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Genes involved in tissue and organ development
Origin of endoderm and the midgut
Ecdysone-regulated genomic networks in Drosophila: Midgut gene expression during metamorphosis
The midgut is derived from the anterior and posterior midgut primordia during the process of gastrulation [Images]. It should be kept in mind that the most terminal aspects of the embryo are fated to become gut endoderm. The terminal system (torso), regulating tailless and huckebein are responsible for this fate determination. Gastrulation is the defining event of gut morphogenesis. The anterior midgut is formed from the anterior midgut primordium; the posterior midgut is derived from the posterior midgut primordium, and the midgut proper is derived from endodermal cells that migrate from both anterior and posterior primordia. The gut is enshrouded in mesoderm which forms vascular musculature around the gut, and is also responsible for creating the gastric ceca. Overlying mesoderm communicates with the gut by secreted factors and through contact.
Dpp has a prime function during endoderm induction in Drosophila. Dpp is secreted from the outer cell layer of the embryonic midgut (the visceral mesoderm) where its main source of expression in parasegment ps7 depends directly on the homeotic gene Ultrabithorax. In the same cell layer, Dpp stimulates expression of another extracellular signal, Wingless (Wg), in a neighboring parasegment that, in turn, feeds back to ps7 to stimulate Ubx expression. Thus, Dpp is part of a "parautocrine" feedback loop for Ubx (i.e., an autocrine feedback loop based partly on paracrine action that sustains its own expression through Dpp and Wg). Dpp also spreads to the inner layer of the embryonic midgut, the endoderm, where it synergizes with Wg to induce expression of the homeotic gene labial (lab). To achieve this, Dpp locally elevates the endodermal expression levels of Drosophila D-Fos with which it cooperates to induce lab. Differentiation of various cell types in the larval gut depends on these inductive effects of Dpp and Wg (Bienz, 1997 and references).
A secondary signal has been discovered with a permissive role in this process; it comes from 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 one another 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. Since up-regulation of vein requires dpp and wg, Vein is considered a secondary signal of Dpp and Wg. Vein stimulates homeotic gene expression in both cell layers of the midgut (Szüts, 1998).
Ecdysone-regulated genomic networks in Drosophila: Midgut gene expression during metamorphosis
During insect metamorphosis, each tissue displays a unique physiological and morphological response to the steroid hormone 20-hydroxyecdysone (ecdysone). Gene expression was assayed in five tissues during metamorphosis onset. Larval-specific tissues display major changes in genome-wide expression profiles, whereas tissues that survive into adulthood display few changes. In one larval tissue, the salivary gland, a computational approach was used to identify a regulatory motif and a cognate transcription factor involved in regulating a set of coexpressed genes. During the metamorphosis of another tissue, the midgut, genes encoding factors from the hedgehog, Notch, EGF, dpp, and wingless pathways are activated by the ecdysone regulatory network. Mutation of the ecdysone receptor abolishes their induction. Cell cycle genes are also activated during the initiation of midgut metamorphosis, and they are also dependent on ecdysone signaling. These results establish multiple new connections between the ecdysone regulatory network and other well-studied regulatory networks (Li, 2003).
Developmental patterns of gene expression were studied from five different tissues and organs: central nervous system (CNS), wing imaginal disc (WD), larval epidermis and attached connective tissue (ED), midgut (MG), and salivary gland (SG), during late larval and early prepupal development when ecdysone triggers metamorphosis. At these stages of development, the five tissues display very different morphological and physiological responses to ecdysone. The wing imaginal disc responds to the hormone by initiating evagination, or unfolding, as it changes from a compact epithelial bilayer to an extended appendage. The salivary glands secrete glue proteins that are used to immobilize the puparium during metamorphosis. The cuticle attached to the larval epidermis undergoes a process of hardening and tanning to form the pupal case. The central nervous system (CNS) displays little morphological change during the late third instar ecdysone pulse, but the animal displays changes in behavior and in neurosecretory status. The two major types of cells in the larval midgut, larval epidermal cells and adult epidermal progenitor cells (midgut imaginal islands), respond in opposite ways to ecdysone. The larval epidermal cells initiate the process of programmed cell death, while the imaginal cells proliferate and form the adult midgut (Li, 2003).
One tissue, the midgut, was selected to assay during its complete metamorphosis, which occurs from 18 hr before puparium formation (BPF) to 12 hr APF. During this 30 hr period, eleven time points were examined as the larval midgut is destroyed and replaced with the adult midgut. The two major cell types present in this organ are distinguishable by size. The larval epithelial cells are large, with decondensed polyploid nuclei, and undergo programmed cell death in response to ecdysone. Embedded among the larval cells are small diploid imaginal midgut cells, which proliferate in response to the hormone to form the adult epithelial cells. Additionally, the midgut contains relatively small numbers of muscle, tracheal, and endocrine cells (Li, 2003).
