Midgut Morphogenesis Research Articles (Page 1)

Herniation, rotation, looping, and retraction of the midgut occur sequentially during midgut morphogenesis. Recent studies have demonstrated the importance of mechanical forces arising from the differential growth between the midgut and mesentery in the formation of small intestinal loops. However, the roles of mechanics and differential growth in the overall process remain unclear. In this study, we developed a computational model of midgut morphogenesis based on continuum mechanics. We showed that the protrusion, rotation, and retraction of the midgut can emerge sequentially because of temporal changes in differential growth. The midgut was modeled as a hyperelastic tube with a Gaussian shape. The differential growth of the midgut and mesentery was modeled by the spatial variation in spontaneous plastic deformation. The hyperelastic tube developed a protrusion by compression-induced deformation, suggesting that other external forces are not necessary for midgut herniation prior to rotation. Appropriate differential growth induced a rotation of the tube. A less-growing mesentery attempts to face inward to minimize the tensile forces, which causes tube twisting and results in midgut rotation. Excess differential growth may cause the retraction of the midgut before the formation of small intestinal loops. The results of this study will serve as reference in future studies on embryology and tissue engineering.

Disruption of morphogenesis, an essential process in organismal development, can lead to disruption of biological processes, reduction in fitness, or even death of an organism. The roles of lethal giant larvae (Lgl) protein in maintaining tissue organization have been studied extensively in mammals, but little is known about this gene's roles in promoting correct tissue morphogenesis in insects. In this study, we identified an Lgl ortholog in Locusta migratoria. RT-qPCR results revealed that LmLgl was constitutively expressed during third, fourth, and fifth instar nymphs. Furthermore, LmLgl showed highest expression in the ovary followed by wing pads, midgut, hindgut, Malpighian tubules, and foregut of the third-instar nymphs. To examine the role of LmLgl in L. migratoria development, RNA interference was performed during nymphal stages. Silencing of LmLgl increased body size but decreased bodyweight by 9.0%. Histological sections of the midgut revealed abnormal large masses of disordered epithelial cells in dsLmLgl-injected nymphs. In addition, downregulation of LmLgl transcript levels significantly altered the morphological structure in midgut, resulting in the formation of tumor-like structures. Our results indicated that LmLgl may act as a tumor-suppressor gene, which plays an essential role in maintaining a normal morphological structure in the midgut of L. migratoria. Our results also suggest that LmLgl may be explored as a potential target for developing dsRNA-based biological pesticides for managing insect pests.

ABSTRACTDevelopment of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.

Mesenchymal-to-epithelial transition (MET) converts cells from migratory mesenchymal to polarized epithelial states. Despite its importance for both normal and pathological processes, very little is known about the regulation of MET in vivo. Here we exploit midgut morphogenesis in Drosophila melanogaster to investigate the mechanisms underlying MET. We show that down-regulation of the EMT transcription factor Serpent is required for MET, but not sufficient, as interactions with the surrounding mesoderm are also essential. We find that midgut MET relies on the secretion of specific laminins via the CopII secretory pathway from both mesoderm and midgut cells. We show that secretion of the laminin trimer containing the Wingblister α-subunit from the mesoderm is an upstream cue for midgut MET, leading to basal polarization of αPS1 integrin in midgut cells. Polarized αPS1 is required for the formation of a monolayered columnar epithelium and for the apical polarization of αPS3, Baz, and E-Cad. Secretion of a distinct LamininA-containing trimer from midgut cells is required to reinforce the localization of αPS1 basally, and αPS3 apically, for robust repolarization. Our data suggest that targeting these MET pathways, in conjunction with therapies preventing EMT, may present a two-pronged strategy toward blocking metastasis in cancer.

In a large-scale RNAi screen in Tribolium castaneum for genes with knock-down phenotypes in the larval somatic musculature, one recurring phenotype was the appearance of larval muscle fibers that were significantly thinner than those in control animals. Several of the genes producing this knock-down phenotype corresponded to orthologs of Drosophila genes that are known to participate in myoblast fusion, particularly via their effects on actin polymerization. A new gene previously not implicated in myoblast fusion but displaying a similar thin-muscle knock-down phenotype was the Tribolium ortholog of Nostrin, which encodes an F-BAR and SH3 domain protein. Our genetic studies of Nostrin and Cip4, a gene encoding a structurally related protein, in Drosophila show that the encoded F-BAR proteins jointly contribute to efficient myoblast fusion during larval muscle development. Together with the F-Bar protein Syndapin they are also required for normal embryonic midgut morphogenesis. In addition, Cip4 is required together with Nostrin during the profound remodeling of the midgut visceral musculature during metamorphosis. We propose that these F-Bar proteins help govern proper morphogenesis particularly of the longitudinal midgut muscles during metamorphosis.

