Distortion of proximodistal information causes JNK-dependent apoptosis in Drosophila wing.

Distinct and evolutionarily conserved signal-transduction cascades mediate the survival or death of cells during development. The c-Jun amino-terminal kinases (JNKs) of the mitogen-activated protein kinase superfamily are involved in apoptotic signalling in various cultured cells. However, the role of the JNK pathway in development is less well understood. In Drosophila, Decapentaplegic (Dpp; a homologue of transforming growth factor-beta) and Wingless (Wg; a Wnt homologue) proteins are secretory morphogens that act cooperatively to induce formation of the proximodistal axis of appendages. Here we show that either decreased Dpp signalling in the distal wing cells or increased Dpp signalling in the proximal wing cells causes apoptosis. Inappropriate levels of Dpp signalling lead to aberrant morphogenesis in the respective wing zones, and these apoptotic zones are also determined by the strength of the Wg signal. Our results indicate that distortion of the positional information determined by Dpp and Wg signalling gradients leads to activation of the JNK apoptotic pathway, and the consequent induction of cell death thereby maintains normal morphogenesis.     

Axial patterning in cephalochordates and the evolution of the organizer

The organizer of the vertebrate gastrula is an important signalling centre that induces and patterns dorsal axial structures. Although a topic of long-standing interest, the evolutionary origin of the organizer remains unclear. Here we show that the gastrula of the cephalochordate amphioxus expresses dorsal/ventral (D/V) patterning genes (for example, bone morphogenetic proteins (BMPs), Nodal and their antagonists) in patterns reminiscent of those of their vertebrate orthlogues, and that amphioxus embryos, like those of vertebrates, are ventralized by exogenous BMP protein. In addition, Wnt-antagonists (for example, Dkks and sFRP2-like) are expressed anteriorly, whereas Wnt genes themselves are expressed posteriorly, consistent with a role for Wnt signalling in anterior/posterior (A/P) patterning. These results suggest evolutionary conservation of the mechanisms for both D/V and A/P patterning of the early gastrula. In light of recent phylogenetic analyses placing cephalochordates basally in the chordate lineage, we propose that separate signalling centres for patterning the D/V and A/P axes may be an ancestral chordate character.     

brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes

Growth and patterning of the Drosophila wing is controlled in part by the long-range organizing activities of the Decapentaplegic protein (Dpp). Dpp is synthesized by cells that line the anterior side of the anterior/posterior compartment border of the wing imaginal disc. From this source, Dpp is thought to generate a concentration gradient that patterns both anterior and posterior compartments. Among the gene targets that it regulates are optomotor blind (omb), spalt (sal), and daughters against dpp (dad). We report here the molecular cloning of brinker (brk), and show that brk expression is repressed by dpp. brk encodes, a protein that negatively regulates Dpp-dependent genes. Expression of brk in Xenopus embryos indicates that brk can also repress the targets of a vertebrate homologue of Dpp, bone morphogenetic protein 4 (BMP-4). The evolutionary conservation of Brk function underscores the importance of its negative role in proportioning Dpp activity.     

The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila.

Wingless (Wg) is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. We demonstrate here that the protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled (fz)/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. We also show that dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes.     

Evidence that Hensen's node is a site of retinoic acid synthesis.

Hensen's node of amniotes, like the Spemann organizer of amphibians, can induce a second body axis when grafted into a host embryo. The avian node, as well as several midline structures originating from it (notochord, floor plate), can also induce digit pattern duplications when grafted into the chick wing bud. We report here that the equivalent of Hensen's node from mouse is an effective inducer of digits in the chick wing bud. Tissues anterior and posterior to the node also evoke pattern duplications, but with a significantly lower efficiency. The finding that the murine node operates in an avian wing bud suggests that the same inducing agent(s) function in both primary and secondary embryonic fields and have been conserved during vertebrate evolution. Digit pattern duplications are also evoked by local administration of all-trans-retinoic acid. This similarity raises the possibility that Hensen's node is a source of retinoic acid. The mouse node is capable of synthesizing retinoic acid from its biosynthetic precursor all-trans-retinol at a substantially higher rate than either anterior or posterior tissues.     

