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Infection with the avian polyomavirus, BFDV, selectively affects myofibril structure in embryonic chick ventricle cardiomyocytes.

Embryonic cardiomyocytes can both beat and divide. They assemble cardiac muscle-specific proteins into sarcomeric myofibrils and contract. In addition, they periodically synthesize DNA, complete mitosis, disassemble sarcomeric myofibrils in the area of the mitotic spindle, assemble cytoplasmic isoform-specific proteins into a cleavage furrow contractile ring, undergo cytokinesis, and then reform sarcomeric myofibrils in daughter cells. Little is known about how embryonic cardiomyocytes disassemble their myofibrils as they traverse the cell cycle and divide. In the present study, beating embryonic avian ventricular cardiomyocytes in primary culture were stimulated to initiate DNA synthesis without subsequent mitosis or cytokinesis by infection with the lytic avian polyomavirus, Budgerigar Fledgling Disease Virus (BFDV). Within 48 hours, infected, adherent cardiomyocytes disassemble most of their sarcomeric myofibrils, retaining cardiac myosin only in thin myofibrils with disrupted sarcomeric periodicity and in amorphous nonfibrillar pools. By 72 hours, infected cardiomyocytes contain no myofibrils and no longer react with antibodies to cardiac myosin. In contrast, infected cardiomyocytes continue to display cytoplasmic myosin localized in stress-fiber-like-structures in adherent cells, or in disrupted fibers and dispersed pools in detaching cells. Infected cardiomyocytes also continue to display interphase-like arrays of polymerized microtubules, even when rounded-up just prior to lysis. These results suggest that polyomavirus infection may provide a useful model system for further study of the regulation of myofibrils disassembly in embryonic cardiomyocytes.

Animals↗

The flightless I protein localizes to actin-based structures during embryonic development.

The product of the flightless I gene is predicted to provide a link between molecules of an as yet unidentified signal transduction pathway and the actin cytoskeleton. Previous work has shown that weak and severe mutations of the flightless I locus in Drosophila melanogaster cause disruption in the indirect flight muscles and in embryonic cellularization events, respectively, indicative of a regulatory role for the flightless I protein in cytoskeletal rearrangements. A C-terminal domain within flightless I with significant homology to the gelsolin-like family of actin-binding proteins has been identified, but evidence of a direct interaction between endogenous flightless I and actin remains to be shown. In the present study, chick, mouse and Drosophila melanogaster embryos have been examined and the localization of flightless I investigated in relation to the actin cytoskeleton. It is shown that flightless I localization is coincident with actin-rich regions in parasympathetic neurons harvested from chicks, in mouse blastocysts and in structures associated with cellularization in Drosophila melanogaster.

3T3 Cells↗

Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo.

Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. We report that mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. We conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes.

Animals↗

An amphioxus netrin gene is expressed in midline structures during embryonic and larval development.

Members of the netrin gene family have been identified in vertebrates, Drosophila and Caenorhabditis elegans and found to encode secreted molecules involved in axon guidance. Here I use the conserved function of netrins in triploblasts, coupled with the phylogenetic position of amphioxus (the closest living relative of the vertebrates), to investigate the evolution of an axon guidance cue in chordates. A single amphioxus netrin gene was isolated by PCR and cDNA library screening and named AmphiNetrin. The predicted AmphiNetrin protein showed high identity to other netrin family members but differed in that the third of three EGF repeats found in other netrins was absent. Molecular phylogene-tic analysis showed that despite the absent EGF repeat AmphiNetrin is most closely related to the vertebrate netrins. AmphiNetrin expression was identified in embryonic notochord and floor plate, a pattern similar to that of vertebrate netrin-1 expression. AmphiNetrin expression was also identified more widely in the posterior larval brain, and in the anterior extension of the notochord that underlies the anterior of the amphioxus brain. All of these areas of expression are correlated with developing axon trajectories: The floor plate with ventrally projecting somatic motor neurons and Rohde cell projections, the posterior brain with the ventral commissure and primary motor centre and the anterior extension of the notochord with ventrally projecting neurons associated with the median eye. Amphioxus is naturally cyclopaedic and also lacks the ventral brain cells that the induction of which results in the splitting of the vertebrate eye field and, when missing, result in cyclopaedia. These cells normally express netrins required for developing axon tracts in the brain, and the expression of AmphiNetrin in the anterior extension of the notochord underlying the brain may explain how amphioxus is able to maintain ventral guidance cues while lacking these cells.