The gastrointestinal tract is an essential system of organs required for nutrient absorption. As a simple tube early in development, the primitive gut is patterned along its anterior-posterior axis into discrete compartments with unique morphologies relevant to their functions in the digestive process. These morphologies are acquired gradually through development as the gut is patterned by tissue interactions, both molecular and mechanical in nature, involving all three germ layers. With a focus on midgut morphogenesis, we review work in the chick embryo demonstrating how these molecular signals and mechanical forces sculpt the developing gut tube into its mature form. In particular, we highlight two mechanisms by which the midgut increases its absorptive surface area: looping and villification. Additionally, we review the differentiation and patterning of the intestinal mesoderm into the layers of smooth muscle that mechanically drive peristalsis and the villification process itself. Where relevant, we discuss the mechanisms of chick midgut morphogenesis in the context of experimental data from other model systems.

Embryo formation requires tight regulation and coordination of adhesion in multiple cell types. By undertaking imaging, three-dimensional (3D) reconstructions and genetic analysis during posterior midgut morphogenesis in Drosophila, we find a new requirement for the conserved fibroblast growth factor (FGF) signaling pathway in the maintenance of epithelial cell adhesion through FGF modulation of zygotic E-cadherin. During Drosophila gastrulation, primordial germ cells (PGCs) are transported with the posterior midgut while it undergoes dynamic cell shape changes. In embryos mutant for the FGF signaling pathway components Branchless and Breathless, zygotic E-cadherin is not targeted to adherens junctions, causing midgut pocket collapse, which impacts on PGC movement. We find that the ventral midline also requires FGF signaling to maintain cell-cell adhesion. We show that FGF signaling regulates the distribution of zygotic E-cadherin during early embryonic development to maintain cell-cell adhesion in the posterior midgut and the ventral midline, a role that is likely crucial in other tissues undergoing active cell shape changes with higher adhesive needs.

Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis

Tissue-autonomous EcR functions are required for concurrent organ morphogenesis in the Drosophila embryo

In Drosophila, trunk visceral mesoderm, a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. We now extend these findings by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts feedback effect on trachea, ectodermal derivatives.

The subdivision of the lateral mesoderm into a visceral (splanchnic) and a somatic layer is a crucial event during early mesoderm development in both arthropod and vertebrate embryos. In Drosophila, this subdivision leads to the differential development of gut musculature versus body wall musculature. Here we report that biniou, the sole Drosophila representative of the FoxF subfamily of forkhead domain genes, has a key role in the development of the visceral mesoderm and the derived gut musculature. biniou expression is activated in the trunk visceral mesoderm primordia downstream of dpp, tinman, and bagpipe and is maintained in all types of developing gut muscles. We show that biniou activity is essential for maintaining the distinction between splanchnic and somatic mesoderm and for differentiation of the splanchnic mesoderm into midgut musculature. biniou is required not only for the activation of differentiation genes that are expressed ubiquitously in the trunk visceral mesoderm but also for the expression of dpp in parasegment 7, which governs proper midgut morphogenesis. Activation of dpp is mediated by specific Biniou binding sites in a dpp enhancer element, which suggests that Biniou serves as a tissue-specific cofactor of homeotic gene products in visceral mesoderm patterning. Based upon these and other data, we propose that the splanchnic mesoderm layers in Drosophila and vertebrate embryos are homologous structures whose development into gut musculature and other visceral organs is critically dependent on FoxF genes.

