Mitotic domains reveal early commitment of cells in Drosophila embryos

In embryos of Drosophila melanogaster all the nuclei in the syncytial egg divide with global synchrony during the first 13 mitotic cycles. But with cellularization in the 14th cycle, global mitotic synchrony ceases. Starting about one hour into the 14th interphase, at least 25 'mitotic domains', which are clusters of cells united by locally synchronous mitosis, partition the embryo blastoderm surface into a complex fine-scale pattern. These mitotic domains, which are constant from one embryo to the next, fire in the same temporal sequence in every embryo. Some domains consist of a single cell cluster straddling the ventral or dorsal midline. Most consist of two separate cell clusters that occupy mirror-image positions on the bilaterally symmetric embryo. Others comprise a series of members present not only as bilateral pairs but also as metameric repeats. Thus a domain can consist of either one, two, or many (if metamerically reiterated) clusters of contiguous cells. Within each cluster, mitosis starts in a single cell or in a small number of interior cells then spreads wave-like, in all directions, until it stops at the domain boundary. Each domain occupies a specific position along the anteroposterior axis--as determined by the expression pattern of the engrailed protein, and along the dorsoventral axis--as determined by cell count from the ventral midline. The primordia of certain larval structures appear to consist solely of the cells of one specific mitotic domain. Moreover, cells in at least some mitotic domains share specific morphogenetic traits, distinct from those of cells in adjacent domains. These traits include cell shape, spindle orientation, and participation by all the cells of a domain in an invagination. The specialized behaviors of the various mitotic domains transform the monolayer cell sheet of the blastoderm into the multilayered gastrula. I conclude that the fine-scale partitioning of the newly cellularized embryo into mitotic domains is an early manifestation of the commitment of cells to specific developmental fates.     

Fate mapping of Drosophila embryonic mitotic domain 20 reveals that the larval visual system is derived from a subdomain of a few cells.

In an attempt to study the fates of cells in the dorsal head region of Drosophila embryos at gastrulation, we used the photoactivated gene expression system to mark small numbers of cells in selected mitotic domains. We found that mitotic domain 20, which is a cluster of approximately 30 cells on the dorsal posterior surface, gives rise to various ectodermal cell types in the head, including dorsal pouch epithelium, the optic lobe, and head sensory organs, including Bolwig's organ, the larval photoreceptor organ. We found that the optic lobe and larval photoreceptors share the same origin of a few adjacent cells near the center of mitotic domain 20, suggesting that within the mitotic domain, there is a subdomain from which the larval visual system is specified. In addition to the components of the larval visual system, this central region of mitotic domain 20 also generates a part of the eye-antennal disc placode; cells that gives rise to the adult visual system. We also observed that a significant amount of cell death occurred within this domain and used cell ablation experiments to determine the ability of the embryo to compensate for cell loss.     

The prethalamus is established during gastrulation and influences diencephalic regionalization

The vertebrate neural plate contains distinct domains of gene expression, prefiguring the future brain areas. In this study, we draw an extended expression map of the rostral neural plate that reveals discrete domains inside the presumptive posterior forebrain. We show, by fate mapping, that these well-defined cell populations will develop into specific diencephalic regions. To address whether these early subterritories are already committed to restricted identities, we began to analyse the consequences of ablation and transplantation of these specific cell populations. We found that precursors of the prethalamus are already specified and irreplaceable at late gastrula stage, because ablation of these cells results in loss of prethalamic markers. Moreover, when transplanted into the ectopic environment of the presumptive hindbrain, these cells still pursue their prethalamic differentiation program. Finally, transplantation of these precursors, in the rostral-most neural epithelium, induces changes in cell identity in the surrounding host forebrain. This cell–non-autonomous property led us to propose that these committed prethalamic precursors may play an instructive role in the regionalization of the developing diencephalon.     

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.     