Amino Acid Sequence↗

The basic-helix-loop-helix transcription factor HAND2 directly regulates transcription of the atrial naturetic peptide gene.

The HAND basic Helix-Loop-Helix (bHLH) transcription factors are essential for the development of heart and extra embryonic structures. Although essential for embryonic development, the molecular pathways in which HAND factors participate are poorly understood. In efforts to identify downstream transcriptional targets, we have determined that HAND2 regulates the transcription of the Atrial Naturetic Peptide (ANP) gene. Results show that ANP expression is reduced in HAND2 null mice. Transactivation assays show significant transcriptional upregulation of ANP by HAND2 and cotransfection experiments using HAND2 and E12 suggest that an E-protein/HAND heterodimer is the likely trans -acting complex. The required cis -elements reside within a 258bp proximal region that contains three evolutionarily conserved Ebox consensus sites. Surprisingly, mutations in these three sites suggest HAND2 activity is DNA-binding independent. In addition, HAND2 and the homeobox factor Nkx2.5 exhibit transcriptional synergy in the regulation of ANP. Taken together, this data shows that HAND2 is an upstream transcriptional regulator of ANP expression, and furthermore HAND2 can synergistically interact with Nkx2.5, showing a functional relationship between HAND2 and Nkx2.5 supporting the genetic observation, that mice null for both HAND2 and Nkx2.5 lack ventricle specification.

Animals↗

A novel NK-related mouse homeobox gene: expression in central and peripheral nervous structures during embryonic development.

We have identified three novel mouse homeobox genes that are related to the Drosophila NK gene family. Two genes without direct homologues in Drosophila were designated Nkx-5.1 and Nkx-5.2; the third gene Nkx-1.1 constitutes the mouse homologue to NK1.Nkx-5.1 and Nkx-5.2 are closely linked on mouse chromosome 7, whereas Nkx-1.1 is located on a different chromosome. Here, we report the spatiotemporal expression pattern of Nkx-5.1 during prenatal mouse development. Nkx-5.1 gene activity begins at Embryonic Day 10.5 in the developing ear, the neural tube, and dorsal root ganglia. It continues to be active throughout prenatal life in discrete regions of the brain with an anterior border in the ventral diencephalon at the optic chiasma and expression domains in mesencephalon, metencephalon, and myelencephalon. At midgestation, Nkx-5.1 is also expressed in mesenchyme of the head and branchial arches, and in some cranial ganglia, as well as in derivatives of neural crest, such as the truncus sympathicus and myenteric ganglia. The time pattern of Nkx-5.1 expression and its confinement to primarily postmitotic cells of the central and peripheral nervous system suggest that Nkx-5.1 may play a role in the specification of neuronal cell types.

Amino Acid Sequence↗

Effects of dideoxyforskolin on proteoglycan synthesis and structure in embryonic chick chondrocyte cultures.

1,9-Dideoxyforskolin inhibits proteoglycan synthesis and xyloside-initiated glycosaminoglycan (GAG) synthesis in chick embryo chondrocytes. Dideoxyforskolin does not affect the length of xyloside-initiated GAG chains secreted into the medium but chains from the dense proteoglycan secreted into the medium appear slightly longer. Incorporation of labeled serine into the dense proteoglycan and subsequent digestion with Pronase revealed a dramatic decrease in percent of total radioactivity associated with GAG chains in the proteoglycan from cultures treated with forskolin or dideoxyforskolin. These observations suggest that these diterpenes have a specific inhibitory effect on chain initiation reactions and thus may be useful tools in the study of proteoglycan synthesis and processing.

Animals↗

Genomic structure and embryonic expression of zebrafish lysyl hydroxylase 1 and lysyl hydroxylase 2.

Collagen biosynthesis in both invertebrates and vertebrates is critically dependent upon the activity of lysyl hydroxylase (LH) enzymes. In humans, mutations in the genes encoding LH1 and LH2 have been shown to cause two distinct connective tissue disorders, Ehlers-Danlos (Type VIA) and Bruck syndromes. While the biochemical properties of these enzymes have been intensively studied, their embryonic patterns of expression and developmental roles remain unknown. We now present the cloning and analyses of the genes encoding LH1 and LH2 in the zebrafish, Danio rerio. We find these genes to be similarly organized to other vertebrate lh (plod) genes, including the presence of an alternatively spliced exon in lh2. We also examine the mRNA expression patterns of lh1 and lh2 during embryogenesis and find them to exhibit unique and dynamic patterns of expression. These results strongly suggest that LH enzymes are not merely housekeeping enzymes, but play distinct developmental roles. The identification of these genes in the zebrafish, a genetic model organism whose development is well characterized, now provides the basis for the establishment of the first animal models for both Ehlers-Danlos (Type VIA) and Bruck syndromes.

