The contribution made by a single somite to the vertebral column: experimental evidence in support of resegmentation using the chick-quail chimaera model

The somitic involvement in the formation of the vertebral column was examined using the chick-quail chimaera model. Single cervical somites from quail donor embryos were transplanted into similarly staged chick host embryos. Following further incubation, serial sections of variously staged embryos were stained with the Feulgen reaction to distinguish the two cell populations. Quail cells were generally located within a delimited region in one half of each of the two adjacent vertebrae, as well as in the intervening disc. The horizontal plane of division through each vertebra passed approximately through the centre of the body and divided the neural arch into rostral and caudal halves through the rostral border of the caudal notch. These results give support to the controversial theory of resegmentation, in which it was suggested that there is an apparent realignment of segmentation between the somite stage and the subsequent vertebral stage of development.     

Evidence for resegmentation in the formation of the vertebral column using the novel approach of retroviral-mediated gene transfer

We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite.     

The ontogeny of allorecognition in a colonial hydroid and the fate of early established chimeras

Colonies of the marine hydroid, Hydractinia, are able to discriminate between their own tissues and those belonging to unrelated conspecifics. We have studied the ontogeny of this allorecognition system by a series of allogeneic transplantations along a developmental gradient, including two-cell-stage embryos, 8 h morulae, planula larvae and metamorphosed polyps. Allograft acceptance of incompatible tissue was observed in all embryonic and larval stages, whereas metamorphosed polyps rejected incompatible transplanted allografts. Most of the chimeras established at the two-cell-stage, although composed of two allogeneic, incompatible entities with mismatching allorecognition loci, developed normally and remained stable through metamorphosis. The results of post metamorphic transplantation assays among the chimeras and the naive ramets, suggested that both incompatible genotypes were still represented in the chimera despite the onset of alloimmune maturation. The naive colonies always rejected each other. Chimeras established from later embryonic and larval stages did not develop into adult chimeric entities, but rather separated immediately post metamorphosis. We thus show that (1) allorecognition in this species matures during metamorphosis and (2) genetically incompatible entities may coexist in one immunologically mature, chimeric soma, provided that they were grafted early enough in ontogeny.     

Somite development without influence of the surface ectoderm in the chick embryo: the compartments of a somite responsible for distal rib development

In the development of the somite, signals from neighboring tissues have been suggested to play critical roles. We have found that when interaction between the ectoderm and the somite is blocked by inserting a piece of polyethylene terephatalate film between them in 2-day-chicken embryo, one of the derivatives of somite, the distal rib, did not form. We examined somite development after the operation, to know the correlation between somite development and distal rib formation. In the operated embryo, the dermomyotome was medio-laterally shorter than in the normal embryo, and Pax3 and Sim1 expressions that are seen in the lateral part of normal dermomyotomes were not found, suggesting that the lateral part of the dermomyotome was missing. Although the sclerotome appeared to be normal in its histology and Pax1 expression pattern in the operated embryo, we could not detect the expression of either Scleraxis nor γ-FBP that are expressed in the cells around the boundaries between the adjacent dermomyotomes in normal embryos. Thus, under the influence of surface ectoderm, the lateral part of dermomyotome and/or the mesenchyme around rostral and caudal edges of dermomyotomes are suggested to play an important role in the distal rib development.     

The exchange hypothesis for the formation of chromatid aberrations: an experimental test using bleomycin

The association between bleomycin-induced chromatid aberrations and BUdR-label exchange between sister chromatids was investigated in order to evaluate Revell's exchange hypothesis for the formation of chromatid aberrations. The results of this study indicate that a larger than expected proportion of chromatid breaks can be accounted for by the exchange hypothesis though not all breaks are the result of incomplete exchange.     

The nature of nurture and the future of evodevo: toward a theory of developmental evolution

This essay has three parts. First, I posit that much research in contemporary evodevo remains steeped in a traditional framework that views traits and trait differences as being caused by genes and genetic variation, and the environment as providing an external context in which development and evolution unfold. Second, I discuss three attributes of organismal development and evolution, broadly applicable to all organisms and traits that call into question the usefulness of gene- and genome-centric views of development and evolution. I then focus on the third and main aim of this essay and ask: what conceptual and empirical opportunities exist that would permit evodevo research to transcend the traditional boundaries inherited from its parent disciplines and to move toward the development of a more comprehensive and realistic theory of developmental evolution? Here, I focus on three conceptual frameworks, the theory of facilitated variation, the theory of evolution by genetic accommodation, and the theory of niche construction. I conclude that combined they provide a rich, interlocking framework within which to revise existing and develop novel empirical approaches toward a better understanding of the nature of developmental evolution. Examples of such approaches are highlighted, and the consequences of expanding existing frameworks are discussed.     

