Expression Patterns, Genomic Conservation and Input Into Developmental Regulation of the GGDEF/EAL/HD-GYP Domain Proteins in Streptomyces

Background: Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods: We have investigated expression stability in mouse embryos from mid to late gestation (E11.5–E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes (ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results: Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1, RPL13A and PAK1IP1 are the strongest performing genes overall. HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable (CDC40, HTATSF1). Conclusion: AP3D1, RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.

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Signaling Modulations of miR-206-3p in Tooth Morphogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms21155251 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5251

Author(s):

Sanjiv Neupane ◽

Yam Prasad Aryal ◽

Tae-Young Kim ◽

Chang-Yeol Yeon ◽

Chang-Hyeon An ◽

...

Keyword(s):

Gene Expression ◽

Expression Pattern ◽

Ex Vivo ◽

Developmental Regulation ◽

Expression Patterns ◽

Tooth Germ ◽

Culture Method ◽

Tooth Pulp ◽

Altered Expression ◽

Tooth Morphogenesis

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that post-transcriptionally regulate gene expression in organisms. Most mammalian miRNAs influence biological processes, including developmental changes, tissue morphogenesis and the maintenance of tissue identity, cell growth, differentiation, apoptosis, and metabolism. The miR-206-3p has been correlated with cancer; however, developmental roles of this miRNA are unclear. In this study, we examined the expression pattern and evaluated the developmental regulation of miR-206-3p during tooth morphogenesis using ex-vivo culture method. The expression pattern of miR-206-3p was examined in the epithelium and mesenchyme of developing tooth germ with stage-specific manners. Perturbation of the expression of miR-206-3p clearly altered expression patterns of dental-development–related signaling molecules, including Axin2, Bmp2, Fgf4, Lef1 and Shh. The gene expression complemented with change in cellular events including, apoptosis and proliferation which caused altered crown and pulp morphogenesis in renal-capsule–calcified teeth. Especially, mislocalization of β-Catenin and SMAD1/5/8 were observed alongside dramatic alterations in the expression patterns of Fgf4 and Shh. Overall, our data suggest that the miR-206-3p regulate the cellular physiology during tooth morphogenesis through modulation of the Wnt, Bmp, Fgf, and Shh signaling pathways to form proper tooth pulp and crown.

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A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

10.1101/133470 ◽

2017 ◽

Cited By ~ 2

Author(s):

Chiara Sinigaglia ◽

Daniel Thiel ◽

Andreas Hejnol ◽

Evelyn Houliston ◽

Lucas Leclère

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Developmental Regulation ◽

Expression Patterns ◽

Urea Solution ◽

Similar Reduction ◽

In Situ Detection ◽

Precise Expression ◽

Significant Health

AbstractIn situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care.We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

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Evolutionary conservation of the structure and expression of alternatively spliced Ultrabithorax isoforms from Drosophila.

Genetics ◽

10.1093/genetics/136.3.965 ◽

1994 ◽

Vol 136 (3) ◽

pp. 965-977

Author(s):

H M Bomze ◽

A J López

Keyword(s):

Alternative Splicing ◽

Protein Function ◽

Developmental Regulation ◽

Expression Patterns ◽

Proximal Region ◽

Amino Acid Sequences ◽

Drosophila Virilis ◽

Protein Isoforms ◽

Developmentally Regulated ◽

Alternatively Spliced

Abstract In Drosophila melanogaster, alternatively spliced mRNAs from the homeotic gene Ultrabithorax (Ubx) encode a family of structurally distinct homeoprotein isoforms. The developmentally regulated expression patterns of these isoforms suggest that they have specialized stage- and tissue-specific functions. To evaluate the functional importance of UBX isoform diversity and gain clues to the mechanism that regulates processing of Ubx RNAs, we have investigated whether the Ubx RNAs of other insects undergo similar alternative splicing. We have isolated and characterized Ubx cDNA fragments from D. melanogaster, Drosophila pseudoobscura, Drosophila hydei and Drosophila virilis, species separated by as much as 60 million years of evolution, and have found that three aspects of Ubx RNA processing have been conserved. (1) These four species exhibit identical patterns of optional exon use in a region adjacent to the homeodomain. (2) These four species produce the same family of UBX protein isoforms with identical amino acid sequences in the optional exons, even though the common amino-proximal region has undergone substantial divergence. The nucleotide sequences of the optional exons, including third positions of rare codons, have also been conserved strongly, suggesting functional constraints that are not limited to coding potential. (3) The tissue- and stage-specific patterns of expression of different UBX isoforms are identical among these Drosophila species, indicating that the developmental regulation of the alternative splicing events has also been conserved. These findings argue for an important role of alternative splicing in Ubx function. We discuss the implications of these results for models of UBX protein function and the mechanism of alternative splicing.

