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- Embryonic and larval developmental stages of African giant catfish Heterobranchus bidorsalis (Geoffroy Saint Hilaire, 1809) (Teleostei, Clariidae). [Journal Article]
- Springerplus 2014.:677.
The dearth of African giant catfish Heterobranchus bidorsalis seeds poses great threat to its aquaculture and biodiversity, hence detailed knowledge and understanding of its embryology is indispensable for its artificial propagation and conservation programmes. Photomicrographs of extruded oocyte through all developmental cell stages of live embryo to larval stage are documented with the aid of a light microscope. The optical transparency of the developing embryo enabled us to describe its deep structures, distinctive features and characterize the stages pictorially. Extruded oocyte had a mean diameter of 1 ± 0.1 mm, ~20% increase when hydrated, and bounded by double thin perivitelline membranes. The first mitotic cleavage occurred at 69 min post-fertilization (pf) resulting in 2, 4 (2 × 2 array of cells), 8 (2 × 4), 16 (4 × 4), 32 (4 × 8), 64 (2 × 4 × 8) blastomeres, then developed to morula, blastula and gastrula stages. Blastula was featured by formation of enveloping layer and yolk syncytial layer. Onset of epiboly at 3 h 57 min depicted the commencement of gastrula while closure of blastopore at 11 h 8 min marked its completion. Neurulation period was distinct from segmentation where organogenesis was fully active. Embryo sudden muscular contraction was noticed at ~17 h pf, increased prior to hatching with caudal locomotion firstly at 42 s interval. Heartbeat of embryo commenced at ~1 h before its unique eclosion at average of 72 beats/min while first larva emerged at 21 h at a controlled temperature of 28.5 ± 0.5°C. Mean total length (TL) of larvae and their pouch thickness were 5 ± 1 mm and 0.05 ± 0.02 mm respectively. 1 -day old larvae revealed 8 distinctive neuromeres and by day 3, epicanthus folds of the eyes were fully uncovered; and thereafter commenced exogenous feeding. At day 4, larvae recorded mean TL of 9 ± 1 mm and 15 caudal fin rays. The fin bifurcation to dorsal and adipose fins was observed at third and half weeks post-hatchability with the dorsal fin length to adipose fin was 1.7:1. This study, for the first time, presents significant morpho-sequential developmental stages of H. bidorsalis and registers its unique form of eclosion.
- Cadherins as regulators of neuronal polarity. [JOURNAL ARTICLE]
- Cell Adh Migr 2014 Nov 14.:0.
A compelling amount of data is accumulating about the polyphonic role of neuronal cadherins during brain development throughout all developmental stages, starting from the involvement of cadherins in the organization of neurulation up to synapse development and plasticity. Recent work has confirmed that specifically N-cadherins play an important role in asymmetrical cellular processes in developing neurons that are at the basis of polarity. In this review we will summarize recent data, which demonstrate how N-cadherin orchestrates distinct processes of polarity establishment in neurons.
- Mapping the dynamic expression of Wnt11 and the lineage contribution of Wnt11-expressing cells during early mouse development. [JOURNAL ARTICLE]
- Dev Biol 2014 Nov 20.
Planar cell polarity (PCP) signaling is an evolutionarily conserved mechanism that coordinates polarized cell behavior to regulate tissue morphogenesis during vertebrate gastrulation, neurulation and organogenesis. In Xenopus and zebrafish, PCP signaling is activated by non-canonical Wnts such as Wnt11, and detailed understanding of Wnt11 expression has provided important clues on when, where and how PCP may be activated to regulate tissue morphogenesis. To explore the role of Wnt11 in mammalian development, we established a Wnt11 expression and lineage map with high spatial and temporal resolution by creating and analyzing a tamoxifen-inducible Wnt11-CreER BAC (bacterial artificial chromosome) transgenic mouse line. Our short- and long-term lineage tracing experiments indicated that Wnt11-CreER could faithfully recapitulate endogenous Wnt11 expression, and revealed for the first time that cells transiently expressing Wnt11 at early gastrulation were fated to become specifically the progenitors of the entire endoderm. During mid-gastrulation, Wnt11-CreER expressing cells also contribute extensively to the endothelium in both embryonic and extraembryonic compartments, and the endocardium in all chambers of the developing heart. In contrast, Wnt11-CreER expression in the myocardium starts from late-gastrulation, and occurs in three transient, sequential waves: first in the precursors of the left ventricular (LV) myocardium from E7.0 to 8.0; subsequently in the right ventricular (RV) myocardium from E8.0 to 9.0; and finally in the superior wall of the outflow tract (OFT) myocardium from E8.5 to 10.5. These results provide formal genetic proof that the majority of the endocardium and myocardium diverge by mid-gastrulation in the mouse, and suggest a tight spatial and temporal control of Wnt11 expression in the myocardial lineage to coordinate with myocardial differentiation in the first and second heart field progenitors to form the LV, RV and OFT. The insights gained from this study will also guide future investigations to decipher the role of non-canonical Wnt/PCP signaling in endoderm development, vasculogenesis and heart formation.
- Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification. [Journal Article]
- Theriogenology 2015 Jan 15; 83(2):238-245.e2.
This study was designed to test a vitrification method in Atlantic salmon spermatozoa and determine the capacity of seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium consisted of a standard buffer for fish spermatozoa (Cortland medium) + 10% DMSO + 2% BSA + 0.13-M sucrose + SP at concentrations of 30% (G30), 40% (G40), or 50% (G50). Fresh sperm was used as a control. To freeze the samples, 30-μL suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. The resulting spheres were placed in cryotubes for storage in liquid nitrogen. The cryotubes with the vitrified spermatozoa were thawed by placing them in a water bath at 37 °C for 45 seconds. After thawing, the following sperm quality parameters were determined by flow cytometry: DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling), plasma membrane integrity (SYBR-14/PI, staining technique), and mitochondrial membrane potential (JC-1 staining). An optical microscope was used to assess subjectively sperm motility, whereas fertility was determined by the presence of neurulation using five replicates per treatment in a sample of 30 eggs. Spermatozoa quality variables were preserved best when the highest concentration of SP (50%) was used (DNA fragmentation, 9.2%; plasma membrane integrity, 98.6%; mitochondrial membrane integrity, 47.2%; motility, 44.1%; and fertility, 46.2%).
- Development of the vertebrate tailbud. [Journal Article]
- Wiley Interdiscip Rev Dev Biol 2015 Jan; 4(1):33-44.
The anatomical tailbud is a defining feature of all embryonic chordates, including vertebrates that do not end up with a morphological tail. Due to its seamless continuity with trunk tissues, the tailbud is often overlooked as a mere extension of the body axis; however, the formation of the tail from the tailbud undoubtedly involves unique and distinct mechanisms for forming axial tissues, such as the secondary neurulation process that generates the tailbud-derived spinal cord. Tailbud formation in the frog Xenopus laevis has been demonstrated to involve interaction of three posterior regions of the embryo that first come into alignment at the end of gastrulation, and molecular models for tailbud outgrowth and patterning have been proposed. While classical studies of other vertebrate models, such as the chicken, initially appeared to draw incompatible conclusions, molecular studies have subsequently shown the involvement of at least some similar genetic pathways. Finally, there is an emerging consensus that at least some vertebrate tailbud cells are multipotent progenitors with the ability to form tissues normally derived from different germ layers- a trait normally associated with regeneration of complex appendages, or stem-like cells. WIREs Dev Biol 2015, 4:33-44. doi: 10.1002/wdev.163 CONFLICT OF INTEREST: The author has declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.
- Comparative Genomic Analysis of slc39a12/ZIP12: Insight into a Zinc Transporter Required for Vertebrate Nervous System Development. [Journal Article]
- PLoS One 2014; 9(11):e111535.
The zinc transporter ZIP12, which is encoded by the gene slc39a12, has previously been shown to be important for neuronal differentiation in mouse Neuro-2a neuroblastoma cells and primary mouse neurons and necessary for neurulation during Xenopus tropicalis embryogenesis. However, relatively little is known about the biochemical properties, cellular regulation, or the physiological role of this gene. The hypothesis that ZIP12 is a zinc transporter important for nervous system function and development guided a comparative genetics approach to uncover the presence of ZIP12 in various genomes and identify conserved sequences and expression patterns associated with ZIP12. Ortholog detection of slc39a12 was conducted with reciprocal BLAST hits with the amino acid sequence of human ZIP12 in comparison to the human paralog ZIP4 and conserved local synteny between genomes. ZIP12 is present in the genomes of almost all vertebrates examined, from humans and other mammals to most teleost fish. However, ZIP12 appears to be absent from the zebrafish genome. The discrimination of ZIP12 compared to ZIP4 was unsuccessful or inconclusive in other invertebrate chordates and deuterostomes. Splice variation, due to the inclusion or exclusion of a conserved exon, is present in humans, rats, and cows and likely has biological significance. ZIP12 also possesses many putative di-leucine and tyrosine motifs often associated with intracellular trafficking, which may control cellular zinc uptake activity through the localization of ZIP12 within the cell. These findings highlight multiple aspects of ZIP12 at the biochemical, cellular, and physiological levels with likely biological significance. ZIP12 appears to have conserved function as a zinc uptake transporter in vertebrate nervous system development. Consequently, the role of ZIP12 may be an important link to reported congenital malformations in numerous animal models and humans that are caused by zinc deficiency.
