Modularity and dissociation in the evolution of gene expression territories in development

SUMMARY Modularity is a salient feature of development and crucial to its evolution. This paper extends modularity to include the concept of gene expression territory, as established for sea urchin embryos. Territories provide a mechanism for partitioning of the cells of a rapidly developing embryo into functional units of a feeding larva. Territories exhibit the characteristics of modules. The paper asks if the embryo and the nonfeeding larva of the direct-developing sea urchin Heliocidaris erythrogramma are organized into gene expression territories, and if its territories correspond to the canonical territories of the pluteus. An analysis of cell lineage and gene expression data for H. erythrogramma shows that skeletogenic cell, coelomic, and vegetal plate gene expression territories are conserved, although they arise from cell lineages distinct from those of the pluteus, and the overall morphology of the larva differs from that of a pluteus. The ectoderm, as in indirect developers, is divided into territories. However, the oral ectodermal territory characteristic of the pluteus is absent in H. erythrogramma. Oral ectoderm is restored in hybrids of H. erythrogramma eggs fertilized by Heliocidaris tuberculata sperm. This indicates that embryonic modules evolve by changes in expression of dominant regulatory genes within territories and that entire modules can be eliminated in evolution of embryos.     

Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation

The relocalisation of some genes to positions outside chromosome territories, and the visible decondensation or unfolding of interphase chromatin, are two striking facets of nuclear reorganisation linked to gene activation that have been assumed to be related to each other. Here, in a study of nuclear reorganisation around the Hoxd cluster, we suggest that this may not be the case. Despite its very different genomic environment from Hoxb, Hoxd also loops out from its chromosome territory, and unfolds, upon activation in differentiating embryonic stem (ES) cells and in the tailbud of the embryo. However, looping out and decondensation are not simply two different manifestations of the same underlying change in chromatin structure. We show that, in the limb bud of the embryonic day 9.5 embryo, where Hoxd is also activated, there is visible decondensation of chromatin but no detectable movement of the region out from the chromosome territory. During ES cell differentiation, decondensed alleles can also be found inside of chromosome territories, and loci that have looped out of the territories can appear to still be condensed. We conclude that evolutionarily conserved chromosome remodelling mechanisms, predating the duplication of mammalian Hox loci, underlie Hox regulation along the rostrocaudal embryonic axis. However, we suggest that separate modes of regulation can modify Hoxd chromatin in different ways in different developmental contexts.     

Cell type specification during sea urchin development

Recent discoveries indicate that cell lineages and fates play a key role in the establishment of spatially restricted gene expression during sea urchin development. Unique sets of founder cells generate five territories of gene expression by means of an invariant pattern of complete cleavage. Cell lineage analysis demonstrates that the second embryonic axis, the oral-aboral axis, is specified with reference to the first cleavage plane. In the undisturbed embryo, clones that contribute to one territory or another begin to appear at the third cleavage, and founder cell segregation to all five territories is completed by the sixth cleavage. Founder cell segregation is a key feature of mechanisms that establish the spatially defined gene activity of sea urchin embryogenesis.     

Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming

The ability of cloned embryos to sustain full-term development depends on the ability of the recipient ooplasm to reprogram the donor cell genome. As the nuclear architecture has recently emerged as a key-factor in the regulation of gene expression, we questioned whether early embryos obtained from transfer of ES metaphasic chromosomes into mouse ooplasm would adopt the somatic or embryonic type of nuclear organization. We have particularly focused on the arrangement of chromosomal territories with respect to the nucleolar compartment, and the pericentric heterochromatin domains called chromocenters. We found that nuclear transfer triggers profound chromatin rearrangements including the dispersion of the donor cell chromocenters components. These rearrangements lead to a typical 1-cell pronuclear organization, namely a radial arrangement of the chromosome territories with centromeres attached to the nucleoli, which adopt the compact fibrillar structure of nucleolar precursor bodies (NPBs). Subsequently, during the second cycle, the cloned embryos undergo further reorganization with the establishment of new chromocenters, clustered in one part of the nucleus, as during normal embryogenesis. We could also establish that the adequate distribution of chromosomal territories at the pronuclear stage seems important for the development until blastocyst.     

Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

Background  

Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals.     

Hindbrain patterning: Krox20 couples segmentation and specification of regional identity.

We have previously demonstrated that inactivation of the Krox20 gene led to the disappearance of its segmental expression territories in the hindbrain, the rhombomeres (r) 3 and 5. We now performed a detailed analysis of the fate of prospective r3 and r5 cells in Krox20 mutant embryos. Genetic fate mapping indicates that at least some of these cells persist in the absence of a functional Krox20 protein and uncovers the requirement for autoregulatory mechanisms in the expansion and maintenance of Krox20-expressing territories. Analysis of even-numbered rhombomere molecular markers demonstrates that in Krox20-null embryos, r3 cells acquire r2 or r4 identity, and r5 cells acquire r6 identity. Finally, study of embryonic chimaeras between Krox20 homozygous mutant and wild-type cells shows that the mingling properties of r3/r5 mutant cells are changed towards those of even-numbered rhombomere cells. Together, these data demonstrate that Krox20 is essential to the generation of alternating odd- and even-numbered territories in the hindbrain and that it acts by coupling the processes of segment formation, cell segregation and specification of regional identity.     

Conservative segregation of maternally inherited CS histone variants in larval stages of sea urchin development

Three sets of histone variants are coexisting in the embryo at larval stages of sea urchin's development: the maternally inherited cleavage stage variants (CS) expressed during the two initial cleavage divisions, the early histone variants, which are recruited into embryonic chromatin from middle cleavage stages until hatching and the late variants, that are fundamentally expressed from blastula stage onward. Since the expression of the CS histones is confined to the initial cleavage stages, these variants represent a very minor proportion of the histones present in the plutei larvae, whereas the late histone variants are predominant. To determine the position of these CS in the embryonic territories, we have immunolocalized the CS histone variants in plutei larvas harvested 72 h post-fertilization. In parallel, we have pulse labeled the DNA replicated during the initial cleavage cycle with bromodeoxyuridine (BrdU) and its position was further determined in the plutei larvas by immunofluorescence. We have found that the CS histone variants were segregated to specific territories in the plutei. The position in which the CS histone variants were found to be segregated was consistent with the position in which the DNA molecules that were replicated during the initial cleavage divisions were localized. These results strongly suggest that a specification of embryonic nuclei occurs at the initial cleavage divisions which is determined by a chromatin organized by CS histone variants.     

Differentiation of human embryonic stem cells induces condensation of chromosome territories and formation of heterochromatin protein 1 foci

Abstract Human embryonic stem cells (hES) are unique in their pluripotency and capacity for self-renewal. Therefore, we have studied the differences in the level of chromatin condensation in pluripotent and all-trans retinoic acid-differentiated hES cells. Nuclear patterns of the Oct4 (6p21.33) gene, responsible for hES cell pluripotency, the C-myc (8q24.21) gene, which controls cell cycle progression, and HP1 protein (heterochromatin protein 1) were investigated in these cells. Unlike differentiated hES cells, pluripotent hES cell populations were characterized by a high level of decondensation for the territories of both chromosomes 6 (HSA6) and 8 (HSA8). The Oct4 genes were located on greatly extended chromatin loops in pluripotent hES cell nuclei, outside their respective chromosome territories. However, this phenomenon was not observed for the Oct4 gene in differentiated hES cells, for the C-myc gene in the cell types studied. The high level of chromatin decondensation in hES cells also influenced the nuclear distribution of all the variants of HP1 protein, particularly HP1α, which did not form distinct foci, as usually observed in most other cell types. Our experiments showed that unlike C-myc , the Oct4 gene and HP1 proteins undergo a high level of decondensation in hES cells. Therefore, these structures seem to be primarily responsible for hES cell pluripotency due to their accessibility to regulatory molecules. Differentiated hES cells were characterized by a significantly different nuclear arrangement of the structures studied.     

