, and can complement genome-sequencing projects that are producing a high number of genes without ascribed functions.