18 research outputs found
Germline Transgenic Methods for Tracking Cells and Testing Gene Function During Regeneration in the Axolotl
The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum(axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible
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Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution
We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called ādorsal.ā On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other.</p
Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution
We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called ādorsal.ā On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other
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Cellular and molecular mechanisms underlying tissue elongation of the developing egg in Drosophila melanogaster
The cellular and molecular mechanisms generating the diversity of animal morphologies are still a relatively little explored subject within developmental biology. While changes in cell number help with the growth of tissue, the orientation of cell divisions, migrations, cell rearrangements or changes in cell shape can explain how anisotropies in tissue shape are formed during development. It remains unclear whether the current set of described behaviors can account for all of the diverse morphologies we see in extant metazoan species.To better understand the mechanisms underlying tissue morphogenesis, my Ph.D. dissertation focused on the development of the ellipsoid egg in Drosophila melanogaster as a new, emerging model system to study tissue elongation. Developing egg chambers (follicles) in Drosophila originate as a sphere and initially grow isotropically but elongate during oogenesis to form an ellipsoid egg. The cellular and molecular mechanisms underlying follicle elongation were not known, although genetic evidence suggested interactions between the follicle epithelium and the surrounding extracellular matrix (ECM) are somehow involved.To elucidate how tissue elongation occurs in the Drosophila ovary, I utilized live imaging of developing follicles ex vivo and discovered that the follicle unexpectedly undergoes several revolutions of polarized, circumferential global tissue rotation - relative to the surrounding ECM - during the major elongation phase of oogenesis. Follicles with epithelia mutant for an Integrin receptor or Collagen IV, an ECM molecule, fail to rotate and result in round eggs. We found that Collagen IV fibrils become circumferentially planar polarized during the rotation phase but become misoriented in non-rotating `round egg' mutants. Furthermore, acute degradation of Collagen IV rounds previously elongated follicles, suggesting that follicle rotation polarizes a fibrillar matrix that constrains the growing egg in a `molecular corset', generating its ellipsoid shape. Global tissue rotation is thus a novel morphogenetic behavior, using polarized cell motility to propagate planar polarity information in synchrony to the ECM to control tissue shape.Many new questions developed from the discovery of global tissue rotation. It is unclear how the follicle epithelium responds to a revolving tissue and a mechanically constraining ECM as the tissue grows and elongates. Do conventional behaviors of tissue elongation like polarized cell intercalation, cell elongation or cell division occur in the Drosophila follicle? Chapter 3 aims to further our understanding of the cellular basis of follicle elongation by developing and performing preliminary quantitative morphometric analysis on the follicle epithelium during the elongation phase. In addition, I examine other known round egg mutants to determine whether they too regulate global tissue rotation or whether these genes regulate additional morphogenetic behaviors necessary for egg elongation.My final chapter investigates how planar cell polarity (PCP) and global tissue rotation are established in the Drosophila follicle. How does molecular planar polarity arise? Does this occur before the onset of polarized follicle rotation? Is there an activation signal to initiate global tissue rotation and from where does this signal originate? I perform an in silico enhancer trap screen to identify developmental signaling pathways and other genes that may coincide with the major elongation phase and has led to the identification of the Notch/Delta signaling pathway as a putative regulator of follicle PCP and global tissue rotation, potentially providing an activation signal from the germline.The findings from my Ph.D. dissertation provide a new framework to think about the cellular and molecular mechanisms underlying tissue elongation of the developing egg, but moreover, challenge current perspectives on metazoan morphogenesis. Global tissue rotation is a novel polarized morphogenetic behavior required for tissue elongation, but the cellular output of this movement remains ambiguous, perhaps because of the follicle's closed topology as an epithelial chamber. It is the first collective cell migration with an obvious individual cell polarity and tissue polarity, but with no obvious collective cell polarity with leader and follower cells. It also raises the possibility that different mechanisms of PCP establishment and propagation may occur in different tissues. These and other issues indicate that continued studies of Drosophila egg elongation will provide novel and exciting insights to our understanding of tissue polarity, morphogenesis and development in a variety of organisms
