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1.  Differential requirement of bone morphogenetic protein receptors Ia (ALK3) and Ib (ALK6) in early embryonic patterning and neural crest development 
Background
Bone morphogenetic proteins regulate multiple processes in embryonic development, including early dorso-ventral patterning and neural crest development. BMPs activate heteromeric receptor complexes consisting of type I and type II receptor-serine/threonine kinases. BMP receptors Ia and Ib, also known as ALK3 and ALK6 respectively, are the most common type I receptors that likely mediate most BMP signaling events. Since early expression patterns and functions in Xenopus laevis development have not been described, we have addressed these questions in the present study.
Results
Here we have analyzed the temporal and spatial expression patterns of ALK3 and ALK6; we have also carried out loss-of-function studies to define the function of these receptors in early Xenopus development. We detected both redundant and non-redundant roles of ALK3 and ALK6 in dorso-ventral patterning. From late gastrula stages onwards, their expression patterns diverged, which correlated with a specific, non-redundant requirement of ALK6 in post-gastrula neural crest cells. ALK6 was essential for induction of neural crest cell fate and further development of the neural crest and its derivatives.
Conclusions
ALK3 and ALK6 both contribute to the gene regulatory network that regulates dorso-ventral patterning; they play partially overlapping and partially non-redundant roles in this process. ALK3 and ALK6 are independently required for the spatially restricted activation of BMP signaling and msx2 upregulation at the neural plate border, whereas in post-gastrula development ALK6 exerts a highly specific, conserved function in neural crest development.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-016-0101-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-016-0101-5
PMCID: PMC4717534  PMID: 26780949
BMP receptor; ALK3; ALK6; Xenopus; Dorso-ventral patterning; Neural crest
2.  Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration 
Background
The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals.
Results
Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish.
Conclusions
The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration.
doi:10.1186/s12861-016-0102-4
PMCID: PMC4719692  PMID: 26787303
Vertebral column; Caudal fin; Mineral apposition; Bone; Fluorescence imaging; Calcium; Hydroxyapatite; Alizarin red S
3.  Identification of differential microRNA expression during tooth morphogenesis in the heterodont dentition of miniature pigs, SusScrofa 
Background
It has been found that microRNAs (miRNAs) play important roles in the regulation of tooth development, and most likely increase the complexity of the genetic network, thus lead to greater complexity of teeth. But there has been no research about the key microRNAs associated with tooth morphogenesis based on miRNAs expression profiles. Compared to mice, the pig model has plentiful types of teeth, which is similar with the human dental pattern. Therefore, we used miniature pigs as large-animal models to investigate differentially expressed miRNAs expression during tooth morphogenesis in the early developmental stages of tooth germ.
Results
A custom-designed miRNA microarray with 742 miRNA gene probes was used to analyze the expression profiles of four types of teeth at three stages of tooth development. Of the 591 detectable miRNA transcripts, 212 miRNAs were continuously expressed in all types of tooth germ, but the numbers of miRNA transcript among the four different types of teeth at each embryonic stage were statistically significant differences (p < 0.01). The hierarchical clustering and principal component analysis results suggest that the miRNA expression was globally altered by types and temporal changes. By clustering analysis, we predicted 11 unique miRNA sequences that belong to mir-103 and mir-107, mir-133a and mir-133b, and mir-127 isomiR families. The results of real-time reverse-transcriptase PCR and in situ hybridization experiments revealed that five representative miRNAs may play important roles during different developmental stages of the incisor, canine, biscuspid, and molar, respectively.
Conclusions
The present study indicated that these five miRNAs, including ssc-miR-103 and ssc-miR-107, ssc-miR-133a and ssc-miR-133b, and ssc-miR-127, may play key regulatory roles in different types of teeth during different stages and thus may play critical roles in tooth morphogenesis during early development in miniature pigs.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0099-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0099-0
PMCID: PMC4696248  PMID: 26715101
Odontogenesis; Morphogenesis; Microarray analysis; In situ hybridization; Miniature swine
4.  A co-culture assay of embryonic zebrafish hearts to assess migration of epicardial cells in vitro 
Background
The vertebrate heart consists of three cell layers: the innermost endothelium, the contractile myocardium and the outermost epicardium. The epicardium is vital for heart development and function, and forms from epicardial progenitor cells (EPCs), which migrate to the myocardium during early development. Disruptions in EPC migration and epicardium formation result in a number of cardiac malformations, many of which resemble congenital heart diseases in humans. Hence, it is important to understand the mechanisms that influence EPC migration and spreading in the developing heart. In vitro approaches heretofore have been limited to monolayer epicardial cell cultures, which may not fully capture the complex interactions that can occur between epicardial and myocardial cells in vivo.
