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Rapid induction of a hyperciliated phenotype in zinc-arrested sea urchin embryos by theophylline.

Zinc ions, present since fertilization, will arrest embryos of the sea urchin Tripneustes gratilla at the hatched blastula stage, but such embryos lack long cilia, the usual characteristic of animalized embryos. Eventually these embryos will express a minimal long cilia phenotype, with a mean population < 1.4 times that of control blastulae, but only after contemporaneous control embryos gastrulate. Theophylline will rapidly but minimally animalize embryos when added after hatching, but its inductive ability decreases as the embryos gastrulate. Theophylline-animalized embryos produce cilia whose mean population length is > 1.5 times that of control blastulae. At any point from the time of hatching up until control complete gastrulation, theophylline added to zinc-arrested embryos will induce a hyperciliated phenotype with a mean population length nearly 3 times that of control blastulae. The elongation of cilia is immediate and significantly exceeds the rate of normal ciliary regeneration. Initially requiring the presence of theophylline, the hyperciliated phenotype becomes stable as control embryos begin gastrulation and, when deciliated, the induced embryos will regenerate hyperlong cilia in the absence of theophylline. The time dependence for both induction and phenotype stability would suggest that certain timing mechanisms are still operative in zinc-arrested embryos. This inducible system should facilitate studies of length control during ciliary elongation and regeneration.

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Activation of pmar1 controls specification of micromeres in the sea urchin embryo.

pmar1 is a transcription factor in the paired class homeodomain family that was identified and found to be transcribed in micromeres beginning at the fourth cleavage of sea urchin development [Dev. Biol. 246 (2002), 209]. Based on in situ data, molecular perturbation studies, and QPCR data, the recently published gene regulatory network (GRN) model for endomesoderm specification [Science 295 (2002) 1669; Dev. Biol. 246 (2002), 162] places pmar1 early in the micromere specification pathway, and upstream of two important micromere induction signals. The goal of this study was to test these three predictions of the network model. A series of embryo chimeras were produced in which pmar1 activity was perturbed in one cell that was transplanted to control hosts. At the fourth cleavage, micromeres bearing altered pmar1 activity were combined with a normal micromereless host embryo. If beta-catenin signaling is blocked, the micromeres remain unspecified and are unable to signal to the host cells. When such beta-catenin-blocked micromeres also express Pmar1, all observed micromere functions are rescued. The rescue includes expression of the primary mesenchyme cell (PMC) differentiation program, expression and execution of the Delta signal to induce secondary mesoderm cell (SMC) specification in macromere progeny, and expression of the early endomesoderm induction signal necessary for full specification of the endoderm. Additionally, Pmar1 expressed mosaically from inserted DNA constructs causes induction of ectopic Endo 16 in adjacent cells, demonstrating further that Pmar1 controls expression of the early endomesoderm induction signal. Based on these experiments, Pmar1 is an important transcription factor necessary for initiating the micromere specification program and for the expression of two inductive signals produced by micromeres. Each of the tests we describe supports the placement and function of Pmar1 in the endomesoderm GRN model.

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Limb deformity proteins: role in mesodermal induction of the apical ectodermal ridge.

During early limb development, distal tip ectoderm is induced by the underlying mesenchyme to form the apical ectodermal ridge. Subsequent limb growth and patterning depend on reciprocal signaling between the mesenchyme and ridge. Mice that are homozygous for mutations at the limb deformity (ld) locus do not form a proper ridge and the anteroposterior axis of the limb is shortened. Skeletal analyses reveal shortened limbs that involve loss and fusion of distal bones and digits, defects in both anteroposterior and proximodistal patterning. Using molecular markers and mouse-chick chimeras we examined the ridge-mesenchymal interactions to determine the origin of the ld patterning defects. In the ld ridge, fibroblast growth factor 8 (Fgf8) RNA is decreased and Fgf4 RNA is not detected. In the ld mesenchyme, Sonic hedgehog (Shh), Evx1 and Wnt5a expression is decreased. In chimeras between ld ectoderm and wild-type mesenchyme, a ridge of normal morphology and function is restored, Fgf8 and Shh are expressed normally, Fgf4 is induced and a normal skeletal pattern arises. These results suggest that the ld mesenchyme is unable to induce the formation of a completely functional ridge. This primary defect causes a disruption of ridge function and subsequently leads to the patterning defects observed in ld limbs. We propose a model in which ridge induction requires at least two phases: an early competence phase, which includes induction of Fgf8 expression, and a later differentiation phase in which Fgf4 is induced and a morphological ridge is formed. Ld proteins appear to act during the differentiation phase.