A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer.

The mid/hindbrain (MHB) junction can act as an organizer to direct the development of the midbrain and anterior hindbrain. In mice, Otx2 is expressed in the forebrain and midbrain and Gbx2 is expressed in the anterior hindbrain, with a shared border at the level of the MHB organizer. Here we show that, in Gbx2-/- mutants, the earliest phenotype is a posterior expansion of the Otx2 domain during early somite stages. Furthermore, organizer genes are expressed at the shifted Otx2 border, but not in a normal spatial relationship. To test whether Gbx2 is sufficient to position the MHB organizer, we transiently expressed Gbx2 in the caudal Otx2 domain and found that the Otx2 caudal border was indeed shifted rostrally and a normal appearing organizer formed at this new Otx2 border. Transgenic embryos then showed an expanded hindbrain and a reduced midbrain at embryonic day 9.5-10. We propose that formation of a normal MHB organizer depends on a sharp Otx2 caudal border and that Gbx2 is required to position and sharpen this border.     

The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling

The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.     

Posterior-to-anterior transformation in engrailed wing imaginal disks of Drosophila

Segments in the Drosophila adult are divided into clonally distinct anterior and posterior compartments. Mutations at the engrailed locus can affect the pattern of cuticular structures in the posterior compartments of segments, but have no obvious effect on anterior structures; for example, bristles that are normally seen only on the anterior wing margin in wild-type flies can be found on the posterior margin of engrailed wings. These and clonal analysis data led to the hypothesis that engrailed causes a transformation of posterior to anterior identity in the wing cells. Despite some striking examples of this transformation, a common engrailed phenotype is the disruption or elimination of posterior pattern elements, without a clear replacement by anterior structures; this, together with indications that localized cell death can mimic some of the observed posterior-to-anterior transformations, has led some investigators to question the original engrailed hypothesis. Recently, monoclonal antibodies displaying region-specific binding patterns on the wing imaginal disk have been described, and one of these antibodies in particular provides a novel probe for the engrailed phenotype in the larval precursors of the adult wing. Here I compare the antibody binding patterns on engrailed and wild-type wing disks. The results strongly support the notion that engrailed mutations cause a posterior-to-anterior transformation in these cells.     

Type IV collagens regulate BMP signalling in Drosophila

Dorsal-ventral patterning in vertebrate and invertebrate embryos is mediated by a conserved system of secreted proteins that establishes a bone morphogenetic protein (BMP) gradient. Although the Drosophila embryonic Decapentaplegic (Dpp) gradient has served as a model to understand how morphogen gradients are established, no role for the extracellular matrix has been previously described. Here we show that type IV collagen extracellular matrix proteins bind Dpp and regulate its signalling in both the Drosophila embryo and ovary. We provide evidence that the interaction between Dpp and type IV collagen augments Dpp signalling in the embryo by promoting gradient formation, yet it restricts the signalling range in the ovary through sequestration of the Dpp ligand. Together, these results identify a critical function of type IV collagens in modulating Dpp in the extracellular space during Drosophila development. On the basis of our findings that human type IV collagen binds BMP4, we predict that this role of type IV collagens will be conserved.     

A zone of non-proliferating cells at a lineage restriction boundary in Drosophila

The use of X-ray-induced mitotic recombination to genetically mark individual cells and their descendants during development has led to the discovery of lineage restriction boundaries in Drosophila imaginal disks, dividing the disks into areas called compartments. Clones of cells initiated after a given developmental stage are unable to grow across these boundaries, even if provided with a growth rate advantage over the remaining cells. It has been suggested that cells within compartments are distinguished by the differential activation of selector genes and that the lineage restrictions are maintained by adhesivity differences between the cells in different compartments, but other mechanisms have not been ruled out. Recently a discrete population of cells with unusual permeability properties has been described along an intersegmental lineage restriction boundary in Oncopeltus, suggesting that a lineage restriction could be maintained by a zone of cells which present a barrier to clone growth. Here we demonstrate by autoradiography the presence of a narrow zone of non-proliferating cells (ZNC) coincident with the presumptive wing margin in the Drosophila wing disk, and suggest that this could account for the observed lineage restriction between presumptive dorsal and ventral surfaces of the wing. As the anterior/posterior compartment boundary does not coincide with a ZNC, the results indicate that different lineage boundaries may be maintained by different mechanisms.     