Results
Here we describe a novel in vitro co-culture assay for assessing epicardial cell migration using embryonic zebrafish hearts. We isolated donor hearts from embryonic zebrafish carrying an epicardial-specific fluorescent reporter after epicardial cells were present on the heart. These were co-cultured with recipient hearts expressing a myocardial-specific fluorescent reporter, isolated prior to EPC migration. Using this method, we can clearly visualize the movement of epicardial cells from the donor heart onto the myocardium of the recipient heart. We demonstrate the utility of this method by showing that epicardial cell migration is significantly delayed or absent when myocardial cells lack contractility and when myocardial cells are deficient in tbx5 expression.
Conclusions
We present a method to assess the migration of epicardial cells in an in vitro assay, wherein the migration of epicardial cells from a donor heart onto the myocardium of a recipient heart in co-culture is monitored and scored. The donor and recipient hearts can be independently manipulated, using either genetic tools or pharmacological agents. This allows flexibility in experimental design for determining the role that target genes/signaling pathways in specific cell types may have on epicardial cell migration.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0100-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0100-y
PMCID: PMC4696273  PMID: 26715205
Epicardial cell; Epicardium; Proepicardium; Myocardial cell; Myocardium; Migration; Co-culture
5.  Transcriptome profiling of bovine inner cell mass and trophectoderm derived from in vivo generated blastocysts 
Background
This study describes the generation and analysis of the transcriptional profile of bovine inner cell mass (ICM) and trophectoderm (TE), obtained from in vivo developed embryos by using a bovine-embryo specific array (EmbryoGENE) containing 37,238 probes.
Results
A total of 4,689 probes were differentially expressed between ICM and TE, among these, 2,380 and 2,309 probes were upregulated in ICM and TE tissues, respectively (P ≤ 0.01, FC ≥ 2.0, FDR: 2.0). Ontological classification of the genes predominantly expressed in ICM emerged a range of functional categories with a preponderance of genes involved in basal and developmental signaling pathways including P53, TGFβ, IL8, mTOR, integrin, ILK, and ELF2 signalings. Cross-referencing of microarray data with two available in vitro studies indicated a marked reduction in ICM vs. TE transcriptional difference following in vitro culture of bovine embryos. Moreover, a great majority of genes that were found to be misregulated following in vitro culture of bovine embryos were known genes involved in epigenetic regulation of pluripotency and cell differentiation including DNMT1, GADD45, CARM1, ELF5 HDAC8, CCNB1, KDM6A, PRDM9, CDX2, ARID3A, IL6, GADD45A, FGFR2, PPP2R2B, and SMARCA2. Cross-species referencing of microarray data revealed substantial divergence between bovine and mouse and human in signaling pathways involved in early lineage specification.
Conclusions
The transcriptional changes occur during ICM and TE lineages specification in bovine is greater than previously understood. Therefore, this array data establishes a standard to evaluate the in vitro imprint on the transcriptome and to hypothesize the cross-species differences that allow in vitro acquisition of pluripotent ICM in human and mice but hinder that process in bovine.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0096-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0096-3
PMCID: PMC4683974  PMID: 26681441
Bovine; In vivo blastocyst; Inner cell mass; Trophectoderm; Transcriptome
6.  Cellular dynamics underlying regeneration of appropriate segment number during axolotl tail regeneration 
Background
Salamanders regenerate their tails after amputation anywhere along their length. How the system faithfully reconstitutes the original number of segments and length is not yet known.