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Molecular evolution and developmental expression of melanin pathway genes in Lepidoptera

10.1101/2020.03.11.987669 ◽

2020 ◽

Author(s):

Muktai Kuwalekar ◽

Riddhi Deshmukh ◽

Ajay Padvi ◽

Krushnamegh Kunte

Keyword(s):

Molecular Evolution ◽

Developmental Stages ◽

Developmental Regulation ◽

Expression Patterns ◽

Gene Tree ◽

Purifying Selection ◽

Developmental Expression ◽

Model Organisms ◽

Sequence Evolution ◽

Melanin Pathway

ABSTRACTPigmentation is involved in a wide array of biological functions across insect orders, including body patterning, thermoregulation, and immunity. The melanin pathway, in particular, has been characterized in several species. However, molecular evolution of the genes involved in this pathway is poorly characterized, and their roles in pigmentation of early developmental stages are just beginning to be explored in non-model organisms. We traced the molecular evolution of six melanin pathway genes in 53 species of Lepidoptera covering butterflies and moths, and representing over 100 million years of diversification. We compared the rates of synonymous and nonsynonymous substitutions within and between these genes to study signatures of selection at the level of individual sites, genes, and branches of the gene tree. We found that molecular evolution of all six genes was governed by strong purifying selection. Yet, a number of sites showed signs of being under positive selection, including in the highly conserved domain regions of three genes. Further, we traced the expression of these genes across developmental stages, tissues, and sexes in the Papilio polytes butterfly using a developmental transcriptome dataset. We observed that the expression patterns of the genes in P. polytes largely reflected their known tissue-specific function in other species. The expression of sequentially acting genes in the melanin pathway was correlated. Interestingly, four out of six melanin pathway genes (ebony, pale, aaNAT, and DDC) showed a sexually dimorphic pattern of developmental heterochrony; i.e., females showed peak activity much earlier in pupal development compared to that of males. Our evolutionary and developmental analyses suggest that the vast diversity of wing patterning and pigmentation in Lepidoptera may have been aided largely by differential developmental regulation of genes in a highly conserved pathway, in which the sequence evolution of individual genes is highly constrained.

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B-box Proteins in Arachis duranensis: Genome-Wide Characterization and Expression Profiles Analysis

Agronomy ◽

10.3390/agronomy10010023 ◽

2019 ◽

Vol 10 (1) ◽

pp. 23 ◽

Cited By ~ 4

Author(s):

Hanqi Jin ◽

Mengge Xing ◽

Chunmei Cai ◽

Shuai Li

Keyword(s):

Developmental Regulation ◽

Expression Profiles ◽

Transcriptional Profiling ◽

Expression Patterns ◽

Promoter Regions ◽

Essential Information ◽

Cis Acting ◽

Arachis Duranensis ◽

Gene Structures ◽

Wild Peanut

B-box (BBX) proteins are important factors involved in plant growth and developmental regulation, and they have been identified in many species. However, information on the characteristics and transcription patterns of BBX genes in wild peanut are limited. In this study, we identified and characterized 24 BBX genes from a wild peanut, Arachis duranensis. Many characteristics were analyzed, including chromosomal locations, phylogenetic relationships, and gene structures. Arachis duranensis B-box (AdBBX) proteins were grouped into five classes based on the diversity of their conserved domains: I (3 genes), II (4 genes), III (4 genes), IV (9 genes), and V (4 genes). Fifteen distinct motifs were found in the 24 AdBBX proteins. Duplication analysis revealed the presence of two interchromosomal duplicated gene pairs, from group II and IV. In addition, 95 kinds of cis-acting elements were found in the genes’ promoter regions, 53 of which received putative functional predictions. The numbers and types of cis-acting elements varied among different AdBBX promoters, and, as a result, AdBBX genes exhibited distinct expression patterns in different tissues. Transcriptional profiling combined with synteny analysis suggests that AdBBX8 may be a key factor involved in flowering time regulation. Our study will provide essential information for further functional investigation of AdBBX genes.

