Induction of a noggin-like gene by ectopic DV interaction during planarian regeneration

In previous studies, we have shown that dorsoventral (DV) interaction evokes not only blastema formation, but also morphogenetic events similar to those that occur in regeneration. However, it is still unclear what kinds of signal molecules are involved in the DV interaction. To investigate the signal systems involved in the DV interaction, we focused on a noggin-like gene (Djnlg) identified by the planarian EST project. Djnlg is the first noggin homologue isolated from an invertebrate. In DjNLG, the positions of nine cysteine residues which may be essential for dimer formation were well conserved, but overall, the amino acid sequence of DjNLG did not show high similarity to the sequences of vertebrate Noggins. Expression of Djnlg was observed only in the proximal region of the branch structures in the brain of intact planarians, suggesting that Djnlg may have a role in pattern formation in the brain. Interestingly, transient strong expression of Djnlg was observed in the amputated region of regenerating planarians. Djnlg-expressing cells were detected beneath the muscle 9 h after amputation and were then detected in the ventral subepidermal region of the blastema. The induction of Djnlg expression by amputation was not affected by X-ray irradiation, even though the stem cells were completely eliminated, implying the existence of signal-producing cells which may provide a positional cue to the stem cells. In DV reversed grafting, expression of Djnlg was strongly induced in the DV boundary between the host and donor. These results suggest that ectopic DV interaction may induce expression of Djnlg in the positional cue-producing cells, and that it might be involved in stimulation of blastema formation as well as DV patterning of the body.     

Dorso-ventral asymmetric functions of teashirt in Drosophila eye development depend on spatial cues provided by early DV patterning genes

The teashirt (tsh) gene has dorso-ventral (DV) asymmetric functions in Drosophila eye development: promoting eye development in dorsal and suppressing eye development in ventral by Wingless mediated Homothorax (HTH) induction [Development 129 (2002) 4271]. We looked for DV spatial cues required by tsh for its asymmetric functions. The dorsal Iroquois-Complex (Iro-C) genes and Delta (Dl) are required and sufficient for the tsh dorsal functions. The ventral Serrate (Ser), but not fringe (fng) or Lobe (L), is required and sufficient for the tsh ventral function. We propose that DV asymmetric function of tsh represents a novel tier of DV pattern regulation, which takes place after the spatial expression patterns of early DV patterning genes are established in the eye.     

Dorsal–ventral patterning of the neural tube: A tale of three signals

Development of the vertebrate nervous system begins with the acquisition of neural identity from the midline dorsal‐ectodermal cells of the gastrulating embryos. The subsequent progressive specification of the neural plate along its anterior–posterior and dorsal–ventral (DV) axes allows the generation of the tremendous variety of neuronal and glial cells that compose the vertebrate central nervous system (CNS). Studies on the development of the spinal cord, the anatomically simplest part of the CNS, have generated most of our current knowledge on the signaling events and the genetic networks that orchestrate the DV patterning of the neural plate. In this review, we discuss the recent advances in our understanding of these events and highlight unresolved questions. We focused our attention on the activity and the integration of the three main instructive cues: Sonic hedgehog, the Wnts and the Bone Morphogenetic Proteins, giving particular attention to the less well understood dorsal signaling events. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

The effect of retinoic acid on positional memory in the dorsoventral axis of regenerating axolotl limbs