NGF mRNA expression in developing cutaneous epithelium related to innervation density

To determine if the initial level of NGF mRNA in developing cutaneous epithelium is correlated with its final innervation density, we measured the concentration of NGF mRNA in the epithelia of the maxillary, mandibular and ophthalmic territories of trigeminal ganglion in the embryonic mouse. At the onset of neuronal death in the ganglion there were marked differences in the concentration of NGF mRNA in these epithelia: the level was highest in the epithelium of the densely innervated maxillary territory, it was lower in the epithelium of the moderately innervated mandibular territory and was lowest in the epithelium of the sparsely innervated ophthalmic territory. These regional differences in the level of NGF mRNA during the early stages of target field innervation suggest that the level of NGF production in target field cells, rather than regional differences in the access of innervating neurons to NGF, governs the number of neurons that survive. Because the same percentage cell death occurs in each of the subsets of trigeminal neurons that innervate the maxillary, mandibular and ophthalmic territories, regional differences in NGF synthesis are not responsible for establishing differences in innervation density, rather they maintain differences that arise earlier in development.     

Whole eyes reconstituted from embryonic half anlagen: alterations in donor-derived territories in Xenopus pigment chimerae

Grafts from pigmented donor embryonic eye rudiments into albino hosts were used to chart i) fates of local cell groups in three positions in whole eye rudiments, and ii) alterations in graft-derived territories when the posterior half of the rudiment was ablated. Small pigmented patches of graft-derived tissue were conspicuous in albino embryos and tadpoles, enabling us to directly monitor their location and size in the eyes of living animals. The three (right eye) positions marked by pigmented grafts were dorsal (12 o'clock), anterior (3 o'clock), and anteroventral (5 o'clock). Control transplants reared without secondary ablation produced black sector territories in pigment retinal epithelium and iris at corresponding 12 o'clock or 2 o'clock or 4 o'clock positions on the larval eyeball. In the experimental series posterior half-anlagen were ablated. The remaining anterior half-anlagen, each containing a pigmented graft, reconstituted spherical larval eyeballs of reduced size. During healing, donor-derived pigmented sector territories remained coherent, but were altered in position and size compared to controls. Dorsal cells (from 12 o'clock grafts) appeared to move rapidly along the newly formed cut edge into the wound and went on to form expanded black sectors in posterior eye regions. More gradually, sector territories of anterior cells (3 o'clock grafts) and anteroventral cells (5 o'clock grafts) shifted toward dorsal in a counterclockwise direction. Thus all three types of graft derived pigment territories were altered in eye anlage fragments as they healed to form half-size spherical eyes.     

Quantitative motion analysis of subchromosomal foci in living cells using four-dimensional microscopy

The motion of subchromosomal foci and of whole chromosome territories in live human cell nuclei was investigated in four-dimensional space-time images. Visualization of subchromosomal foci was achieved by incorporating Cy3-dUTP into the nuclear DNA of two different cell types after microinjection. A subsequent segregation of the labeled cell nuclei led to the presence of only a few labeled chromosome territories on a background of nonlabeled chromatin (. Hum. Genet. 102:241-251). This procedure yielded many distinct signals in a given cell nucleus. Motion analysis in four-dimensional space-time images was performed using single-particle tracking and a statistical approach to the detection of a possible directional motion of foci relative to the center of mass of a chromosome territory. The accuracy of the analysis was tested using simulated data sets that closely mirrored the experimental setup and using microparticles of known size. Application of the analysis tools to experimental data showed that mutual diffusion-like movements between foci located on different chromosomes were more pronounced than inside the territories. In the time range observed, movements of individual foci could best be described by a random diffusion process. The statistical test for joint directed motion of several foci inside chromosome territories revealed that foci occasionally switched from random to directional motion inside the territories.     

