Using Quantitative Real-Time PCR to Detect MicroRNA Expression Profile During Embryonic Stem Cell Differentiation.

Quantitative real-time PCR (qRT-PCR) is a reliable method to determine and monitor microRNA (miRNA) expression profiles in different cells, tissues, and organisms. Although there are several different strategies in performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequencing and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of quantitative real-time PCR for determining miRNA expression profiles during mouse embryonic stem cell differentiation.

Functional and Comparative Genomics of Hoxa2 Gene cis-Regulatory Elements: Evidence for Evolutionary Modification of Ancestral Core Element Activity.

is an evolutionarily conserved developmental regulatory gene that functions to specify rhombomere (r) and pharyngeal arch (PA) identities throughout the Osteichthyes. Japanese medaka () like orthologous genes from other osteichthyans, is expressed during embryogenesis in r2-7 and PA2-7, whereas the paralogous medaka pseudogene, , is expressed in noncanonical domains, including the pectoral fin buds. To understand the evolution of -regulatory element (CRE) control of gene expression, we conducted eGFP reporter gene expression studies with extensive functional mapping of several conserved CREs upstream of medaka and in transient and stable-line transgenic medaka embryos.

Nuclear progestin receptor (pgr) knockouts in zebrafish demonstrate role for pgr in ovulation but not in rapid non-genomic steroid mediated meiosis resumption.

Progestins, progesterone derivatives, are the most critical signaling steroid for initiating final oocyte maturation (FOM) and ovulation, in order to advance fully-grown immature oocytes to become fertilizable eggs in basal vertebrates. It is well-established that progestin induces FOM at least partly through a membrane receptor and a non-genomic steroid signaling process, which precedes progestin triggered ovulation that is mediated through a nuclear progestin receptor (Pgr) and genomic signaling pathway. To determine whether Pgr plays a role in a non-genomic signaling mechanism during FOM, we knocked out Pgr in zebrafish using transcription activator-like effector nucleases (TALENs) and studied the oocyte maturation phenotypes of Pgr knockouts (Pgr-KOs).

Chemical dispersant potentiates crude oil impacts on growth, reproduction, and gene expression in Caenorhabditis elegans.

The economic, environmental, and human health impacts of the deepwater horizon (DWH) oil spill have been of significant concern in the general public and among scientists. This study employs parallel experiments to test the effects of crude oil from the DWH oil well, chemical dispersant Corexit 9500A, and dispersant-oil mixture on growth and reproduction in the model organism Caenorhabditis elegans. Both the crude oil and the dispersant significantly inhibited the reproduction of C.

Monitoring microRNA expression during embryonic stem-cell differentiation using quantitative real-time PCR (qRT-PCR).

Quantitative real-time PCR (qRT-PCR) is a reliable method to determine and monitor microRNA (miRNA) expression profiles in different cells, tissues, and organisms. Although there are several different strategies for performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequence and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of TaqMan-based real-time PCR for determining miRNA expression profiles during mouse embryonic stem-cell differentiation.

Spatio-temporal patterns of Hox paralog group 3-6 gene expression during Japanese medaka (Oryzias latipes) embryonic development.

Clustered Hox genes encode transcription factors that pattern the regional identities of tissues along the anterior-posterior (A-P) axis of animals during embryonic development. They are expressed along the A-P axis collinear with their physical location within a cluster. Several studies have examined the expression of teleost Hox genes from paralog groups (PG) 1-4 in the rhombomeres of the hindbrain and the anterior pharyngeal arches.

Role of Hox PG2 genes in Nile tilapia pharyngeal arch specification: implications for gnathostome pharyngeal arch evolution.

Phylogenetic reconstructions suggest that the ancestral osteichthyan Hox paralog group 2 gene complement was composed of two genes, Hoxa2 and b2, both of which have been retained in tetrapods, but only one of which functions as a selector gene of second pharyngeal arch identity (PA2). Genome duplication at the inception of the teleosts likely generated four Hox PG2 genes, only two of which, hoxa2b and b2a, have been preserved in zebrafish, where they serve as functionally redundant PA2 selector genes. Evidence from our laboratory has shown that other telelosts, specifically striped bass and Nile tilapia, harbor three transcribed Hox PG2 genes, hoxa2a, a2b, and b2a, with unspecified function(s).

Embryonic development and skeletogenesis of the pharyngeal jaw apparatus in the cichlid Nile tilapia (Oreochromis niloticus).