People afflicted with certain rheumatological auto-immune diseases produce autoantibodies directed against a select group of proteins such as the La auto-antigen. Biochemical studies have revealed La to be a promiscuous RNA-binding protein that appears to play a role in a variety of intracellular activities such as processing and/or transport of RNA polymerase III precursor transcripts and translational regulation from internal ribosome entry sites (IRES). We have previously identified an RNA-binding protein that is a Drosophila melanogaster homolog of La (D-La) and shown that early transcript accumulation throughout the embryo is later refined to be most prevalent in the visceral mesoderm, gut, gonads and salivary glands. Here we report the first in vivo genetic characterization of a La homolog in a multicellular eukaryote. Lethality was observed in homozygous larvae harboring a small chromosomal deletion that removed the D-La gene, which was rescued by an inducible D-La cDNA transgene. This implies that D-La confers essential functions for larval development. In addition, loss of D-La function gives rise to defects in embryonic midgut morphogenesis; one of the midgut defects correlates with loss of Ultrabithorax ( Ubx ) expression along the second midgut constriction. Finally, genetic interactions between chromosomal deficiencies that remove D-La and certain Ubx alleles were demonstrated in adults. Our results support the hypothesis that D-La provides essential functions for proper Drosophila development and imply that the conserved La family of proteins may perform critical developmental functions in higher eukaryotes.

The Hox gene family of transcription factors constitutes candidate regulators in the molecular cascade of events that governs establishment of normal terminal differentiation along the duodenum to colon axis. One member of this family, Hoxa5, displays a dynamic pattern of expression during gut development. Hoxa5 transcripts are present in midgut mesenchyme at the time of remodeling, supporting a role for this gene in digestive tract specification. To study the role of Hoxa5 in proper intestinal development and maturation, we examined whether Hoxa5 mutant mice exhibit any defect in this process. We report here that even though Hoxa5 is not required for midgut morphogenesis, its loss of function perturbs the acquisition of adult mode of digestion, which normally is temporally coordinated with the process of spontaneous weaning. Impaired maturation of the digestive tract might be related to altered specification of intestinal epithelial cells. Our findings provide evidence that Hoxa5 expression in the gut mesoderm is important for the region-specific differentiation of the adjacent endoderm.

We have isolated mutations in the Drosophila melanogaster gene glass bottom boat (gbb), which encodes a TGF-beta signaling molecule (formerly referred to as 60A) with highest sequence similarity to members of the bone morphogenetic protein (BMP) subgroup including vertebrate BMPs 5-8. Genetic analysis of both null and hypomorphic gbb alleles indicates that the gene is required in many developmental processes, including embryonic midgut morphogenesis, patterning of the larval cuticle, fat body morphology, and development and patterning of the imaginal discs. In the embryonic midgut, we show that gbb is required for the formation of the anterior constriction and for maintenance of the homeotic gene Antennapedia in the visceral mesoderm. In addition, we show a requirement for gbb in the anterior and posterior cells of the underlying endoderm and in the formation and extension of the gastric caecae. gbb is required in all the imaginal discs for proper disc growth and for specification of veins in the wing and of macrochaete in the notum. Significantly, some of these tissues have been shown to also require the Drosophila BMP2/4 homolog decapentaplegic (dpp), while others do not. These results indicate that signaling by both gbb and dpp may contribute to the development of some tissues, while in others, gbb may signal independently of dpp.

decapentaplegic (dpp) is a Transforming Growth Factor beta (TGF-beta)-related growth factor that controls multiple developmental processes in Drosophila. To identify components involved in dpp signaling, we carried out a genetic screen for dominant enhancer mutations of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. We recovered new alleles of tkv, punt, Mothers against dpp (Mad) and Medea (Med), all of which are known to mediate dpp signaling. We also recovered mutations in the 60A gene which encodes another TGF-beta-related factor in Drosophila. DNA sequence analysis established that all three 60A alleles were nonsense mutations in the prodomain of the 60A polypeptide. These mutations in 60A caused defects in midgut morphogenesis and fat body differentiation. We present evidence that when dpp signaling is compromised, lowering the level of 60A impairs several dpp-dependent developmental processes examined, including the patterning of the visceral mesoderm, the embryonic ectoderm and the imaginal discs. These results provide the first in vivo evidence for the involvement of 60A in the dpp pathway. We propose that 60A activity is required to maintain optimal signaling capacity of the dpp pathway, possibly by forming biologically active heterodimers with Dpp proteins.