Amino Acid Sequence↗

The effects of two different decalcification procedures on size and structure of embryonic epithelial tissue in objects prepared for light microscopy.

Electrolytic decalcification is a very fast and effective method for removing calcium compounds from bones with minimum damage to tissues. Changes of dimension of tissues in histological sections prepared from specimens decalcified by immersion in a formic acid solution and sections prepared from specimens treated in an electrolytic decalcifier were studied. Heads of mouse foetuses were cut in half, decalcified by one of the above-mentioned methods and embedded in histowax. Dimensional changes of skin, tongue and nasal epithelia in histological sections were evaluated by t-test. Significant shrinking and other unwanted effects of decalcification, such as acidophilia of nuclei, were found in objects decalcified by both methods. No significant differences in the effects of the two methods on tissue dimensions were demonstrated. It is concluded that both decalcification methods are equivalent from the qualitative point of view.

Animals↗

The fine structure of embryonic chick skeletal muscle cells differentiated in vitro.

Dissociated myoblasts from 12-day chick embryos were cultured in monolayer, and the differentiation of skeletal muscle cells was studied by electron microscopy. The results have revealed a striking ultrastructural similarity between the in vivo and the in vitro developing muscle, particularly with respect to the myofibrils and sarcoplasmic reticulum. This study demonstrates that all the characteristic organelles of mature skeletal muscle can develop in vitro in the absence of nerves.

Animals↗

Plasma 17beta-estradiol levels and ovarian interstitial cell structure in embryonic Japanese quail.

Plasma concentrations of 17beta-estradiol (E2) and left ovarian histology were investigated by light and electron microscopy in female Japanese quail from Day 10 of embryonic development through Day 7, posthatch. Plasma E2 levels remained relatively constant (102 to 140 pg/mL) in the embryo followed by a sharp decrease posthatch (47 to 70 pg/mL). Beginning on Day 10 of incubation, cells in the medullary portion (medullary cell; MC) of the left ovaries exhibited ultrastructural evidence of steroidogenic capability. The MC had numerous lipid droplets in close proximity to the smooth endoplasmic reticulum (SER). Mitochondria were also observed in the vicinity of the lipid droplets and SER. On Days 10 and 12, the cristae of the inner mitochondrial membranes were of a lamellar configuration; the cristae of some mitochondria in MC had a tubular appearance by Day 14. These data document relative ontogenic changes in ovarian morphology and plasma E2 levels during the early developmental period in female Japanese quail. These data further support the role of this steroid in sexual differentiation.

Animals↗

Comparison of implantation and early development of human embryos fertilized in vitro versus in vivo using transvaginal ultrasound.

Several reports in the literature suggest delayed implantation of in vitro-fertilized human embryos compared to in vivo-fertilized eggs. The use of high-frequency transvaginal transducers for early detection of pregnancy has allowed the identification of the gestational sac with very low serum human chorionic gonadotropin (beta-hCG) levels. Thus, the present study evaluated whether retarded implantation can be identified using this novel technology. We studied 13 single pregnancies after in vitro fertilization (IVF) and 14 pregnancies after artificial insemination either by husband (n = 6) or donor (n = 8). In the IVF patients, oocytes were retrieved 35 hours after hCG administration. Embryo transfer occurred approximately 48 hours after retrieval. Artificial insemination was performed 24 and 48 hours after hCG administration. Transvaginal ultrasound scans and serum beta-hCG levels were evaluated every 3 days starting day 12 post-hCG administration. Serum beta-hCG levels rose in parallel when in vitro- and in vivo-fertilized embryos were compared. Similarly, there was no difference between groups in the mean time needed to detect early embryonic structures, such as the embryonic sac, yolk sac, and heartbeats, or the growth rate of the gestational sac. In conclusion, there was no difference in detecting implantation and early embryonic development of human embryos fertilized in vivo versus in vitro as ascertained by ultrasound scans and serum beta-hCG levels. An embryonic sac is detected 23-24 days after hCG administration in pregnancies achieved by assisted reproductive techniques.

Amnion↗