Paracrine Wingless signalling controls self-renewal of Drosophila intestinal stem cells

In the Drosophila midgut, multipotent intestinal stem cells (ISCs) that are scattered along the epithelial basement membrane maintain tissue homeostasis by their ability to steadily produce daughters that differentiate into either enterocytes or enteroendocrine cells, depending on the levels of Notch activity. However, the mechanisms controlling ISC self-renewal remain elusive. Here we show that a canonical Wnt signalling pathway controls ISC self-renewal. The ligand Wingless (Wg) is specifically expressed in the circular muscles next to ISCs, separated by a thin layer of basement membrane. Reduced function of wg causes ISC quiescence and differentiation, whereas wg overexpression produces excessive ISC-like cells that express high levels of the Notch ligand, Delta. Clonal analysis shows that the main downstream components of the Wg pathway, including Frizzled, Dishevelled and Armadillo, are autonomously required for ISC self-renewal. Furthermore, epistatic analysis suggests that Notch acts downstream of the Wg pathway and a hierarchy of Wg/Notch signalling pathways controls the balance between self-renewal and differentiation of ISCs. These data suggest that the underlying circular muscle constitutes the ISC niche, which produce Wg signals that act directly on ISCs to promote ISC self-renewal. This study demonstrates markedly conserved mechanisms regulating ISCs from Drosophila to mammals. The identification of the Drosophila ISC niche and the principal self-renewal signal will facilitate further understanding of intestinal homeostasis control and tumorigenesis.     

Distalization of the Drosophila leg by graded EGF-receptor activity

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-beta (TGF-beta) and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. Here I revise our understanding of limb development in flies and show that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals.     

The caudal limit of Otx2 expression positions the isthmic organizer.

The homeobox gene Otx2 is expressed in the anterior neural tube with a sharp limit at the midbrain/hindbrain junction (the isthmic organizer). Otx2 inactivation experiments have shown that this gene is essential for the development of its expression domain. Here we investigate whether the caudal limit of Otx2 expression is instrumental in positioning the isthmic organizer and in specifying midbrain versus hindbrain fate, by ectopically expressing Otx2 in the presumptive anterior hindbrain using a knock-in strategy into the En1 locus. Transgenic offspring display a cerebellar ataxia. Morphological and histological studies of adult transgenic brains reveal that most of the anterior cerebellar vermis is missing, whereas the inferior colliculus is complementarily enlarged. During early neural pattern formation expression of the midbrain markers Wnt1 and Ephrin-A5, the isthmic organizer markers Pax2 and Fgf-8 and the hindbrain marker Gbx2 are shifted caudally in the presumptive hindbrain territory. These findings show that the caudal limit of Otx2 expression is sufficient for positioning the isthmic organizer and encoding caudal midbrain fate within the mid/hindbrain domain.     

Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud

In recent years there has been considerable interest in the role of retinoic acid (RA) in vertebrate-limb pattern formation. When RA is applied to the anterior of the chick wing bud, a mirror-image duplication of the limb pattern develops that is identical to the pattern resulting from grafts of posterior tissue (zone of polarizing activity, or ZPA). It has been proposed that position along the anterior-posterior axis in the chick limb is specified by a gradient of a diffusible factor produced by the ZPA. The ZPA-mimicking action of RA has led to the hypothesis that exogenously applied RA acts by providing graded spatial information across the anterior-posterior limb axis. An alternative interpretation is that RA changes anterior cells into ZPA cells, which in turn provide the actual pattern-duplicating stimulus; there is already some preliminary evidence that this occurs. A hybrid interpretation has also been suggested whereby ZPA cells are formed in response to RA exposure and then begin to release retinoids that act as graded spatial cues. We have used a functional assay to test anterior chick wing-bud cells for ZPA activity after exposure to RA. The results of our studies indicate that the action of RA is to change anterior cells into ZPA cells. Further, our results indicate that it is unlikely that RA-treated anterior cells then begin producing RA in such a way as to provide a graded positional signal.     