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De novo induction of a gene product during heterologous epithelial--mesenchymal interactions in vitro.

Mesenchymal specification of epithelial cytodifferentiation and morphogenesis has been considered to be a general feature of various epithelial-mesenchymal interacting systems (e.g., salivary gland, mammary gland, feather, hair, and tooth morphogenesis). In contrast, we have demonstrated that a mesenchyme can be induced by a heterologous epithelium to synthesize in quantity a specific gene product(s) unorthodox to the organ from which the mesenchyme was taken. Stage 22-23 avian limb bud epithelium induced 17-day embryonic mouse tooth mesenchyme to differentiate into cartilage. Peptide analysis (cyanogen bromide cleavage after purification of extracted collagen chains) demonstrated that heterologous tissue recombinations produced type II collagen [alpha(II)](3) (i.e., cartilage-type) in addition to type I collagen [alpha(I)](2)alpha(2). Intact or reconstituted mouse molar tooth organs synthesized type I collagen and type I trimer [alpha(I)](3) collagen. Immunohistochemical criteria using anti-type II collagen antibodies identified type II collagen in cartilage-like matrix within the mesenchymal component of heterologous tissue recombinants. Cartilage has never been described during in vivo or in vitro tooth tissue differentiation or associated with the pathology of dental papilla mesenchyme. These results support the hypothesis that epithelial-mesenchymal interactions during embryonic development can selectively induce de novo synthesis of unique gene products.

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Zebrafish nodal-related 2 encodes an early mesendodermal inducer signaling from the extraembryonic yolk syncytial layer.

Nodal-related factors have been implicated in mesodermal and neural patterning, and left-right asymmetry, in mouse, frog, and chicken embryos. We describe the isolation and characterization of zebrafish nodal-related 2 (znr2). znr2 is expressed at low levels maternally, and zygotic transcripts localize to dorsal blastomeres at MBT. Slightly later, znr2 is also expressed dorsally in the extraembryonic yolk syncytial layer (YSL). During early gastrulation, znr2 expression expands to include deep and superficial cells in the entire marginal zone and YSL. During shield stages, expression is primarily localized to superficial noninvoluting cells of the organizer called dorsal forerunners. Znr2 misexpression in whole fish embryos expands or duplicates dorsoanterior and axial cell fates. Furthermore, Znr2 overexpression exclusively in the YSL, a region implicated in endogenous mesodermal induction, causes broadened or duplicated gsc expression in the overlying blastoderm. Functional comparison of Znr2 and another recently identified zebrafish nodal-related factor, Znr1/Cyclops, reveals distinct inductive properties of each ligand. Znr2 efficiently induces organizer-type dorsoanterior mesodermal and endodermal markers, but only weakly, if at all, neural markers. In contrast, while Znr1/Cyclops reproducibly induces mesodermal and neural markers, it is an inefficient inducer of organizer-type mesoderm. Our results suggest that znr2 encodes a robust mesendodermal inducer that signals nonautonomously during the earliest stages of embryonic patterning, and that part of this activity arises from within the YSL.

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Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus.

Previous gain-of-function assays in Xenopus have demonstrated that Xwnt-3a can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. We report that expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. We further demonstrate that the ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants is dependent upon a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants was found to be dependent on endogenous Wnt signaling. We conclude that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways.

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Functional intertwining of Dpp and EGFR signaling during Drosophila endoderm induction.

Endoderm induction in Drosophila is mediated by the extracellular signals Decapentaplegic (Dpp) and Wingless (Wg). We discovered a secondary signal with a permissive role in this process, namely Vein, a neuregulin-like ligand that stimulates the epidermal growth factor receptor (EGFR) and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. Experiments based on lack of function and ectopic stimulation of Dpp and EGFR signaling show that these two pathways are functionally interdependent and that they synergize with each other, revealing functional intertwining. The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras, the secondary signal of Dpp. We discuss the potential significance of why an inductive process might use a secondary signal whose function is intertwined with that of the primary signal.

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Timing and competence of neural crest formation.

Neural crest cells can be induced by an interaction between neural plate and ectoderm. To clarify the timing and nature of these inductive interactions, we have examined the time of competence of the neural plate to become neural crest as well as the time of neural fold specification. The neural plate is competent to respond to inductive interactions with the nonneural ectoderm for a limited period, rapidly losing its responsive ability after stage 10. In contrast, nonneural ectoderm from numerous stages retains the ability to induce neural crest cells from competent neural plate. When neural folds are explanted to test their ability to produce neural crest without further tissue interactions, we find that folds derived from all rostrocaudal levels of the open neural plate are already specified to express the neural crest marker Slug. However, additional signals may be required for maintenance of Slug expression, since the transcript is later down-regulated in vitro in the absence of tissue interactions. Taken together, these results suggest that there are multiple stages of neural crest induction. The earliest induction must have occurred by the end of gastrulation, since the newly formed neural fold population is already specified to form neural crest. However, isolated neural folds eventually down-regulate Slug, suggesting a second phase that maintains neural crest formation. Thus, induction of the neural crest may involve multiple and sustained tissue interactions.