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Prototype chicken galectins revisited: characterization of a third protein with distinctive hydrodynamic behaviour and expression pattern in organs of adult animals

Biochemical Journal ◽

10.1042/bj20070419 ◽

2007 ◽

Vol 409 (2) ◽

pp. 591-599 ◽

Cited By ~ 40

Author(s):

Herbert Kaltner ◽

Dolores Solís ◽

Jürgen Kopitz ◽

Martin Lensch ◽

Michaela Lohr ◽

...

Keyword(s):

Sequence Similarity ◽

Developmental Regulation ◽

Expression Patterns ◽

Cloning And Expression ◽

Proximal Promoter ◽

Divergent Evolution ◽

Protein Ligands ◽

Hydrodynamic Behaviour ◽

Intestinal Protein ◽

Embryonic Skin

Prototype galectins are versatile modulators of cell adhesion and growth via their reactivity to certain carbohydrate and protein ligands. These functions and the galectins' marked developmental regulation explain their attractiveness as models to dissect divergent evolution after gene duplication. Only two members have so far been assumed to constitute this group in chicken, namely the embryonic muscle/liver form {C-16 or CLL-I [16 kDa; chicken lactose lectin, later named CG-16 (chicken galectin-16)]} and the embryonic skin/intestine form (CLL-II or C-14; later named CG-14). In the present study, we report on the cloning and expression of a third prototype CG. It has deceptively similar electrophoretic mobility compared with recombinant C-14, the protein first isolated from embryonic skin, and turned out to be identical with the intestinal protein. Hydrodynamic properties unusual for a homodimeric galectin and characteristic traits in the proximal promoter region set it apart from the two already known CGs. Their structural vicinity to galectin-1 prompts their classification as CG-1A (CG-16)/CG-1B (CG-14), whereas sequence similarity to mammalian galectin-2 gives reason to refer to the intestinal protein as CG-2. The expression profiling by immunohistochemistry with specific antibodies discerned non-overlapping expression patterns for the three CGs in several organs of adult animals. Overall, the results reveal a network of three prototype galectins in chicken.

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Transcriptome profiling reveals the developmental regulation of NaCl-treated Forcipomyia taiwana eggs

BMC Genomics ◽

10.1186/s12864-021-08096-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Mu-En Chen ◽

Mong-Hsun Tsai ◽

Hsiang-Ting Huang ◽

Ching-Chu Tsai ◽

Mei-Ju Chen ◽

...

Keyword(s):

De Novo ◽

Developmental Regulation ◽

Life Stage ◽

Expression Patterns ◽

Transcriptome Profiling ◽

Functional Domain ◽

Pest Species ◽

Alternative Tactics ◽

Biting Midge ◽

Blood Sucking

Abstract Background The biting midge, Forcipomyia taiwana, is one of the most annoying blood-sucking pests in Taiwan. Current chemical control methods only target the adult, not the immature stages (egg to pupa), of F. taiwana. Discovering new or alternative tactics to enhance or replace existing methods are urgently needed to improve the effectiveness of F. taiwana control. The egg is the least understood life stage in this pest species but may offer a novel point of control as addition of NaCl to the egg environment inhibits development. Thus, the objective of this study was to use RNA profiling to better understand the developmental differences between wild-type melanized (black) and NaCl-induced un-melanized (pink), infertile F. taiwana eggs. Results After de novo assembly with Trinity, 87,415 non-redundant transcripts (Ft-nr) with an N50 of 1099 were obtained. Of these, 26,247 (30%) transcripts were predicted to have long open reading frames (ORFs, defined here as ≥300 nt) and 15,270 (17.5%) transcripts have at least one predicted functional domain. A comparison between two biological replicates each of black and pink egg samples, although limited in sample size, revealed 5898 differentially expressed genes (DEGs; 40.9% of the transcripts with long ORFs) with ≥2-fold difference. Of these, 2030 were annotated to a Gene Ontology biological process and along with gene expression patterns can be separated into 5 clusters. KEGG pathway analysis revealed that 1589 transcripts could be assigned to 18 significantly enriched pathways in 2 main categories (metabolism and environmental information processing). As expected, most (88.32%) of these DEGs were down-regulated in the pink eggs. Surprisingly, the majority of genes associated with the pigmentation GO term were up-regulated in the pink egg samples. However, the two key terminal genes of the melanin synthesis pathway, laccase2 and DCE/yellow, were significantly down-regulated, and further verified by qRT-PCR. Conclusion We have assembled and annotated the first egg transcriptome for F. taiwana, a biting midge. Our results suggest that down-regulation of the laccase2 and DCE/yellow genes might be the mechanism responsible for the NaCl-induced inhibition of melanization of F. taiwana eggs.