河南鲤科鱼类咽骨咽齿的比较研究(二)

<正> 本文记述了密鲴亚科鱼类4属4种,鲤卿亚科鱼类2属2种,现分述如下:I密鲴亚科:咽骨较宽,前后角明显。骨长为骨宽的2.5～4.4倍。体长为骨长的14.3～17.0倍。前肢稍长于后肢。前无齿突顶端尖;后无齿突侧扁,顶端园钝。咽齿1～3列,侧扁,先端尖不带钩,内列齿6～7枚,咀嚼面发达。咽齿1列以上者,外列齿细长而弱,容易     

The organizer factors Chordin and Noggin are required for mouse forebrain development

In mice, there is evidence suggesting that the development of head and trunk structures is organized by distinctly separated cell populations. The head organizer is located in the anterior visceral endoderm (AVE) and the trunk organizer in the node and anterior primitive streak. In amphibians, Spemann's organizer, which is homologous to the node, partially overlaps with anterior endoderm cells expressing homologues of the AVE markers cerberus, Hex and Hesx1. For mice, this raises the question of whether the AVE and node are independent of each other, as suggested by their anatomical separation, or functionally interdependent as is the case in amphibians. Chordin and Noggin are secreted bone morphogenetic protein (BMP) antagonists expressed in the mouse node, but not in the AVE. Here we show that mice double-homozygous mutants that are for chordin and noggin display severe defects in the development of the prosencephalon. The results show that BMP antagonists in the node and its derivatives are required for head development.     

鸡胚早期发育中组织者的起源及其诱导能力

本研究利用活性荧光染料ＤｉＩ，对鸡胚的期和期胚盘进行标记，以确定组织者的部位。并将组织者区域的细胞移植到宿主胚胎中，验证其诱导次级胚胎的能力。结果表明，期、期胚盘的组织者细胞位于胚盘中轴的后端，其细胞的性质已被决定，能够诱导次级胚胎的发育，本研究结果支持原条的发育与组织者细胞性质不是受下胚层诱导的观点。     

Local inhibition and long-range enhancement of Dpp signal transduction by Sog

Extracellular gradients of signalling molecules can specify different thresholds of gene activity in development. A gradient of Decapentaplegic (Dpp) activity subdivides the dorsal ectoderm of the Drosophila embryo into amnioserosa and dorsal epidermis. The proteins Short gastrulation (Sog) and Tolloid (Tld) are required to shape this gradient. Sog has been proposed to form an inhibitory complex with either Dpp or the related ligand Screw, and is subsequently processed by the protease Tld. Paradoxically, Sog appears to be required for amnioserosa formation, which is specified by peak Dpp signalling activity. Here we show that the misexpression of sog using the even-skipped stripe-2 enhancer redistributes Dpp signalling in a mutant background in which dpp is expressed throughout the embryo. Dpp activity is diminished near the Sog stripe and peak Dpp signalling is detected far from this stripe. However, a tethered form of Sog suppresses local Dpp activity without augmenting Dpp activity at a distance, indicating that diffusion of Sog may be required for enhanced Dpp activity and consequent amnioserosa formation. The long-distance stimulation of Dpp activity by Sog requires Tld, whereas Sog-mediated inhibition of Dpp does not. The heterologous Dpp inhibitor Noggin inhibits Dpp signalling but fails to augment Dpp activity. These results suggest an unusual strategy for generating a gradient threshold of growth-factor activity, whereby Sog and its protease specify peak Dpp signalling far from a localized source of Sog.