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Mechanisms specifying area fate in cortex include cell-cycle-dependent decisions and the capacity of progenitors to express phenotype memory.

Progenitor cells in the early developing cerebral cortex produce neurons destined for discrete functional areas in response to specific inductive signals. Using lineage analysis, we show that cortical progenitor cells at different fetal ages retain the memory of an area-specific inductive signal received in vivo, even though they may pass through as many as two cell cycles in the absence of the signal in culture. When exposed to inductive signals in vitro, only those progenitors that progress through at least one complete cell cycle alter their areal phenotype. Our findings suggest that induction of an areal phenotype is linealy inherited, with the phenotype specified prior to the final cell cycle.

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Conserved patterns of cell movements during vertebrate gastrulation.

Vertebrate embryogenesis entails an exquisitely coordinated combination of cell proliferation, fate specification and movement. After induction of the germ layers, the blastula is transformed by gastrulation movements into a multilayered embryo with head, trunk and tail rudiments. Gastrulation is heralded by formation of a blastopore, an opening in the blastula. The axial side of the blastopore is marked by the organizer, a signaling center that patterns the germ layers and regulates gastrulation movements. During internalization, endoderm and mesoderm cells move via the blastopore beneath the ectoderm. Epiboly movements expand and thin the nascent germ layers. Convergence movements narrow the germ layers from lateral to medial while extension movements elongate them from head to tail. Despite different morphology, parallels emerge with respect to the cellular and genetic mechanisms of gastrulation in different vertebrate groups. Patterns of gastrulation cell movements relative to the blastopore and the organizer are similar from fish to mammals, and conserved molecular pathways mediate gastrulation movements.

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Alternative morphs and plasticity of vulval development in a rhabditid nematode species.

The nematode species Rhabditis sp. SB347 (Family Rhabditidae) in standard culture conditions displays two developmental morphs with distinct modes of sexual reproduction: (1). females and males that develop through four feeding juvenile ("larval") stages; (2). self-fertile protandric hermaphrodites that develop through an obligatory non-feeding third juvenile stage, the "dauer" larva. In females and males, somatic gonad development begins in the first larval stage, whereas in hermaphrodites it is delayed to the second larval stage. Vulval development also differs between females and hermaphrodites: (1). the P8.p cell divides in females but stays undivided in hermaphrodites; (2). the number, timing, and source of inductive signals from the gonad to the vulval precursor cells differ between the two morphs. These results show that discrete vulva developmental routes can be adopted by animals of the same genotype.

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Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway.

A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. Long-range effects of hedgehog (hh) signaling have been suggested to result either from local induction of a secondary diffusible signal or from the direct action of the highly diffusible carboxy-terminal product of HH autoproteolytic cleavage. Here we provide evidence that the long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product. We also show that pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. Our results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members.

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A connectionist model of development.

We present a phenomenological modeling framework for development. Our purpose is to provide a systematic method for discovering and expressing correlations in experimental data on gene expression and other developmental processes. The modeling framework is based on a connectionist or "neural net" dynamics for biochemical regulators, coupled to "grammatical rules" which describe certain features of the birth, growth, and death of cells, synapses and other biological entities. We outline how spatial geometry can be included, although this part of the model is not complete. As an example of the application of our results to a specific biological system, we show in detail how to derive a rigorously testable model of the network of segmentation genes operating in the blastoderm of Drosophila. To further illustrate our methods, we sketch how they could be applied to two other important developmental processes: cell cycle control and cell-cell induction. We also present a simple biochemical model leading to our assumed connectionist dynamics which shows that the dynamics used is at least compatible with known chemical mechanisms.

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An anterior signalling centre in Xenopus revealed by the homeobox gene XHex.