We investigated the effect of retinoic acid (RA) on pattern regulation in the dorsoventral (DV) axis of regenerating axolotl limbs. Half and double half dorsal and ventral zeugopodia (lower arms or legs) were amputated through their distal ends, and 4 days later the animals were injected intraperitoneally with 50 (large animals) or 100 (small animals) micrograms RA/g body wt. Half and double half dorsal and ventral zeugopodia of uninjected axolotls, and sham-operated zeugopodia of untreated and RA-treated limbs served as controls. Skeletal patterns and the DV muscle patterns of control and experimental regenerates were then analyzed. Sham-operated zeugopodia of uninjected animals regenerated normally. Sham-operated, RA-treated zeugopodia regenerated normally with proximodistal duplications. Sixty percent of uninjected control dorsal half zeugopodia, 80% of control ventral half zeugopodia, and 100% of control double dorsal and double ventral zeugopodia regenerated distally, but the regenerates did not reconstitute the muscle pattern of the missing half. Thirty-eight percent of RA-treated ventral half zeugopodia and 78% of RA-treated double ventral zeugopodia failed to regenerate distally. Of those cases that did regenerate distally, none regenerated the muscle pattern of the missing half. By contrast, 100% of RA-treated dorsal half zeugopodia regenerated distally and all completed the normal DV muscle pattern. Forty-one percent of RA-treated double dorsal zeugopodia failed to regenerate, but of the remainder that did regenerate, 50% completed the normal DV muscle pattern. These represented eight cases, six of which regenerated single limbs, and two of which regenerated twin limbs, each with a normal DV muscle pattern. We interpret these data to mean that RA ventralizes the positional memory of blastema cells in the DV axis.     

Dorsoventral differences in cAMP levels and correlated changes in the subcellular distribution of the PKA catalytic domain,provide further evidence that PKA signaling coordinates dorsoventral patterning of the otocyst

Dorsoventral (DV) patterning of the otocyst gives rise to formation of the morphologically and functionally complex membranous labyrinth composed of unique dorsal and ventral sensory organs. DV patterning results from extracellular signaling by secreted growth factors, which presumably form reciprocal concentration gradients across the DV axis of the otocyst. Previous work suggested a model in which two important growth factors, bone morphogenetic protein (BMP) and SHH, undergo crosstalk through an intersecting pathway to coordinate DV patterning. cAMP‐dependent protein kinase A (PKA) lies at the heart of this pathway. Here, we provide further evidence that PKA signaling coordinates DV patterning, showing that both BMPs and SHH regulate cAMP levels, with BMPs increasing levels in the dorsal otocyst and SHH decreasing levels in the ventral otocyst. This, in turn, results in regional changes in the subcellular distribution of the catalytic domain of PKA, as well as DV regulation of PKA activity, increasing it dorsally and decreasing it ventrally. These new results fill an important gap in our previous understanding of how ligand signaling acts intracellularly during otocyst DV patterning and early morphogenesis, thereby initiating the series of events leading to formation of the inner ear sensory organs that function in balance and hearing.     

Noggin is required for correct guidance of dorsal root ganglion axons

Members of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal-ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter.     

Physiological Reorganization and Post-Traumatic Regeneration in Stylonychia mytilus: Reflections on Developmental Constraints and the Evolutionary Origin of Alternative Modes of Asexual Morphogenesis

We investigated development of cortical ciliature in Stylonychia mytilus during starvation-induced physiological reorganization, and during regeneration following amputation of the anterior part of the cell. Cortical reorganization in the two processes is generally similar. The posterior part of the adoral zone of membranelles is resorbed and replaced with newly assembled membranelles. The pre-existing set of ventral cirri and dorsal bristles is entirely resorbed and replaced with new ones. Regenerants exhibit posterior displacement of the frontal-ventral-transverse cirri primordium and the undulating membrane primordium, and recruit basal bodies from ectopic locations for the development of these ciliature. This illustrates flexibility in the initiation site of ciliary primordia, and opportunism in utilizing building blocks. Such morphogenetic versatility of hypotrichs provides the basis for the operation of a global control of pattern formation, which governs cortical reorganization in dividers, and additionally, in the absence of the prerequisites for binary fission, alternative modes of cortical development such as physiological reorganization or regeneration. These considerations suggest that the three processes are homologous and that physiological reorganization and regeneration have evolved from binary fission. In physiological reorganization and regeneration, the micro- and macronuclei reorganize to resemble that in binary fission; these nuclear events are considered evolutionary relics of the nuclear development of binary fission. Tetrahymena also exhibits such morphogenetic flexibility; stomatogenesis is under global control, so that asexual cells can replace its oral apparatus without undergoing binary fission. Paramecium , on the other hand, adopts a more rigid strategy in relying heavily on pre-existing structures for morphogenetic cues; this could have imposed constraints in the exploration of alternative modes of asexual development.     