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Germ Cell Expression of an Isolated Human Endogenous Retroviral Long Terminal Repeat of the HERV-K/HTDV Family in Transgenic Mice

Journal of Virology ◽

10.1128/jvi.73.12.9976-9983.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9976-9983 ◽

Cited By ~ 11

Author(s):

Armelle E. Casau ◽

Joe E. Vaughan ◽

Guillermina Lozano ◽

Arnold J. Levine

Keyword(s):

Transgenic Mice ◽

Germ Cell ◽

Insertional Mutagenesis ◽

Germ Line ◽

Developmental Regulation ◽

Expression Patterns ◽

Germ Cell Tumors ◽

Open Reading Frames ◽

Teratocarcinoma Cell ◽

Reverse Transcription Pcr

ABSTRACT In contrast to most other human endogenous retroviral families, various HERV-K members have open reading frames that code for functional viral proteins which can form noninfectious particles in some germ cell tumors. The HERV-K viral genes are highly transcribed in germ cell tumors but are transcribed to lower or undetectable levels in most other tissue and tumor types. To further analyze the expression patterns of these proviruses, long terminal repeats (LTRs) were isolated from the human genome and used in reporter gene assays. Expression of some HERV-K LTRs was found to be high in human and murine germ cell tumors (testicular teratocarcinomas) and low in non-germ-cell tumors. Furthermore, upon differentiation of a teratocarcinoma cell line, the expression of an active LTR dropped dramatically, suggesting developmental regulation of these proviral LTRs. Transgenic mice harboring an active LTR driving lacZ expression were generated and analyzed. Adult mouse testes showed the highest levels of expression, and the transgene staining appeared to be restricted primarily to the more undifferentiated spermatocytes. Most other tissues analyzed revealed very low or undetectable levels of expression both by reverse transcription-PCR and by Northern blot analysis. Whether the restricted expression of HERV-K in germ cells and in germ cell-derived tumors is of significant importance during development or tumorigenesis remains to be elucidated. Germ line expression of these viruses would allow for their expansion and movement, while somatic repression would ensure limited insertional mutagenesis and misexpression in an individual.

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Developmentally regulated Shh expression is robust to TAD perturbations

10.1101/609941 ◽

2019 ◽

Cited By ~ 4

Author(s):

Iain Williamson ◽

Lauren Kane ◽

Paul S. Devenney ◽

Eve Anderson ◽

Fiona Kilanowski ◽

...

Keyword(s):

Developmental Regulation ◽

Expression Patterns ◽

In Situ Hybridisation ◽

Detectable Effect ◽

Chromatin Conformation ◽

Fluorescence In Situ Hybridisation ◽

Developmentally Regulated ◽

Enhancer Activity ◽

Chromosome Conformation

AbstractTopologically Associating Domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD – creating internal deletions, deleting CTCF sites including those at TAD boundaries, as well as larger deletions and inversions of TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used the investigate changes in chromatin conformation that result from these manipulations. Our data suggest that the substantial alteration of TAD structure has no readily detectable effect on Shh expression patterns during development – except where enhancers are deleted - and results in no detectable phenotypes. Only in the case of a larger deletion of one TAD boundary could some ectopic influence of the Shh limb enhancer be detected on a gene - Mnx1 in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.

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