BACKGROUND: Signals from anterior endodermal cells that express the homeobox gene Hex initiate development of the most rostral tissues of the mouse embryo. The dorsal/anterior endoderm of the Xenopus gastrula, which expresses Hex and the putative head-inducing gene cerberus, is proposed to be equivalent to the mouse anterior endoderm. Here, we report the origin and signalling properties of this population of cells in the early Xenopus embryo. RESULTS: Xenopus anterior endoderm was found to derive in part from cells at the centre of the blastocoel floor that express XHex, the Xenopus cognate of Hex. Like their counterparts in the mouse embryo, these Hex-expressing blastomeres moved to the dorsal side of the Xenopus embryo as gastrulation commenced, and populated deep endodermal adjacent to Spemann's organiser. Experiments involving the induction of secondary axes confirmed that XHex expression was associated with anterior development. Ventral misexpression of XHex induced ectopic cerberus expression and conferred anterior signalling properties to the endoderm. Unlike the effect of misexpressing cerberus, these signals could not neuralise overlying ectoderm. CONCLUSIONS: XHex expression reveals the unexpected origin of an anterior signalling centre in Xenopus, which arises in part from the centre of the blastula and localises to the deep endoderm adjacent to Spemann's organiser. Signals originating from these endodermal cells impart an anterior identity to the overlying ectoderm, but are insufficient for neural induction. The anterior movement of Hex-expressing cells in both Xenopus and mouse embryos suggests that this process is a conserved feature of vertebrate development.

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Cell lineage and the induction of second nervous systems in amphibian development.

The amphibian nervous system has long been thought to arise from within a large (greater than 10(3) population of dorsal ectodermal cells, that would otherwise differentiate only as epidermis, as a result of inductive signals from underlying dorsal mesoderm at gastrulation. It has recently been claimed, however, that small cell groups are set aside much earlier in amphibian development, as the sole founders of particular portions or 'compartments' of the body plan. This would imply a dramatic re-interpretation of classical experiments where a second dorsal blastoporal lip, grafted to the ventral side of a gastrula containing 10,000 or more cells, causes there the development of a second central nervous system (CNS) in host tissue. We show that such surgically induced second nervous systems are made by host cells which have lineages separate from those that contribute to the original CNS from the 32-cell stage. Thus neural induction can occur as traditionally supposed, by assignment of ectodermal cell fate in relation to dorsal mesoderm during gastrulation. We discuss the implications of this for the recent proposals about early compartmentation in vertebrate development.

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The retinoblastoma gene family in differentiation and development.

The retinoblastoma (Rb) tumor suppressor gene and its close relatives p107 and p130 are best known for their function in the control of cell cycle progression. In recent years, however, a new role for these proteins has been emerging as they have been linked with regulation of terminal differentiation of many tissues and cell types. In fact, Rb and its family members have been shown to be involved in multiple stages of the differentiation process including irreversible exit from the cell cycle, protection from apoptosis, induction of cell type specific gene expression and maintenance of the post-mitotic state. They also play a critical role in assuring the orderly progression through all these stages of differentiation.

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Gli1 is a target of Sonic hedgehog that induces ventral neural tube development.

The vertebrate zinc finger genes of the Gli family are homologs of the Drosophila gene cubitus interruptus. In frog embryos, Gli1 is expressed transiently in the prospective floor plate during gastrulation and in cells lateral to the midline during late gastrula and neurula stages. In contrast, Gli2 and Gli3 are absent from the neural plate midline with Gli2 expressed widely and Gli3 in a graded fashion with highest levels in lateral regions. In mouse embryos, the three Gli genes show a similar pattern of expression in the neural tube but are coexpressed throughout the early neural plate. Because Gli1 is the only Gli gene expressed in prospective floor plate cells of frog embryos, we have investigated a possible involvement of this gene in ventral neural tube development. Here we show that Shh signaling activates Gli1 transcription and that widespread expression of endogenous frog or human glioma Gli1, but not Gli3, in developing frog embryos results in the ectopic differentiation of floor plate cells and ventral neurons within the neural tube. Floor-plate-inducing ability is retained when cytoplasmic Gli1 proteins are forced into the nucleus or are fused to the VP16 transactivating domain. Thus, our results identify Gli1 as a midline target of Shh and suggest that it mediates the induction of floor plate cells and ventral neurons by Shh acting as a transcriptional regulator.

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Stem cell and precursor cell therapy.

Strategies for cell replacement therapy have been guided by the success in the hematopoietic stem cell field. In this review, we discuss the basis of this success and examine whether this stem cell transplant model can be replicated in other systems where stem cell therapy is being evaluated. We conclude that identifying the most primitive stem cell and using it for transplant therapy may not be appropriate in all systems. We suggest alternative strategies such as progenitor cell replacement, inductive factors, bioengineering organs, in utero transplants, or any approach that takes advantage of the unique properties of the tissue and the stem cell type which, are more likely to provide effective functional replacement.

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