Methods
To gain quantitative insight into how the system regenerates the appropriate length, we amputated tails at 4 or 16 myotomes post-cloaca and measured blastema size, cell cycle kinetics via cumulative Bromodeoxyuridine (BrdU) incorporation and the method of Nowakowski, and myotome differentiation rate.
Results
In early stages until day 15, blastema cells were all proliferative and divided at the same rate at both amputation levels. A larger blastema was formed in 4th versus 16th myotome amputations indicating a larger founding population. Myotome differentiation started at the same timepoint in the 4th and 16 th level blastemas. The rate of myotome formation was more rapid in 4th myotome blastemas so that by day 21 the residual blastema from the two amputation levels achieved equivalent size. At that time point, only a fraction of blastema cells remain in cycle, with the 4th myotome blastema harboring double the number of cycling cells as the 16th myotome blastema allowing it to grow faster and further reconstitute the larger number of missing myotomes.
Conclusions
These data suggest that there are two separable phases of blastema growth. The first is level-independent, with cells displaying unrestrained proliferation. In the second phase, the level-specific growth is revealed, where differing fractions of cells remain in the cell cycle over time.
doi:10.1186/s12861-015-0098-1
PMCID: PMC4673748  PMID: 26647066
Salamander; Tail; Regeneration; Size control; Cell cycle; Myotome; Segmentation
7.  Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis 
Background
Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics.
Results
We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection.
Conclusions
HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0097-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0097-2
PMCID: PMC4667476  PMID: 26627605
Transcriptome; Geldanamycin; Bile acid; cyp7a1; Biliary atresia
8.  3D structured illumination microscopy of mammalian embryos and spermatozoa 
Background
Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass.
Results
Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa.
Conclusions
Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa.
doi:10.1186/s12861-015-0092-7
PMCID: PMC4661982  PMID: 26610350
Super-resolution fluorescence microscopy; Mammalian embryos
9.  Positional plasticity in regenerating Amybstoma mexicanum limbs is associated with cell proliferation and pathways of cellular differentiation 
Background
The endogenous ability to dedifferentiate, re-pattern, and re-differentiate adult cells to repair or replace damaged or missing structures is exclusive to only a few tetrapod species. The Mexican axolotl is one example of these species, having the capacity to regenerate multiple adult structures including their limbs by generating a group of progenitor cells, known as the blastema, which acquire pattern and differentiate into the missing tissues. The formation of a limb regenerate is dependent on cells in the connective tissues that retain memory of their original position in the limb, and use this information to generate the pattern of the missing structure. Observations from recent and historic studies suggest that blastema cells vary in their potential to pattern distal structures during the regeneration process; some cells are plastic and can be reprogrammed to obtain new positional information while others are stable. Our previous studies showed that positional information has temporal and spatial components of variation; early bud (EB) and apical late bud (LB) blastema cells are plastic while basal-LB cells are stable. To identify the potential cellular and molecular basis of this variation, we compared these three cell populations using histological and transcriptional approaches.
Results
Histologically, the basal-LB sample showed greater tissue organization than the EB and apical-LB samples. We also observed that cell proliferation was more abundant in EB and apical-LB tissue when compared to basal-LB and mature stump tissue. Lastly, we found that genes associated with cellular differentiation were expressed more highly in the basal-LB samples.
Conclusions
Our results characterize histological and transcriptional differences between EB and apical-LB tissue compared to basal-LB tissue. Combined with our results from a previous study, we hypothesize that the stability of positional information is associated with tissue organization, cell proliferation, and pathways of cellular differentiation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0095-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0095-4
PMCID: PMC4657325  PMID: 26597593
Limb regeneration; Positional information; Plasticity; Intercalation; Differentiation; Extracellular matrix; Microarray
10.  The developmental basis of mesenchymal stem/stromal cells (MSCs) 
Background
Mesenchymal Stem/Stromal Cells (MSCs) define a population of progenitor cells capable of giving rises to at least three mesodermal lineages in vitro, the chondrocytes, osteoblasts and adipocytes. The validity of MSCs in vivo has been questioned because their existence, either as a homogeneous progenitor cell population or as a stem cell lineage, has been difficult to prove. The wide use of primary MSCs in regenerative and therapeutic applications raises ethical and regulatory concerns in many countries. In contrast to hematopoietic stem cells, a parallel concept which carries an embryological emphasis from its outset, MSCs have attracted little interest among developmental biologists and the embryological basis for their existence, or lack thereof, has not been carefully evaluated.