Segregation of lens and olfactory precursors from a common territory: cell sorting and reciprocity of Dlx5 and Pax6 expression

Cranial placodes are focal regions of columnar epithelium next to the neural tube that contribute to sensory ganglia and organs in the vertebrate head, including the olfactory epithelium and the crystalline lens of the eye. Using focal dye labelling within the presumptive placode domain, we show that lens and nasal precursors arise from a common territory surrounding the anterior neural plate. They then segregate over time and converge to their final positions in discrete placodes by apparently directed movements. Since these events closely parallel the separation of eye and antennal primordia (containing olfactory sensory cells) from a common imaginal disc in Drosophila, we investigated whether the vertebrate homologues of Distalless (Dll) and Eyeless (Ey), which determine antennal and eye identity in the fly, play a role in segregation of lens and nasal precursors in the chick. Dlx5 and Pax6 are initially co-expressed by future lens and olfactory cells. As soon as presumptive lens cells acquire columnar morphology all Dlx family members are down-regulated in the placode, while Pax6 is lost in the olfactory region. Lens precursor cells that express ectopic Dlx5 never acquire lens-specific gene expression and are excluded from the lens placode to cluster in the head ectoderm. These results suggest that the loss of Dlx5 is required for cells to adopt a lens fate and that the balance of Pax6 and Dlx expression regulates cell sorting into appropriate placodal domains.     

Numb-Associated Kinase Interacts with the Phosphotyrosine Binding Domain of Numb and Antagonizes the Function of Numb In Vivo

During asymmetric cell division, the membrane-associated Numb protein localizes to a crescent in the mitotic progenitor and is segregated predominantly to one of the two daughter cells. We have identified a putative serine/threonine kinase, Numb-associated kinase (Nak), which interacts physically with the phosphotyrosine binding (PTB) domain of Numb. The PTB domains of Shc and insulin receptor substrate bind to an NPXY motif which is not present in the region of Nak that interacts with Numb PTB domain. We found that the Numb PTB domain but not the Shc PTB domain interacts with Nak through a peptide of 11 amino acids, implicating a novel and specific protein-protein interaction. Overexpression of Nak in the sensory organs causes both daughters of a normally asymmetric cell division to adopt the same cell fate, a transformation similar to the loss of numb function phenotype and opposite the cell fate transformation caused by overexpression of Numb. The frequency of cell fate transformation is sensitive to the numb gene dosage, as expected from the physical interaction between Nak and Numb. These findings indicate that Nak may play a role in cell fate determination during asymmetric cell divisions.     

Differential expression of the human gonadotropin alpha gene in ectopic and eutopic cells.

We have analyzed the regulation of the alpha gonadotropin gene in eutopic placental cells and ectopic tumor cells by constructing a series of plasmid vectors containing alpha genomic 5' flanking DNA placed upstream of the gene encoding the bacterial enzyme chloramphenicol acetyltransferase (CAT). These plasmid DNAs were transfected into a eutopic (JAr) and an ectopic (HeLa) cell line. Both cell types expressed the CAT gene from plasmid constructs containing as much as 1,500 base pairs (bp) and as little as 140 bp of alpha 5' flanking DNA; JAr cells were considerably more efficient than HeLa cells. Ectopic and eutopic cells differed qualitatively in their expression from these alpha-CAT constructs when cells were treated with cAMP or butyrate. Butyrate induced alpha expression in HeLa cells but not in JAr cells, while cAMP induced expression in JAr cells. These results are consistent with and extend previous observations suggesting that there are cell-specific differences in the regulation of alpha gene expression in ectopic and eutopic cells. However, by using deletion constructs of the alpha-CAT gene, we found that the basal expression and cell-specific induction of the alpha gene in ectopic and eutopic cells were dependent on the same 140 bp of alpha 5' flanking DNA. These 140 bp were sequenced and found to contain a 9-bp stretch of DNA homologous with the consensus viral enhancer sequence. Such features of alpha expression common to both ectopic and eutopic cells may be involved in the coordinate expression of the alpha gene and the tumorigenic phenotype observed in each cell type.     