In total, transcripts from a surprisingly large fraction of the genome, >30%, changed significantly during the metamorphosis of the midgut (18 hr BPF to 12 hr APF). Broad classes of temporally separable gene expression patterns are evident. These classes include sets of transcripts that rapidly decrease coincident with onset of programmed cell death in the larval cells, sets that are induced during early or late metamorphosis, and sets of transcripts expressed at highest levels during the middle period of the time course when the larval cells are in the final stages of cell death and the adult cells are rearranging to form new tissue (Li, 2003).
Within these broad classes, specific sets of genes that have related functions and show parallel expression were identified, indicating that they make up gene batteries. Six such examples, included coregulated transcripts that encode proteins found in specific macromolecular complexes, biochemical pathways, organellar functions, and structural components of the cells that compose this tissue. Transcripts encoding proteasome components increase during the ecdysone pulse that triggers the onset of cell death in larval cells. Transcripts encoding glycolytic enzymes rapidly decrease during the initiation of metamorphosis, but gradually resume expression as the imaginal cells proliferate. Vacuolar ATPases shows a pattern similar to the glycolytic enzymes, whereas tubulin- and actin-encoding transcripts peak during the intense period of imaginal cell proliferation and migration as the adult midgut is formed. Transcripts encoding structural components of the peritrophic membrane of the mature larval gut gradually decrease during its replacement with adult tissue (Li, 2003).
The expression patterns were examined of regulatory genes known to be involved in the ecdysone transcriptional hierarchy predicted to control the gene batteries that were identified. Also examined was the expression of genes with known roles in programmed cell death or cell cycle control. The expression of known ecdysone-responsive regulatory genes was consistent with previous observations in midgut. Although the larval midgut is composed of cell types that undergo divergent responses to ecdysone -- apoptosis and cell proliferation -- it was nonetheless possible to detect significant changes in transcript levels from genes encoding proteins involved in both processes. The apoptosis activator gene ark was expressed at 4 hr BPF. E93 and reaper, which encode proteins that serve as critical control points in the commitment to programmed cell death, were expressed at PF, as was the initiator caspase dronc. These midgut expression profiles were compared to those reported for salivary glands at and after 10 hr APF, when a prepupal pulse of ecdysone triggers apoptosis in that tissue; almost the entire genetic cascade was found to be similarly activated in salivary glands and midgut albeit at two distinct periods of development. However, one notable difference was observed at the top level of the cascade. In the salivary gland, E93 is activated by βFTZ-F1, whereas in the midgut the βFTZ-F1 gene is not induced until 6-8 hr after E93 is induced. The regulation of E93 therefore does not depend on βFTZ-F1 in the midgut, but must rely on another as yet unidentified factor(s). During midgut metamorphosis, developmental modulation of transcript levels were also observed for genes encoding DNA polymerases, cyclins, CDCs, and other cell cycle regulators, as well as genes encoding DNA repair proteins such as Hus1, Rad23, and PCNA/Mus209 (Li, 2003).
Which of the genes that are differentially expressed at the onset of midgut metamorphosis require ecdysone signaling? Ecdysone-dependent transcriptional activity was removed using mutant Ecdysone Receptor (EcR) alleles, rescuing null EcR mutants to the third larval instar by using a heat shock-inducible EcR transgene. Gene expression was examined in mutant midguts that were isolated from mutant animals arrested at the end of the third larval stage (stage 2a mutants). 376 (76%) of the 495 genes that are significantly induced during the onset of midgut metamorphosis (18 hr BPF to 2 hr APF) required EcR function, whereas 296 (64%) of 460 transcripts that decline significantly in level during this time period require ecdysone signaling through EcR. Thus, a very large proportion of the genes that are developmentally regulated at the initiation of metamorphosis in this organ are under the control of the transcription factors that mediate the ecdysone signal. However, it does not appear that EcR function is a general requirement for transcription, because a significant fraction of differentially expressed genes are unaffected in EcR mutant tissue (Li, 2003).
Of the several different classes of genes expressed during midgut metamorphosis, the regulation of all genes in the proteasome, tubulin/actin, and lysozyme clusters requires EcR to exhibit their normal changes in developmental expression. However, many genes in the v-ATPase cluster and nearly half the genes in the peritrophin cluster did not require EcR. The downregulation of hexokinase A, 6-phosphofructokinase, and pyruvate kinase genes in the glycolysis pathway were affected in the EcR mutants, while many others in this pathway were not. Hexokinase A, 6-phosphofructokinase, and pyruvate kinase are rate-controlling enzymes in the glycolytic pathway, indicating that their ecdysone dependence is functionally significant. The expression of the numerous known ecdysone receptor target genes such as E75, E74, broad, E23, and DHR3 required EcR as expected. The induction dynamics for the E74 and DHR3 transcription factor genes was as expected, as was their dependence on EcR. In contrast to E74 and DHR3, DHR78 has previously been described to reside upstream of EcR at the top of the ecdysone regulatory hierarchy -- the expression of EcR is dependent on the wild-type function of DHR78. However, DHR78 can also be induced by ecdysone in organ culture. The results demonstrate that DHR78 wild-type induction is indeed dependent on EcR function. Taken together, these data indicate a positive feedback loop between EcR and DHR78 during the onset of metamorphosis in the midgut (Li, 2003).