Methods
This article provides a brief, embryological overview of these three mesoderm cell lineages and offers a framework of ontological rationales for the potential existence of MSCs in vivo.
Results
Emphasis is given to the common somatic lateral plate mesoderm origin of the majority of body’s adipose and skeletal tissues and of the major sources used for MSC derivation clinically. Support for the MSC hypothesis also comes from a large body of molecular and lineage analysis data in vivo.
Conclusions
It is concluded that despite the lack of a definitive proof, the MSC concept has a firm embryological basis and that advances in MSC research can be facilitated by achieving a better integration with developmental biology.
doi:10.1186/s12861-015-0094-5
PMCID: PMC4654913  PMID: 26589542
11.  MiR-21 is required for efficient kidney regeneration in fish 
Background
Acute kidney injury in mammals, which is caused by cardiovascular diseases or the administration of antibiotics with nephrotoxic side-effects is a life-threatening disease, since loss of nephrons is irreversible in mammals. In contrast, fish are able to generate new nephrons even in adulthood and thus provide a good model to study renal tubular regeneration.
Results
Here, we investigated the early response after gentamicin-induced renal injury, using the short-lived killifish Nothobranchius furzeri. A set of microRNAs was differentially expressed after renal damage, among them miR-21, which was up-regulated. A locked nucleic acid-modified antimiR-21 efficiently knocked down miR-21 activity and caused a lag in the proliferative response, enhanced apoptosis and an overall delay in regeneration. Transcriptome profiling identified apoptosis as a process that was significantly affected upon antimiR-21 administration. Together with functional data this suggests that miR-21 acts as a pro-proliferative and anti-apoptotic factor in the context of kidney regeneration in fish. Possible downstream candidate genes that mediate its effect on proliferation and apoptosis include igfbp3 and fosl1, among other genes.
Conclusion
In summary, our findings extend the role of miR-21 in the kidney. For the first time we show its functional involvement in regeneration indicating that fast proliferation and reduced apoptosis are important for efficient renal tubular regeneration.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0089-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0089-2
PMCID: PMC4650918  PMID: 26577279
Kidney regeneration; LNA; miRNA; Teleost killifish; igfbp3; fosl1
12.  Not all the number of skeletal muscle fibers is determined prenatally 
Background
The investigation of skeletal muscle development is of importance in stock farming and biomedicine. It is still ambiguous that whether animals are born with the full set of skeletal muscle fibers or if the number of myofibers continues to increase postnatally.
Results
Here, an inducible lineage-tracing system was employed to monitor the changes of myofiber number in various skeletal muscles during development. We confirm that the total myofiber number of longissimus dorsi, gastrocnemius and rectus femoris is determined prenatally. However, tibialis anterior and extensor digitorum longus have a different development pattern, and their myofiber number still increases in the first postnatal week and then remains stable afterwards.
Conclusions
Our results highlight different development time frames of anatomically distinct skeletal muscles.
doi:10.1186/s12861-015-0091-8
PMCID: PMC4642764  PMID: 26559169
Lineage-tracing system; Myofiber number; Development time frames
13.  Med14 cooperates with brg1 in the differentiation of skeletogenic neural crest 
Background
An intricate gene regulatory network drives neural crest migration and differentiation. How epigenetic regulators contribute to this process is just starting to be understood.
Results
We found that mutation of med14 or brg1 in zebrafish embryos resulted in a cluster of neural crest cell-related defects. In med14 or brg1 mutants, neural crest cells that form the jaw skeleton were specified normally and migrated to target sites. However, defects in their subsequent terminal differentiation were evident. Transplantation experiments demonstrated that med14 and brg1 are required directly in neural crest cells. Analysis of med14; brg1 double mutant embryos suggested the existence of a strong genetic interaction between members of the Mediator and BAF complexes.