The information theory of developmental pruning: Optimizing global network architectures using local synaptic rules

During development, biological neural networks produce more synapses and neurons than needed. Many of these synapses and neurons are later removed in a process known as neural pruning. Why networks should initially be over-populated, and the processes that determine which synapses and neurons are ultimately pruned, remains unclear. We study the mechanisms and significance of neural pruning in model neural networks. In a deep Boltzmann machine model of sensory encoding, we find that (1) synaptic pruning is necessary to learn efficient network architectures that retain computationally-relevant connections, (2) pruning by synaptic weight alone does not optimize network size and (3) pruning based on a locally-available measure of importance based on Fisher information allows the network to identify structurally important vs. unimportant connections and neurons. This locally-available measure of importance has a biological interpretation in terms of the correlations between presynaptic and postsynaptic neurons, and implies an efficient activity-driven pruning rule. Overall, we show how local activity-dependent synaptic pruning can solve the global problem of optimizing a network architecture. We relate these findings to biology as follows: (I) Synaptic over-production is necessary for activity-dependent connectivity optimization. (II) In networks that have more neurons than needed, cells compete for activity, and only the most important and selective neurons are retained. (III) Cells may also be pruned due to a loss of synapses on their axons. This occurs when the information they convey is not relevant to the target population.     

The fittest versus the flattest: experimental confirmation of the quasispecies effect with subviral pathogens

The "survival of the fittest" is the paradigm of Darwinian evolution in which the best-adapted replicators are favored by natural selection. However, at high mutation rates, the fittest organisms are not necessarily the fastest replicators but rather are those that show the greatest robustness against deleterious mutational effects, even at the cost of a low replication rate. This scenario, dubbed the "survival of the flattest", has so far only been shown to operate in digital organisms. We show that "survival of the flattest" can also occur in biological entities by analyzing the outcome of competition between two viroid species coinfecting the same plant. Under optimal growth conditions, a viroid species characterized by fast population growth and genetic homogeneity outcompeted a viroid species with slow population growth and a high degree of variation. In contrast, the slow-growth species was able to outcompete the fast species when the mutation rate was increased. These experimental results were supported by an in silico model of competing viroid quasispecies.     

Epaxial-adaxial-hypaxial regionalisation of the vertebrate somite: evidence for a somitic organiser and a mirror-image duplication

During vertebrate neural tube formation, the initially lateral borders between the neural and epidermal ectoderm fuse to form the definitive dorsal region of the embryo, while the initially dorsally located notochord-floor plate complex is being internalised. Along the definitive dorso-ventral body axis, one can distinguish an epaxial (dorsal to the notochord) and a hypaxial (ventral to the notochord) body region. The mesodermal somites on both sides of the notochord and neural tube give rise to the trunk skeleton and skeletal muscle. Muscle forms from the somite-derived dermomyotomes and myotomes that elongate dorsally and ventrally. Based on gene expression patterns and comparative embryology, it is proposed here that the epaxial (dermo)myotome region in amniote embryos is subdivided into a dorsalmost and a centrally intercalated subregion. The intercalated subregion abuts to the hypaxial (dermo)myotome region that elongates ventrally via the hypaxial somitic bud. The dorsalmost subregion elongates towards the dorsal neural tube and is proposed to derive from an epaxial somitic bud. The dorsalmost and hypaxial somite derivatives share specific gene expression patterns which are distinct from those of the intercalated somite derivatives. The intercalated somite derivatives develop adaxially, i.e. at the level of the notochord-floor plate complex. Thus, the dorsalmost and intercalated (dermo)myotome subregions may be influenced preferentially by signals from the dorsal neural tube and from the notochord-floor plate complex, respectively. These (dermo)myotome subregions are sharply delimited from each other by molecular boundary markers, including Engrailed and Wnts. It thus appears that the molecular network that polarises borders in Drosophila and vertebrate embryogenesis is redeployed during subregionalisation of the (dermo)myotome. It is proposed here that cells within the amniote (dermo)myotome establish polarised borders with organising capacity, and that the epaxial somitic bud represents a mirror-image duplication of the hypaxial somitic bud along such a border. The resulting epaxial-intercalated/adaxial-hypaxial regionalisation of somite derivatives is conserved in vertebrates although the differentiation of sclerotome and myotome starts heterochronically in embryos of different vertebrate groups.     

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.     

The genetic equidistance result: misreading by the molecular clock and neutral theory and reinterpretation nearly half of a century later

In 1963,Margoliash discovered the unexpected genetic equidistance result after comparing cytochrome c sequences from different species.This finding,together with the hemoglobin analyses of Zuckerkandl and Pauling in 1962,directly inspired the ad hoc molecular clock hypothesis.Unfortunately,however,many biologists have since mistakenly viewed the molecular clock as a genuine reality,which in turn inspired Kimura,King,and Jukes to propose the neutral theory of molecular evolution.Many years of studies have found numerous contradictions to the theory,and few today believe in a universal constant clock.What is being neglected,however,is that the failure of the molecular clock hypothesis has left the original equidistance result an unsolved mystery.In recent years,we fortuitously rediscovered the equidistance result,which remains unknown to nearly all researchers.Incorporating the proven virtues of existing evolutionary theories and introducing the novel concept of maximum genetic diversity,we proposed a more complete hypothesis of evolutionary genetics and reinterpreted the equidistance result and other major evolutionary phenomena.The hypothesis may rewrite molecular phylogeny and population genetics and solve major biomedical problems that challenge the existing framework of evolutionary biology.     