Genes encoding factors involved in cell cycle and growth control, and in DNA repair, are also under the control of EcR. In spite of the role of ecdysone in stimulating cell proliferation during metamorphosis, no cell cycle genes have previously been linked to the ecdysone regulatory hierarchy. The induction of the cell cycle regulatory genes CyclinB, cdc2, and CyclinD were all observed to be dependent on EcR function. The rapid induction of cdc2 during the late third instar ecdysone pulse is similar to that observed for direct targets of EcR. The CyclinD gene is also induced at this time, but its maximal induction occurs several hours after that observed for cdc2. Cyclin D promotes cellular growth, whereas Cyclin B/Cdc2 controls G2/M transitions in proliferative cells. The dependence of these three genes on EcR function indicates that ecdysone may control cell proliferation, at least in part, through their regulation. Coordinate with the induction of CyclinB, cdc2, and CyclinD, the induction was observed of DNA polymerase-delta and DNA repair genes such as Rad23, and PCNA/mus209. The induction of these DNA repair and synthesis genes is also EcR dependent. The expression changes of these genes may be the result of the direct action of EcR, or due to the action of factors directly controlled by the ecdysone receptor complex. It is unlikely that the increase in expression of these genes is simply due to increased numbers of proliferative cells because the total number of divisions between 18 hr BPF and PF are few, and not all cell cycle or DNA repair genes showed an increase in expression at the initiation of metamorphosis. For example, the level of CyclinJ, which is known to be required during early embryonic division cycles, is actually reduced in expression from 18 hr BPF to PF. When the expression of cell death genes was examined in EcR mutant tissue, E93 induction was observed as well as induction of the Ark caspase activator and the dronc caspase gene required wild-type function of EcR (Li, 2003).
Factors in several well-studied signaling pathways are induced during midgut metamorphosis. These include Wnt (dishevelled, armadillo, and zeste white 3), TGFβ/BMP (sara, daughters against dpp, and glass bottom boat), EGFR (torpedo/egfr, rhomboid/veinlet, vein, and keren/spitz2), and Notch pathway genes (delta, kuzbanian, suppressor of hairless, E(spl)malpha, and E(spl)mβ). All of these pathways are used during embryonic midgut development, and these data indicate they are reused during midgut metamorphosis. Genes in the Hedgehog signaling pathway (hedgehog, smoothened, and cubitus interruptus) changed significantly as well (Li, 2003).
To determine whether any of the genes in these pathways are expressed as a consequence of ecdysone signaling, the EcR mutant expression data was examined for those genes that were induced during the late third instar ecdysone pulse. The induction of zeste white-3/shaggy, keren/spitz2, kuzbanian, and hedgehog are all dependent on the presence of functional EcR. The induction dynamics of the EGFR ligand gene keren/spitz2, the Notch proteolytic activation factor gene kuzbanian, and the shaggy/zeste white-3 kinase gene are similar to genes that are known direct targets of ecdysone signaling. The induction of hedgehog follows a secondary response pattern, as do genes from the E(spl) complex that are induced in response to Notch activation, although these induction kinetics are also consistent with these genes being partially activated directly by the ecdysone receptor and partially with other factors (i.e., they may be 'early-late' genes). These data show that the regulatory network controlled by ecdysone in midguts includes the activation of known components of the Wnt, EGFR, Hedgehog, and Notch pathways. Notably, ligand production for the EGF, Hedgehog, TGFβ/BMP, and Notch pathways is under control of ecdysone. The specific roles that each of these pathways plays during metamorphosis are currently unknown. These results nonetheless indicate new connections between ecdysone signaling and the activity of several other signaling pathways during the metamorphosis of this organ, either through direct targeting of the ecdysone receptor or through the actions of downstream factors (Li, 2003).
Bienz, M. (1997). Endoderm induction in Drosophila: the nuclear targets of the inducing signals. Curr. Opin. Genet. Dev. 7(5): 683-688
Li, T.-R. and White, K. P. (2003). Tissue-specific gene expression and Ecdysone-regulated genomic networks in Drosophila. Dev. Cell 5: 59-72. 12852852
Szüts, D., Eresh, S. and Bienz, M. (1998). Functional intertwining of Dpp and EGFR signaling during Drosophila endoderm induction. Genes Dev. 12: 2022-2035
Genes involved in tissue development
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