The evolution of a specialized pharyngeal jaw apparatus (PJA) has been argued to be the key evolutionary innovation that allowed the explosive adaptive radiation of cichlid fishes in East African lakes. Subsequent studies together with recent molecular phylogenies have shown that similar innovations evolved independently several times within the teleosts, which poses the questions: (1) how similar are the developmental mechanisms responsible for these changes in divergent taxa and (2) how did such complex features arise independently in evolution? A detailed knowledge of PJA development in cichlids and other teleosts is needed to address these questions. Here, we provide a detailed account of the development of the PJA in one species of cichlid, the Nile tilapia (Oreochromis niloticus), from the early segmentation and patterning of its embryonic precursors - pharyngeal arches 3 to 7 - to its ossification.

Large-scale genome analysis reveals unique features of microRNAs.

Although great progress has been made in identifying microRNAs (miRNAs) and their functions, their essential functional features remain largely unknown. In this study, we systemically investigated the nucleotide and thermodynamic folding distribution characteristics of 3853 miRNAs currently reported for metazoans. We determined that uracil is the dominant nucleotide in both mature and precursor sequences, and that it is particularly enriched at three sites in mature miRNAs: the first, ninth, and the five terminal 3' nucleotides.

Japanese medaka Hox paralog group 2: insights into the evolution of Hox PG2 gene composition and expression in the Osteichthyes.

Hox paralog group 2 (PG2) genes function to specify the development of the hindbrain and pharyngeal arch-derived structures in the Osteichthyes. In this article, we describe the cDNA cloning and embryonic expression analysis of Japanese medaka (Oryzias latipes) Hox PG2 genes. We show that there are only two functional canonical Hox genes, hoxa2a and b2a, and that a previously identified hoxa2b gene is a transcribed pseudogene, psihoxa2b.

Identification of soybean microRNAs and their targets.

The microRNAs (miRNAs) are a newly identified class of small non-protein-coding regulatory RNA. Using comparative genomics, we identified 69 miRNAs belonging to 33 families in the domesticated soybean (Glycine max) as well as five miRNAs in the soybean wild species Glycine soja and Glycine clandestine. TaqMan((R)) MicroRNA Assay analyses demonstrated that these miRNAs were differentially expressed in soybean tissues, with certain classes expressed preferentially in both a spatiotemporal and a tissue-specific manner.

Prolactin-dependent modulation of organogenesis in the vertebrate: Recent discoveries in zebrafish.

The scientific literature is replete with evidence of the multifarious functions of the prolactin (PRL)/growth hormone (GH) superfamily in adult vertebrates. However, little information is available on the roles of PRL and related hormones prior to the adult stage of development. A limited number of studies suggest that GH functions to stimulate glucose transport and protein synthesis in mouse blastocytes and may be involved during mammalian embryogenesis.

Comparative analysis of Hox paralog group 2 gene expression during Nile tilapia (Oreochromis niloticus) embryonic development.

The hindbrain and pharyngeal arch-derived structures of vertebrates are determined, at least in part, by Hox paralog group 2 genes. In sarcopterygians, the Hoxa2 gene alone appears to specify structures derived from the second pharyngeal arch (PA2), while in zebrafish (Danio rerio), either of the two Hox PG2 genes, hoxa2b or hoxb2a, can specify PA2-derived structures. We previously reported three Hox PG2 genes in striped bass (Morone saxatilis), including hoxa2a, hoxa2b, and hoxb2a and observed that only HoxA cluster genes are expressed in PA2, indicative that they function alone or together to specify PA2.

Differential expression of hoxa2a and hoxa2b genes during striped bass embryonic development.

Here, we report the cloning and expression analysis of two previously uncharacterized paralogs group 2 Hox genes, striped bass hoxa2a and hoxa2b, and the developmental regulatory gene egr2. We demonstrate that both Hox genes are expressed in the rhombomeres of the developing hindbrain and the pharyngeal arches albeit with different spatio-temporal distributions relative to one another. While both hoxa2a and hoxa2b share the r1/r2 anterior boundary of expression characteristic of the hoxa2 paralog genes of other species, hoxa2a gene expression extends throughout the hindbrain, whereas hoxa2b gene expression is restricted to the r2-r5 region.

Are genome evolution, organism complexity and species diversity linked?

Fishes represent an extremely diverse group of vertebrates with a deeply rooted evolutionary history. An understanding of their biology is being enriched by advancements in phylogenetic analysis and genomics, which are providing the framework for deciphering their evolutionary relationships and the molecular details that govern their evolution. Recent discoveries about the structure and function of fish genomes suggest the occurrence of large-scale genome level duplications within the stem lineage of the Actinopterygii (ray-finned fishes).

Phylogeny, fossils, and model systems in the study of evolutionary developmental biology.

The emerging field of evolutionary developmental biology (evo-devo) continues to operate largely under a single paradigm. In this paradigm developmental regulatory genes and processes are compared among a collection of "model organisms" selected primarily on the basis of their historical utility in the study of development. This approach has proven to be extremely informative, revealing an unexpected deep evolutionary conservation among developmental genes and genetic systems.