Otx genes are required for tissue specification in the developing eye

Patterning of the vertebrate eye appears to be controlled by the mutual regulation and the progressive restriction of the expression domains of a number of genes initially co-expressed within the eye anlage. Previous data suggest that both Otx1 and Otx2 might contribute to the establishment of the different eye territories. Here, we have analysed the ocular phenotype of mice carrying different functional copies of Otx1 and Otx2 and we show that these genes are required in a dose-dependent manner for the normal development of the eye. Thus, all Otx1(-/-); Otx2(+/-) and 30% of Otx1(+/-); Otx2(+/-) genotypes presented consistent and profound ocular malformation, including lens, pigment epithelium, neural retina and optic stalk defects. During embryonic development, optic vesicle infolding was severely altered and the expression of pigment epithelium-specific genes, such as Mitf or tyrosinase, was lost. Lack of pigment epithelium specification was associated with an expansion of the prospective neural retina and optic stalk territories, as determined by the expression of Pax6, Six3 and Pax2. Later in development the presumptive pigment epithelium region acquired features of mature neural retina, including the generation of Islet1-positive neurones. Furthermore, in Otx1(-/-); Otx2(+/-) mice neural retina cell proliferation, cell differentiation and apoptotic cell death were also severely affected. Based on these findings we propose a model in which Otx gene products are required for the determination and differentiation of the pigment epithelium, co-operating with other eye patterning genes in the determination of the specialised tissues that will constitute the mature vertebrate eye.     

Common and divergent pathways in alternative developmental processes of ascidians

Colonial ascidians offer opportunities to investigate how developmental events are integrated to generate the animal form, since they can develop similar individuals (oozooids from eggs, blastozooids from pluripotent somatic cells) through very different reproductive processes, i.e. embryogenesis and blastogenesis. Moreover, thanks to their key phylogenetic position, they can help in the understanding of the molecular mechanisms of morphogenesis and their evolution in chordates. We review organogenesis of the ascidian neural complex comparing embryos and buds in terms of topology, developmental mechanisms and terminology. We propose a new interpretation of bud territories, and reconsider nervous system development based on recent results suggesting that ascidians have vertebrate placodal and neural-crest-like cells. Comparing embryonic and blastogenic development in Botryllus schlosseri, we propose that the bud has territories with a placodal potentiality, suggesting that chordate ancestors possessed neurogenic placodes, and that the genetic pathways regulating neurogenic placode formation were co-opted for new developmental processes, such as blastogenesis.     

Female receptivity, embryonic diapause, and superfetation in the European badger (Meles meles): implications for the reproductive tactics of males and females

The European badger Meles meles is thought to mate throughout the year with two mating peaks occurring in late winter/spring and summer/autumn. After mating, fertilized ova enter embryonic diapause (delayed implantation) at the blastocyst stage, which lasts up to eleven months. Even if mating is successful, however, the estrous cycle may continue during embryonic diapause, which suggests that female badgers are capable of superfetation (conception during pregnancy). This may increase female fitness by facilitating polyandry, and reduce the risk of infanticide by resident males through paternity confusion. Detailed understanding of female receptivity, specifically the association of superfetation with embryonic diapause, may explain field observations of seemingly inconsistent reproductive tactics of male badgers with regard to, for instance, whether or not they guard mates or defend territories. The combination of embryonic diapause and superfetation may occur in other mustelids; if so, the sociobiology of mustelids will need reevaluating, and the Mustelidae may prove to be a good model taxon for studies of sexual conflict in the reproduction of eutherian mammals.     

Vessel and blood specification override cardiac potential in anterior mesoderm

Organ progenitors arise within organ fields, embryonic territories that are larger than the regions required for organ formation. Little is known about the regulatory pathways that define organ field boundaries and thereby limit organ size. Here we identify a mechanism for restricting heart size through confinement of the developmental potential of the heart field. Via fate mapping in zebrafish, we locate cardiac progenitors within hand2-expressing mesoderm and demonstrate that hand2 potentiates cardiac differentiation within this region. Beyond the rostral boundary of hand2 expression, we find progenitors of vessel and blood lineages. In embryos deficient in vessel and blood specification, rostral mesoderm undergoes a fate transformation and generates ectopic cardiomyocytes. Therefore, induction of vessel and blood specification represses cardiac specification and delimits the capacity of the heart field. This regulatory relationship between cardiovascular pathways suggests strategies for directing progenitor cell differentiation to facilitate cardiac regeneration.