Conclusions
These results suggest a critical role for Mediator and BAF complex function in neural crest development, and may also clarify the nature of defects in some craniofacial abnormalities.
doi:10.1186/s12861-015-0090-9
PMCID: PMC4640375  PMID: 26553192
Neural crest; Mediator complex; BAF complex; Brg1; Craniofacial defect; Med14; Jaw development
14.  Meis2 is essential for cranial and cardiac neural crest development 
Background
TALE-class homeodomain transcription factors Meis and Pbx play important roles in formation of the embryonic brain, eye, heart, cartilage or hematopoiesis. Loss-of-function studies of Pbx1, 2 and 3 and Meis1 documented specific functions in embryogenesis, however, functional studies of Meis2 in mouse are still missing. We have generated a conditional allele of Meis2 in mice and shown that systemic inactivation of the Meis2 gene results in lethality by the embryonic day 14 that is accompanied with hemorrhaging.
Results
We show that neural crest cells express Meis2 and Meis2-defficient embryos display defects in tissues that are derived from the neural crest, such as an abnormal heart outflow tract with the persistent truncus arteriosus and abnormal cranial nerves. The importance of Meis2 for neural crest cells is further confirmed by means of conditional inactivation of Meis2 using crest-specific AP2α-IRES-Cre mouse. Conditional mutants display perturbed development of the craniofacial skeleton with severe anomalies in cranial bones and cartilages, heart and cranial nerve abnormalities.
Conclusions
Meis2-null mice are embryonic lethal. Our results reveal a critical role of Meis2 during cranial and cardiac neural crest cells development in mouse.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0093-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0093-6
PMCID: PMC4636814  PMID: 26545946
Meis2; Neural crest; Persistent truncus arteriosus; Craniofacial skeleton; Cranial nerves
15.  Stable and bicistronic expression of two genes in somite- and lateral plate-derived tissues to study chick limb development 
Background
Components of the limb musculoskeletal system have distinct mesoderm origins. Limb skeletal muscles originate from somites, while the skeleton and attachments (tendons and connective tissues) derive from limb lateral plate. Despite distinct mesoderm origins, the development of muscle, skeleton and attachments is highly coordinated both spatially and temporally to ensure complete function of the musculoskeletal system. A system to study molecular interactions between somitic-derived tissues (muscles) and lateral-plate-derived tissues (skeletal components and attachments) during limb development is missing.
Results
We designed a gene delivery system in chick embryos with the ultimate aim to study the interactions between the components of the musculoskeletal system during limb development. We combined the Tol2 genomic integration system with the viral T2A system and developed new vectors that lead to stable and bicistronic expression of two proteins at comparable levels in chick cells. Combined with limb somite and lateral plate electroporation techniques, two fluorescent reporter proteins were co-expressed in stoichiometric proportion in the muscle lineage (somitic-derived) or in skeleton and their attachments (lateral-plate-derived). In addition, we designed three vectors with different promoters to target muscle cells at different steps of the differentiation process.
Conclusion
Limb somite electroporation technique using vectors containing these different promoters allowed us to target all myogenic cells, myoblasts or differentiated muscle cells. These stable and promoter-specific vectors lead to bicistronic expression either in somitic-derived myogenic cells or lateral plate-derived cells, depending on the electroporation sites and open new avenues to study the interactions between myogenic cells and tendon or connective tissue cells during limb development.
doi:10.1186/s12861-015-0088-3
PMCID: PMC4628273  PMID: 26518454
Electroporation; 2A peptide; Tol2; Muscle; Tendon; Limb; Chick
16.  A loss-of-function and H2B-Venus transcriptional reporter allele for Gata6 in mice 
Background
The GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs.
Results
We have generated a Gata6H2B-Venus knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution.
Conclusions
Expression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6H2B-Venus/H2B-Venus homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0086-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0086-5
PMCID: PMC4619391  PMID: 26498761
GATA6; H2B-Venus; Reporter mice; Endoderm; Cardiac mesoderm; Live Imaging
17.  Sox7 is dispensable for primitive endoderm differentiation from mouse ES cells 
Background
Primitive endoderm is a cell lineage segregated from the epiblast in the blastocyst and gives rise to parietal and visceral endoderm. Sox7 is a member of the SoxF gene family that is specifically expressed in primitive endoderm in the late blastocyst, although its function in this cell lineage remains unclear.