Zili antagonizes Bmp signaling to regulate dorsal-ventral patterning during zebrafish early embryogenesis

Bone morphogenetic protein (Bmp) signaling plays a pivotal role in dorsal-ventral (DV) patterning in vertebrate embryos. Piwi proteins are required for germline and stem cell development. Our previous study demonstrated that Zili, zebrafish Piwil2, inhibits transforming growth factor (TGF)-βsignaling by interacting with Smad4, suggesting a role for zili in Bmp signaling. In the present study, zili-MO or zili mRNA was microinjected into one-cell embryos to knock down or elevate the expression of zili to study the role of zili during early zebrafish embryogenesis. Knockdown of zili inhibited the expression of dorsal marker genes, and enhanced that of ventral marker genes. In contrast, overexpression of zili promoted expression of dorsal marker genes, while it inhibited ventral marker genes. These results suggest that zili regulates DV patterning. The influence of zili on the Bmp pathway was further explored. Knockdown of zili resulted in higher expression levels of bmp2b, and bmp4, the Bmp signaling ligands, and reduced expression of chordin (chd), noggin (nog1), and follistatin (fst), which encode BMP antagonists. Meanwhile, overexpression of zili produced opposite effects. In conclusion, our results indicate that zili regulates dorsal-ventral patterning by antagonizing Bmp signaling during early embryogenesis in zebrafish.     

Epigenetic alterations brought about by lithium treatment disrupt mouse embryo development

The commonly accepted mechanism by which LiCl corsalizes amphibian embryos is a respecification of ventral blastomeres, presumably through realignment of dorsal positional information in the embryo. An alternative mechanism, however, is an epigenetic change in the competence of cells to respond to cues they may be normally exposed to without effect. In order to test this hypothesis, we treated mouse preimplantation embryos, which do not possess any axial positional information, with LiCl, and observed axial abnormalities which must have been elaborated several days after treatment. We interpret this as support for the hypothesis that cellular competence rather than positional information is altered by LiCl, and suggest that this competence may be altered through the action of lithium sensitive enzymes that interact with chromatin. © 1996 Wiley-Liss, Inc.     

Principles of regeneration revealed by the planarian eye

One approach to elucidating the principles of regeneration is to investigate mechanisms that regenerate a target organ. Planarian eyes are discrete, visible structures that are dispensable for viability, making them powerful for studying the logic of regeneration. Fate specification in eye regeneration occurs in stem cells (neoblasts), generating eye progenitors. Eye progenitor production is not responsive to the presence or absence of the eye, with regeneration explained by constant progenitor production in the appropriate positional environment. Eye progenitors display coarse spatial specification. A combination of eye-extrinsic cues and self-organization with differentiated eye cells dictate where migratory eye progenitors target. Finally, guidepost-like cells influence regenerating axons to facilitate the restoration of eye circuitry. These findings from the eye as a case study present a model that explains how regeneration can occur.     

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.     

A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.     

Twisted gastrulation promotes BMP signaling in zebrafish dorsal-ventral axial patterning

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.     