- Embryonic Expression and Function of the Xenopus Ink4d Cyclin D-Dependent Kinase Inhibitor. [JOURNAL ARTICLE]
- Cell Dev Biol 2014 Feb 15; 3(1)
Here we report the cloning and functional characterization of the cyclin D-dependent kinase 4 and 6 (Cdk4/6) inhibitory protein Cdkn2d/p19(Ink4d) of Xenopuslaevis (Xl-Ink4d). Xl-Ink4d is the only Ink4 family gene highly expressed during Xenopus development and its transcripts were detected maternally and during neurulation. The Xl-Ink4d protein has 63% identity to mouse and human Cdkn2d/p19(Ink4d) and its function as a negative regulator of cell cycle traverse is evolutionary conserved. Indeed, Xl-lnk4d can functionally substitute for mouse Cdkn2d in binding to mouse Cdk4 and inhibiting cyclin-D1-dependent CDK4 kinase activity. Further, enforced expression of Xl-lnk4d arrests mouse fibroblasts in the G1 phase of the cell cycle. These findings indicate that CDKN2d/p19(Ink4d) is conserved through vertebrate evolution and suggest Xl-lnk4d may contribute to the development of Xenopuslaevis.
- Cell lineages and fate maps in tunicates: conservation and modification. [Journal Article]
- Zoolog Sci 2014 Oct; 31(10):645-52.
Comparison of features of the cell lineages and fate maps of early embryos between related species is useful in inferring developmental mechanisms and amenable to evolutionary considerations. We present cleavage patterns, cell lineage trees, and fate maps of ascidian and appendicularian embryos side by side to facilitate comparison. This revealed a number of significant differences in cleavage patterns and cell lineage trees, whereas the fate maps were found to be conserved. This fate map similarity can be extended to vertebrates, thus representing the fate map characteristics of chordates. Cleavage patterns and cell lineages may have been modified during evolution without any drastic changes in fate maps. Selective pressures that constrain developmental mechanisms at early embryonic stages might not be so strong as long as embryos are still able to generate a chordate-type fate map. Aquatic chordates share similar fate maps and morphogenetic movements during gastrulation and neurulation, eventually developing into tadpole-shaped larvae. As swimming by tail beats, and not by cilia, is advantageous, selective pressure may maintain the basic elements of the tadpole shape. We also discuss the evolutionary origin of the vertebrate neural crest and the embryonic origin of the appendicularian heart to illustrate the usefulness of cell lineage data. From an evolutionary standpoint, cell lineages behave like other characteristics such as morphology or protein sequences. Both novel and primitive features are present in extant organisms, and it is of interest to identify the relative degree of evolutionary conservation as well as the level at which homology is inferred.
- Junctional neurulation: a unique developmental program shaping a discrete region of the spinal cord highly susceptible to neural tube defects. [Journal Article, Research Support, Non-U.S. Gov't]
- J Neurosci 2014 Sep 24; 34(39):13208-21.
In higher vertebrates, the primordium of the nervous system, the neural tube, is shaped along the rostrocaudal axis through two consecutive, radically different processes referred to as primary and secondary neurulation. Failures in neurulation lead to severe anomalies of the nervous system, called neural tube defects (NTDs), which are among the most common congenital malformations in humans. Mechanisms causing NTDs in humans remain ill-defined. Of particular interest, the thoracolumbar region, which encompasses many NTD cases in the spine, corresponds to the junction between primary and secondary neurulations. Elucidating which developmental processes operate during neurulation in this region is therefore pivotal to unraveling the etiology of NTDs. Here, using the chick embryo as a model, we show that, at the junction, the neural tube is elaborated by a unique developmental program involving concerted movements of elevation and folding combined with local cell ingression and accretion. This process ensures the topological continuity between the primary and secondary neural tubes while supplying all neural progenitors of both the junctional and secondary neural tubes. Because it is distinct from the other neurulation events, we term this phenomenon junctional neurulation. Moreover, the planar-cell-polarity member, Prickle-1, is recruited specifically during junctional neurulation and its misexpression within a limited time period suffices to cause anomalies that phenocopy lower spine NTDs in human. Our study thus provides a molecular and cellular basis for understanding the causality of NTD prevalence in humans and ascribes to Prickle-1 a critical role in lower spinal cord formation.
- Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character. [Journal Article]
- Dev Biol 2014 Nov 15; 395(2):317-30.
The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.