We tested the ability of different integrin alpha subunits to substitute for each other during embryonic development. Two alpha subunits, which form heterodimers with the same betaPS subunit, are expressed in complementary tissues in the Drosophila embryo, with alphaPS1 expressed in the epidermis and endoderm, and alphaPS2 expressed in the mesoderm. As a result the two integrin heterodimers are present on opposite surfaces at sites of interaction between the mesoderm and the other cell layers where they are required for normal development. Using the GAL4 system, we are able to rescue fully the embryonic lethality of an alphaPS2 null mutation with a UAS-alphaPS2 transgene, but only partially with a UAS-alphaPS1 gene, due to partial rescue of both muscle and midgut phenotypes. Similarly we are able to rescue the embryonic/first instar larval lethality of an alphaPS1 null mutation gene with UAS-alphaPS1, but only partially with UAS-alphaPS2. Each UAS-alpha gene, when it contains the cytoplasmic domain from the other alpha subunit, maintains an equivalent ability to rescue its own mutation and cannot fully rescue a mutation in the other alpha. We conclude that the two alpha subunits are not equivalent and have distinct functions which reside in the extracellular domains.

Genetic Analysis of theDrosophilaβ3-Tubulin Gene Demonstrates That the Microtubule Cytoskeleton in the Cells of the Visceral Mesoderm Is Required for Morphogenesis of the Midgut Endoderm

The Drosophila midgut is an excellent system for studying the cell migration, cell-cell communication, and morphogenetic events that occur in organ formation. Genes representative of regulatory gene families common to all animals, including homeotic, TGF beta, and Wnt genes, play roles in midgut development. To find additional regulators of midgut morphogenesis, we screened a set of genomic deficiencies for midgut phenotypes. Fifteen genomic intervals necessary for proper midgut morphogenesis were identified, three contain genes already known to act in the midgut. Three other genomic regions are required for formation of the endoderm or visceral mesoderm components of the midgut. Nine regions are required for proper formation of the midgut constrictions. The E75 ecdysone-induced gene, which encodes a nuclear receptor superfamily member, is the relevant gene in one region and is essential for proper formation of midgut constrictions. E75 acts downstream of the previously known constriction regulators or in parallel. Temporal hormonal control may therefore work in conjunction with spatial regulation by the homeotic genes in midgut development. Another genomic region is required to activate transcription of the homeotic genes Antp and Scr specifically in visceral mesoderm. The genomic regions identified by this screen provide a map to novel midgut development regulators.

The products of the homeotic genes in Drosophila are transcription factors that are necessary to impose regional identity along the anterior-posterior axis of the developing embryo. However, the target genes under homeotic regulation that control this developmental process are largely unknown. We have utilized an immunopurification method to clone target genes of the Antennapedia protein (ANTP). We present here the characterization of centrosomin (cnn), one of the target genes isolated using this approach. The spatial and temporal expression of the cnn gene in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS) of wild-type and homeotic mutant embryos is consistent with the idea that cnn is a homeotic target. In the VM, Antp and abdominal-A (abd-A) negatively regulate cnn, while Ultrabithorax (Ubx) shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Characterization of a cDNA encoding CNN predicts a novel structural protein with three leucine zipper motifs and several coiled-coil domains exhibiting limited homology to the rod portion of myosin. Immunocytochemical results demonstrate that the cnn encoded protein is localized to the centrosome and the accumulation pattern is coupled to the nuclear and centrosome duplication cycles of cleavage. In addition, evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut construction. The centrosomal localization of CNN and the involvement of microtubules in midgut morphogenesis suggest that this protein may participate in mitotic spindle assembly and the mechanics of morphogenesis through an interaction with microtubules, either directly or indirectly.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a growth factor that belongs to the transforming growth factor-beta (TGF-beta) superfamily and that plays a central role in multiple cell-cell signaling events throughout development. Through genetic screens we are seeking to identify other functions that act upstream, downstream or in concert with dpp to mediate its signaling role. We report here the genetic characterization and cloning of Mothers against dpp (Mad), a gene identified in two such screens. Mad loss-of-function mutations interact with dpp alleles to enhance embryonic dorsal-ventral patterning defects, as well as adult appendage defects, suggesting a role for Mad in mediating some aspect of dpp function. In support of this, homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk development and embryonic dorsal-ventral patterning that are very reminiscent of dpp mutant phenotypes. We cloned the Mad region and identified the Mad transcription unit through germline transformation rescue. We sequenced a Mad cDNA and identified three Mad point mutations that alter the coding information. The predicted MAD polypeptide lacks known protein motifs, but has strong sequence similarity to three polypeptides predicted from genomic sequence from the nematode Caenorhabditis elegans. Hence, MAD is a member of a novel, highly conserved protein family.