Expanding the Morphogenetic Repertoire: Perspectives from the Drosophila Egg
Tissue and organ architectures are incredibly diverse, yet our knowledge of the morphogenetic behaviors that generate them is relatively limited. Recent studies have revealed unexpected mechanisms that drive axis elongation in the Drosophila egg, including an unconventional planar polarity signaling pathway,Ā a distinctive type of morphogenetic movement termed āglobal tissue rotation,ā a molecular corset-like role of extracellular matrix, and oscillating basal cellular contractions. We review here what is known about Drosophila egg elongation, compare it to other instances of morphogenesis, and highlight several issues of general developmental relevance
Current Biology, Vol. 13, 2125--2137, December 16, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.cub.2003.11.054 Shroom Induces Apical Constriction
this report we used the undifferentiated cells of sociated with enrichment of apically localized actin very early Xenopus embryos as a source of heterologous filaments and required the small GTPase Rap1 but not and naive epithelial cells in which to study Shroom func- Rho. Endogenous Xenopus shroom was found to be extion (Figure 1A). We show that expression of Shroom is pressed in cells engaged in apical constriction. Consistent with a role for Shroom in organizing apical constriction, sufficient to cause apical constriction in these cells, a disrupting Shroom function resulted in a specific failure novel property for a vertebrate protein. Interestingly, of hingepoint formation, defective neuroepithelial sheet- Shroom does not appear to affect nonpolarized cells bending, and failure of neural tube closure. in these early blastulae. We show that during normal Conclusions: These data demonstrate that Shroom is development, Xenopus shroom is expressed primarily an essential regulator of apical constriction during neu- in cells undergoing apical constriction. We inhibited enrulation. The finding that a single protein can initiate this dogenous Shroom activity with a dominant-negative process in epithelial cells establishes that bending of construct and with an antisense oligonucleotide. Conepithelial sheets may be patterned during development sistent with a role in generating apical constrictions, by the regulation of expression of single genes. Shroom was required specifically for the formation of hingepoints and for bending of the neuroepithelial sheet. Together, the data presented here establish that bend
Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl
The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular
processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools
for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma
mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration.We demonstrate tissue-dependent control
of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use
of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cellcycle
inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly
inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible
Correction: Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution.
Correction: Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution
Genes Expressed with Dorsal or Ventral Domains
<div><p>These genes were chosen as candidate targets of Bmp activation or repression. All embryos are oriented as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040291#pbio-0040291-g002" target="_blank">Figure 2</a> with anterior to the top and left of each panel and dorsal in the top right of each panel, unless otherwise specified. The telotroch or ciliated band is marked by white arrowheads. Expression of <i>dlx</i> at (A) day 2 of development, just after gastrulation and (B) at day 3 of development.</p>
<p>(C) <i>Tbx 2/3</i> expression just after gastrulation, and (D) at day 3 of development.</p>
<p>(E) Expression of <i>hex</i> at day 3 of development (F) Expression of <i>nk2.3/2.5</i> at day 4.</p>
<p>(G) Expression of olig on day 2, just after gastrulation, and (H) at day 3.</p>
<p>(I) <i>poxN</i> expression at day 4 of development.</p>
<p>(J) <i>Pitx</i> expression at day 2 of development, dorsal midline toward the viewer, a glancing optical section through the dorsal-most ectoderm.</p>
<p>(K) <i>Pitx</i> expression at day 4 of development. Note the two domains of expression.</p>
<p>(L) <i>Netrin</i> expression, a transverse section of a post-gastrula embryo at the level of the ciliated band. Note the broad ventral expression of <i>netrin</i>, and (M) the more narrow domain at day 3 of development.</p>
<p>(N) Expression of <i>lim3</i> at day 3 of development.</p>
<p>(O) Expression of <i>mnx</i> at day 2 of development, and (P) at day 4. Note the ventral endodermal expression.</p>
<p>(Q) Expression of <i>mox</i> (also called <i>gax</i>) at day 3 of development, and (R) a close up of the ventral domain at day 3, ventral midline toward the viewer, displaying the metasome and part of the mesosome.</p>
<p>(S) Expression of <i>sim</i> at day 2 of development, and (T) at day 5.</p></div