Lens formation by pigmented epithelial cell reaggregate from dorsal iris implanted into limb blastema in the adult newt

In newt lens regeneration, the dorsal iris has lens forming ability and the ventral iris has no such capability, whereas there is no difference in the morphological criteria. To investigate the real aspects of this characteristic lens regeneration in the newt at the cellular level, a useful model system was constructed by transplanting the dorsal and ventral reaggregate derived from singly dissociated pigmented epithelial cells of the iris into the blastema of the forelimb in the newt. The lens was formed from the dorsal reaggregate with high efficiency, but not from the ventral one. No lens formation was observed in the implantation of the reaggregate into the tissue of the intact limbs. In detailed examination of the process of lens formation from the reaggregate, it was shown that tubular formation was the first step in the rearrangement of cells within the reaggregate. This was followed by depigmentation, vesicle formation with active cell growth, and the final step was lens fiber formation by transdifferentiation of epithelial cells composing the lens vesicle. The process was almost the same as in situ lens regeneration except the reconstitution of the two-layered epithelial structure was embodied as flattened tubular formation in the first step. The present study made it possible for the first time to examine lens forming ability in the reaggregate mixed with dorsal and ventral cells, because the formation of a reaggregate was started from singly dissociated cells of the dorsal and ventral cells of the iris. Mixed reaggregate experiments indicated that the existence of the dorsal cells in a cluster within the reaggregate is important in lens formation, and ventral cells showed an inhibitory effect on the formation. The present study demonstrated that the limb system thus constructed was effective for the analysis of lens formation at the cellular level and made it possible to examine the role of dorsal and ventral cells in lens regeneration.     

Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain.

Regional patterning in the developing mammalian brain is partially regulated by restricted gene expression patterns within the germinal zone, which is composed of stem cells and their progenitor cell progeny. Whether or not neural stem cells, which are considered at the top of the neural lineage hierarchy, are regionally specified remains unknown. Here we show that the cardinal properties of neural stem cells (self-renewal and multipotentiality) are conserved among embryonic cortex, ganglionic eminence and midbrain/hindbrain, but that these different stem cells express separate molecular markers of regional identity in vitro, even after passaging. Neural stem cell progeny derived from ganglionic eminence but not from other regions are specified to respond to local environmental cues to migrate ventrolaterally, when initially deposited on the germinal layer of ganglionic eminence in organotypic slice cultures. Cues exclusively from the ventral forebrain in a 5 day co-culture paradigm could induce both early onset and late onset marker gene expression of regional identity in neural stem cell colonies derived from both the dorsal and ventral forebrain as well as from the midbrain/hindbrain. Thus, neural stem cells and their progeny are regionally specified in the developing brain, but this regional identity can be altered by local inductive cues.     

miRNAs in Newt Lens Regeneration: Specific Control of Proliferation and Evidence for miRNA Networking

Background

Lens regeneration in adult newts occurs via transdifferentiation of the pigment epithelial cells (PECs) of the dorsal iris. The same source of cells from the ventral iris is not able to undergo this process. In an attempt to understand this restriction we have studied in the past expression patterns of miRNAs. Among several miRNAs we have found that mir-148 shows an up-regulation in the ventral iris, while members of the let-7 family showed down-regulation in dorsal iris during dedifferentiation.

Methodology/Principal Findings

We have performed gain- and loss-of–function experiments of mir-148 and let-7b in an attempt to delineate their function. We find that up-regulation of mir-148 caused significant decrease in the proliferation rates of ventral PECs only, while up-regulation of let-7b affected proliferation of both dorsal and ventral PECs. Neither miRNA was able to affect lens morphogenesis or induction. To further understand how this effect of miRNA up-regulation is mediated we examined global expression of miRNAs after up-regulation of mir148 and let-7b. Interestingly, we identified a novel level of mirRNA regulation, which might indicate that miRNAs are regulated as a network.

Conclusion/Significance

The major conclusion is that different miRNAs can control proliferation in the dorsal or ventral iris possibly by a different mechanism. Of interest is that down-regulation of the let-7 family members has also been documented in other systems undergoing reprogramming, such as in stem cells or oocytes. This might indicate that reprogramming during newt regeneration shares common molecular signatures with reprogramming in stem or germ cells. On the other hand that miRNAs can regulate the levels of other miRNAs is a novel level of regulation, which might provide new insights on their function.