Effects of cholecystokinin peptides and neurotensin on dopamine release and metabolism in the rostral and caudal part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat

By means of intracerebral microdialysis effects of cholecystokinin peptides and neurotensin administered via the microdialysis probe have been studied on dopamine release and metabolism in the nucleus accumbens and neostriatum of the halothane anaesthetized male rat. Levels of extra cellular dopamine (DA) and its metabolites 3,4 dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assessed in nuc. accumbens (rostral and caudal part) using high performance liquid chromatography in combination with electrochemical detection.

(1) In the rostral part of the nuc. accumbens CCK-8 (10 and 100 μM), CCK-33 (100 μM) but not CCK-4 (10 and 100 μM) increased the levels of DA in the perfusate without increasing the extracellular levels of DOPAC and HVA. (2) In the caudal nuc. accumbens CCK-8 and CCK-4 in concentrations of 10 μM and 100 μ M of CCK-33 had no effect on DA release and metabolism, since the extracellular levels of DA, DOPAC and HVA were not changed. (3) In the rostral nuc. accumbens perfusion with 10 μM of neurotensin but not with any other concentration of neurotensin (0.01, 0.1, 1 and 100 μM) increased the levels of DA in the extracellular fluid. (4) In the caudal nuc. accumbens a 40 min perfusion with neutrotensin produced a concentration dependent increase of the levels of DA in the perfusate (peak action at 10 μ M) which in this case was associated with increases in the extracellular levels of DOPAC and HVA. (5) By means of receptor autoradiography using (3-[¹²⁵I]iodotyrosyl³) neurotensin it was found that a 40 min perfusion with this radioligand in the rostral nuc. accumbens reached a total volume of 0.051 mm³. The diffusion of the radioligand was limited to the rostral or caudal part of the nuc. accumbens depending upon the site of placement of the dialysis probe.

The results indicate the existence of cholecystokinin (CCK) receptors in the rostral nuc. accumbens, which are sensitive to CCK-8 and CCK-33 but not to CCK-4, and which facilitate DA release without producing any detectable increase in DA metabolites. In contrast, such receptors do not appear to play a similar role in the regulation of DA release in the caudal nuc. accumbens, where DA terminals contain CCK-like immunoreactivity. Furthermore, the results indicate that neurotensin receptors exist both in the rostral and caudal nuc. accumbens, where they inter alia enhance the release of DA. In the caudal nuc. accumbens these effects of neurotensin are also associated with an increase of DA metabolites, possibly suggesting that in this region neurotensin receptors may also control DA synthesis.     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

The concept of developmental reprogramming and the quest for an inclusive theory of evolutionary mechanisms

SUMMARY Evolutionary developmental biology has already made a major contribution to our understanding of evolutionary patterns, notably homology. However, while it has the potential to make an equally important contribution to our understanding of evolutionary mechanisms, and indeed to the integration of mechanism and pattern, it has not yet done so. This paper explores how this potential may be realized. In particular, I focus on the limitations of present-day neo-Darwinian theory, and indicate how a combination of the neo-Darwinian and "evo-devo" approaches provides a more inclusive view of evolutionary mechanisms with greater explanatory power. There is a particular focus on developmental reprogramming, which lies logically between mutation and selection, yet has been neglected in mainstream evolutionary theory. The inclusion of developmental reprogramming in the list of evolutionary mechanisms leads to a view that the direction of evolutionary change is determined by a combination of internal and external factors, rather than being controlled entirely by the environment.     

Morphological chimeras of larvae and adults in a hydrozoan--insights into the control of pattern formation and morphogenesis

In the marine hydroid Hydractinia echinata, metamorphosis transforms the spindle-shaped larva into a primary polyp. It bears a hypostome with a ring of tentacles at its apical end, a gastric region in the middle and stolons at the base. In nature, metamorphosis is induced in response to external stimuli provided by bacteria. These stimuli can be replaced by artificial inducers, one of which is heat shock. Among heat shock treated stages are those undergoing complete metamorphosis but also specimens forming chimeras of different developmental stages. In the chimeric larvae, the posterior is transformed into the apical hypostome of the adult polyp while the anterior part of the larva persists as larval tissue. After transverse sectioning, these stage chimeras regenerate the missing body parts with respect to the nature of the tissue at the wound surface. This shows that the decision to make larva or polyp morphology depends not on the majority of the tissue in the original body section, but on stage specificity within the regenerating animal part. Single cells can escape from this general rule, since RFamide nerve cells which usually differentiate in polyp tissue appear in regenerated larval tails of sectioned stage chimeras. The results indicate that the pattern-forming system of the larva and of the adult have features in common. The primary signals controlling patterning along the anterior-posterior axis in larvae and the apical-basal axis in polyps arethus likelyto be the same while the interpretation of these primary signals by the individual cells changes during metamorphosis.