XMam1, Xenopus Mastermind1, induces neural gene expression in a Notch-independent manner

Mastermind, which is a Notch signal component, is a nuclear protein and is thought to contribute to the transactivation of target genes. Previously we showed that XMam1, Xenopus Mastermind1, was essential in the transactivation of a Notch target gene, XESR-1, and was involved in primary neurogenesis. To examine the function of XMam1 during Xenopus early development in detail, XMam1-overexpressed embryos were analyzed. Overexpression of XMam1 ectopically caused the formation of a cell mass with pigmentation on the surface of embryos and expressed nrp-1. The nrp-1-positive cell mass was produced by XMam1 without expression of the Notch target gene, XESR-1, and not by the activation form of Notch, NICD. The ectopic expression of nrp-1 was not inhibited by co-injection of XMam1 with a molecule known to inhibit Notch signaling. The nrp-1 expression was also recognized in the animal cap injected with XMam1DeltaN, which lacks the basic domain necessary for interacting with NICD and Su(H). These results show that XMam1 has the ability to induce the cell fate into the neurogenic lineage in a Notch-independent manner.     

Drumstick is a zinc finger protein that antagonizes Lines to control patterning and morphogenesis of the Drosophila hindgut

Elongation of the Drosophila embryonic hindgut epithelium occurs by a process of oriented cell rearrangement requiring the genes drumstick (drm) and lines (lin). The elongating hindgut becomes subdivided into domains -- small intestine, large intestine and rectum -- each characterized by a specific pattern of gene expression dependent upon normal drm and lin function. We show that drm encodes an 81 amino acid (10 kDa) zinc finger protein that is a member of the Odd-skipped family. drm expression is localized to the developing midgut-hindgut junction and is required to establish the small intestine, while lin is broadly expressed throughout the gut primordium and represses small intestine fate. lin is epistatic to drm, suggesting a model in which localized expression of drm blocks lin activity, thereby allowing small intestine fate to be established. Further supporting this model, ectopic expression of Drm throughout the hindgut produces a lin phenotype. Biochemical and genetic data indicate that the first conserved zinc finger of Drm is essential for its function. We have thus defined a pathway in which a spatially localized zinc finger protein antagonizes a globally expressed protein, thereby leading to specification of a domain (the small intestine) necessary for oriented cell rearrangement.     

dBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.     

sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation

The appearance of molecular differences between arterial and venous endothelial cells before circulation suggests that genetic factors determine these cell types. We find that vascular endothelial growth factor (vegf) acts downstream of sonic hedgehog (shh) and upstream of the Notch pathway to determine arterial cell fate. Loss of Vegf or Shh results in loss of arterial identity, while exogenous expression of these factors causes ectopic expression of arterial markers. Microinjection of vegf mRNA into embryos lacking Shh activity can rescue arterial differentiation. Finally, activation of the Notch pathway in the absence of Vegf signaling can rescue arterial marker gene expression. These studies reveal a complex signaling cascade responsible for establishing arterial cell fate and suggest differential effects of Vegf on developing endothelial cells.     

Members of the YABBY gene family specify abaxial cell fate in Arabidopsis.

Lateral organs produced by shoot apical and flower meristems exhibit a fundamental abaxial-adaxial asymmetry. We describe three members of the YABBY gene family, FILAMENTOUS FLOWER, YABBY2 and YABBY3, isolated on the basis of homology to CRABS CLAW. Each of these genes is expressed in a polar manner in all lateral organ primordia produced from the apical and flower meristems. The expression of these genes is precisely correlated with abaxial cell fate in mutants in which abaxial cell fates are found ectopically, reduced or eliminated. Ectopic expression of either FILAMENTOUS FLOWER or YABBY3 is sufficient to specify the development of ectopic abaxial tissues in lateral organs. Conversely, loss of polar expression of these two genes results in a loss of polar differentiation of tissues in lateral organs. Taken together, these observations indicate that members of this gene family are responsible for the specification of abaxial cell fate in lateral organs of Arabidopsis. Furthermore, ectopic expression studies suggest that ubiquitous abaxial cell fate and maintenance of a functional apical meristem are incompatible.     