Results
Here we characterize the function of Sox7 in primitive endoderm differentiation using mouse embryonic stem (ES) cells as a model system. We show that ectopic expression of Sox7 in ES cells has a marginal effect on triggering differentiation into primitive endoderm-like cells. We also show that targeted disruption of Sox7 in ES cells does not affect differentiation into primitive endoderm cells in embryoid body formation as well as by forced expression of Gata6.
Conclusions
These data indicate that Sox7 function is supplementary and not essential for this differentiation from ES cells.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0079-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0079-4
PMCID: PMC4609079  PMID: 26475439
ES cells; XEN cells; primitive endoderm; Sox7
18.  Muscleblind-like 1 is required for normal heart valve development in vivo 
Background
Development of the valves and septa of the heart depends on the formation and remodeling of the endocardial cushions in the atrioventricular canal and outflow tract. These cushions are populated by mesenchyme produced from the endocardium by epithelial-mesenchymal transition (EMT). The endocardial cushions are remodeled into the valves at post-EMT stages via differentiation of the mesenchyme and changes in the extracellular matrix (ECM). Transforming growth factor β (TGFβ) signaling has been implicated in both the induction of EMT in the endocardial cushions and the remodeling of the valves at post-EMT stages. We previously identified the RNA binding protein muscleblind-like 1 (MBNL1) as a negative regulator of TGFβ signaling and EMT in chicken endocardial cushions ex vivo.  Here, we investigate the role of MBNL1 in endocardial cushion development and valvulogenesis in Mbnl1∆E3/∆E3 mice, which are null for MBNL1 protein.
Methods
Collagen gel invasion assays, histology, immunohistochemistry, real-time RT-PCR, optical coherence tomography, and echocardiography were used to evaluate EMT and TGFβ signaling in the endocardial cushions, and morphogenesis, ECM composition, and function of the heart valves.
Results
As in chicken, the loss of MBNL1 promotes precocious TGFβ signaling and EMT in the endocardial cushions. Surprisingly, this does not lead to the production of excess mesenchyme, but later valve morphogenesis is aberrant. Adult Mbnl1∆E3/∆E3 mice exhibit valve dysmorphia with elevated TGFβ signaling, changes in ECM composition, and increased pigmentation. This is accompanied by a high incidence of regurgitation across both inflow and outflow valves. Mbnl1∆E3/∆E3 mice also have a high incidence of ostium secundum septal defects accompanied by atrial communication, but do not develop overt cardiomyopathy.
Conclusions
Together, these data indicate that MBNL1 plays a conserved role in negatively regulating TGFβ signaling, and is required for normal valve morphogenesis and homeostasis in vivo.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0087-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0087-4
PMCID: PMC4608261  PMID: 26472242
Muscleblind-like 1; Transforming growth factor β; Epithelial-mesenchymal transition; Endocardial cushions; Heart valves; Mouse
19.  Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation 
Background
Pluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking.
Methods
We manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively.
Results
Here, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo.
Conclusions
Our findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0084-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0084-7
PMCID: PMC4599726  PMID: 26453549
Extragonadal teratoma; Pluripotency; Oct4; Nanog; Brachyury; Inducible expression
20.  Cooperative and independent functions of FGF and Wnt signaling during early inner ear development 
Background
In multiple vertebrate organisms, including chick, Xenopus, and zebrafish, Fibroblast Growth Factor (FGF) and Wnt signaling cooperate during formation of the otic placode. However, in the mouse, although FGF signaling induces Wnt8a expression during induction of the otic placode, it is unclear whether these two signaling pathways functionally cooperate. Sprouty (Spry) genes encode intracellular antagonists of receptor tyrosine kinase signaling, including FGF signaling. We previously demonstrated that the Sprouty1 (Spry1) and Sprouty2 (Spry2) genes antagonize FGF signaling during induction of the otic placode. Here, we investigate cross talk between FGF/SPRY and Wnt signaling during otic placode induction and assess whether these two signaling pathways functionally cooperate during early inner ear development in the mouse.
Methods
Embryos were generated carrying combinations of a Spry1 null allele, Spry2 null allele, β-catenin null allele, or a Wnt reporter transgene. Otic phenotypes were assessed by in situ hybridization, semi-quantitative reverse transcriptase PCR, immunohistochemistry, and morphometric analysis of sectioned tissue.
Results
Comparison of Spry1, Spry2, and Wnt reporter expression in pre-otic and otic placode cells indicates that FGF signaling precedes and is active in more cells than Wnt signaling. We provide in vivo evidence that FGF signaling activates the Wnt signaling pathway upstream of TCF/Lef transcriptional activation. FGF regulation of Wnt signaling is functional, since early inner ear defects in Spry1 and Spry2 compound mutant embryos can be genetically rescued by reducing the activity of the Wnt signaling pathway. Interestingly, we find that although the entire otic placode increases in size in Spry1 and Spry2 compound mutant embryos, the size of the Wnt-reporter-positive domain does not increase to the same extent as the Wnt-reporter-negative domain.
Conclusions
This study provides genetic evidence that FGF and Wnt signaling cooperate during early inner ear development in the mouse. Furthermore, our data suggest that although specification of the otic placode may be globally regulated by FGF signaling, otic specification of cells in which both FGF and Wnt signaling are active may be more tightly regulated.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0083-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0083-8
PMCID: PMC4594887  PMID: 26443994
Inner ear; Otic placode; Sprouty1; Sprouty2; Wnt8a; FGF; β-catenin; Cross talk
21.  Conservation analysis of sequences flanking the testis-determining gene Sry in 17 mammalian species 
Background
Sex determination in mammals requires expression of the Y-linked gene Sry in the bipotential genital ridges of the XY embryo. Even minor delay of the onset of Sry expression can result in XY sex reversal, highlighting the need for accurate gene regulation during sex determination. However, the location of critical regulatory elements remains unknown. Here, we analysed Sry flanking sequences across many species, using newly available genome sequences and computational tools, to better understand Sry’s genomic context and to identify conserved regions predictive of functional roles.
Methods
Flanking sequences from 17 species were analysed using both global and local sequence alignment methods. Multiple motif searches were employed to characterise common motifs in otherwise unconserved sequence.
Results
We identified position-specific conservation of binding motifs for multiple transcription factor families, including GATA binding factors and Oct/Sox dimers. In contrast with the landscape of extremely low sequence conservation around the Sry coding region, our analysis highlighted a strongly conserved interval of ~106 bp within the Sry promoter (which we term the Sry Proximal Conserved Interval, SPCI). We further report that inverted repeats flanking murine Sry are much larger than previously recognised.
Conclusions
The unusually fast pace of sequence drift on the Y chromosome sharpens the likely functional significance of both the SPCI and the identified binding motifs, providing a basis for future studies of the role(s) of these elements in Sry regulation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0085-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0085-6
PMCID: PMC4595323  PMID: 26444262
SRY; Sex determination; Y chromosome; Gene regulation; Testis; Gonad
22.  Evidence for lung epithelial stem cell niches 
Recent studies have identified epithelial stem and progenitor cell populations of the lung. We are just beginning to understand the mechanisms that regulate their homeostatic, regenerative and maladaptive behaviors. Here, we discuss evidence of regulatory niches for epithelial stem cells of the lung.
doi:10.1186/s12861-015-0082-9
PMCID: PMC4574358  PMID: 26376663
23.  The growth pattern of the human intestine and its mesentery 
Background
It remains unclear to what extent midgut rotation determines human intestinal topography and pathology. We reinvestigated the midgut during its looping and herniation phases of development, using novel 3D visualization techniques.
Results
We distinguished 3 generations of midgut loops. The topography of primary and secondary loops was constant, but that of tertiary loops not. The orientation of the primary loop changed from sagittal to transverse due to the descent of ventral structures in a body with a still helical body axis. The 1st secondary loop (duodenum, proximal jejunum) developed intraabdominally towards a left-sided position. The 2nd secondary loop (distal jejunum) assumed a left-sided position inside the hernia before returning, while the 3rd and 4th secondary loops retained near-midline positions. Intestinal return into the abdomen resembled a backward sliding movement. Only after return, the 4th secondary loop (distal ileum, cecum) rapidly “slid” into the right lower abdomen. The seemingly random position of the tertiary small-intestinal loops may have a biomechanical origin.
Conclusions
The interpretation of “intestinal rotation” as a mechanistic rather than a descriptive concept underlies much of the confusion accompanying the physiological herniation. We argue, instead, that the concept of “en-bloc rotation” of the developing midgut is a fallacy of schematic drawings. Primary, secondary and tertiary loops arise in a hierarchical fashion. The predictable position and growth of secondary loops is pre-patterned and determines adult intestinal topography. We hypothesize based on published accounts that malrotations result from stunted development of secondary loops.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0081-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0081-x
PMCID: PMC4546136  PMID: 26297675
24.  Monitoring brain development of chick embryos in vivo using 3.0 T MRI: subdivision volume change and preliminary structural quantification using DTI 
Background
Magnetic resonance imaging (MRI) has many advantages in the research of in vivo embryonic brain development, specifically its noninvasive aspects and ability to avoid skeletal interference. However, few studies have focused on brain development in chick, which is a traditional animal model in developmental biology. We aimed to serially monitor chick embryo brain development in vivo using 3.0 T MRI.
Methods
Ten fertile Hy-line white eggs were incubated and seven chick embryo brains were monitored in vivo and analyzed serially from 5 to 20 days during incubation using 3.0 T MRI. A fast positioning sequence was pre-scanned to obtain sagittal and coronal brain planes corresponding to the established atlas. T2-weighted imaging (T2WI) was performed for volume estimation of the whole brain and subdivision (telencephalon, cerebellum, brainstem, and lateral ventricle [LV]); diffusion tensor imaging (DTI) was used to reflect the evolution of neural bundle structures.
Results
The chick embryos’ whole brain and subdivision grew non-linearly over time; the DTI fractional anisotropy (FA) value within the telencephalon increased non-linearly as well. All seven scanned eggs hatched successfully.
Conclusions
MRI avoids embryonic sacrifice in a way that allows serial monitoring of longitudinal developmental processes of a single embryo. Feasibility for analyzing subdivision of the brain during development, and adding structural information related to neural bundles, makes MRI a powerful tool for exploring brain development.
doi:10.1186/s12861-015-0077-6
PMCID: PMC4513430  PMID: 26208519
Chick embryo; Brain development; In vivo; Magnetic resonance imaging; Diffusion tensor imaging
25.  The pattern of congenital heart defects arising from reduced Tbx5 expression is altered in a Down syndrome mouse model 
Background
Nearly half of all individuals with Down Syndrome (DS) have some type of congenital heart defect (CHD), suggesting that DS sensitizes to CHD but does not cause it. We used a common mouse model of DS, the Ts65Dn mouse, to study the contribution of Tbx5, a known modifier of CHD, to heart defects on a trisomic backgroun. Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10% formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays.
Methods
Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10 % formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays.
Results
We crossed mice that were heterozygous for a Tbx5 null allele with Ts65Dn mice. Mice that were trisomic and carried the Tbx5 mutation (Ts65Dn;Tbx5+/−) had a significantly increased incidence of overriding aorta compared to their euploid littermates. Ts65Dn;Tbx5+/− mice also showed reduced expression of Pitx2, a molecular marker for the left atrium. Transcript levels of the trisomic Adamts1 gene were decreased in Tbx5+/− mice compared to their euploid littermates. Evidence of a valid binding site for TBX5 upstream of the trisomic Adamts1 locus was also shown.
Conclusion
Haploinsufficiency of Tbx5 and trisomy affects alignment of the aorta and this effect may stem from deviations from normal left-right patterning in the heart. We have unveiled a previously unknown interaction between the Tbx5 gene and trisomy, suggesting a connection between Tbx5 and trisomic genes important during heart development.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0080-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0080-y
PMCID: PMC4514943  PMID: 26208718
Heart development; Congenital heart defect; Down syndrome; Trisomy

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