The pan-neural bHLH proteins DEADPAN and ASENSE regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes

Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. Our analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo. genesis 26:77-85, 2000.     

Altered mitotic domains reveal fate map changes in Drosophila embryos mutant for zygotic dorsoventral patterning genes.

The spatial and temporal pattern of mitoses during the fourteenth nuclear cycle in a Drosophila embryo reflects differences in cell identities. We have analysed the domains of mitotic division in zygotic mutants that exhibit defects in larval cuticular pattern along the dorsoventral axis. This is a powerful means of fate mapping mutant embryos, as the altered position of mitotic domains in the dorsoventral pattern mutants correlate with their late cuticular phenotypes. In the mutants twist and snail, which fail to differentiate the ventrally derived mesoderm, mitoses specific to the mesoderm are absent. The lateral mesectodermal domain shows a partial ventral shift in twist mutants but a proportion of ventral cells do not behave characteristically, suggesting that twist has a positive role in the establishment of the mesoderm. In contrast, snail is required to repress mesectodermal fates in cells of the presumptive mesoderm. In the absence of both genes, the mesodermal and the mesectodermal anlage are deleted. Mutations at five loci delete specific pattern elements in the dorsal half of the embryo and cause partial ventralization. Mutations in the genes zerknüllt and shrew affect cell division only in the dorsalmost cells corresponding to the amnioserosa, while the genes tolloid, screw and decapentaplegic (dpp) affect divisions in both the prospective amnioserosa and the dorsal epidermis. We demonstrate that in each of these mutants dorsally placed mitotic domains are absent and this effect is correlated with an expansion and dorsal shift in the position of more ventral domains. The loss of activity in each of the five genes results in qualitatively similar alterations in the mitotic pattern; mutations with stronger ventralizing phenotypes affect increasingly greater subsets of the dorsal cells. Double mutant analysis indicates that these genes act in a concerted manner to specify dorsal fates. The correlation between phenotypic strength and the progressive loss of dorsal pattern elements in the ventralized mutants, suggests that one of these gene products, perhaps dpp, may provide positional information in a graded manner.     

Contractions and expansions of CAG/CTG trinucleotide repeats occur during ectopic gene conversion in yeast,by a MUS81-independent mechanism

CAG/CTG trinucleotide repeat tracts expand and contract at a high rate during gene conversion in Saccharomyces cerevisiae. In order to characterize the mechanism responsible for such rearrangements, we built an experimental system based on the use of the rare cutter endonuclease I-SceI, to study the fate of trinucleotide repeat tracts during meiotic or mitotic (allelic or ectopic) gene conversion. After double-strand break (DSB) induced meiotic recombination, (CAG)(98) and (CAG)(255) are rearranged in 5% and 52% of the gene conversions, respectively, with similar proportions of contractions and expansions. No evidence of a meiotic hot spot activity associated with trinucleotide repeats could be found. When gene conversion is induced by a DSB during mitotic growth of the cells, no rearrangement of the repeat tracts is detected when the donor sequence is allelic to the recipient site of the DSB. However, when the donor sequence is at an ectopic location, frequent contractions and expansions of the repeat tract are found. No crossing-over associated with gene conversion could be detected. Mutants for the MUS81 gene, involved in the resolution of recombination intermediates, show a frequency of rearrangements identical with that of the wild-type strain. We concluded that trinucleotide repeat rearrangements occur frequently during ectopic but not during allelic recombination, by a mechanism that does not require crossover formation.     

The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages.