Midgut development in rat embryos using microcomputed tomography

The development of the mammalian gut was first described more than a century ago. Since then, it has been believed that a series of highly orchestrated developmental processes occur before the intestine achieves its final formation. The key steps include the formation of the umbilicus, the so-called “physiological herniation” of the midgut into the umbilical cord, an intestinal “rotation”, and the “return of the gut” into the abdominal cavity. However, this sequence of events is predominantly based on histological sections of dissected embryos, a 2D technique with methodological limitations. For a better understanding of spatial relationships in the embryo, we utilized microcomputed tomography (µCT), a nondestructive 3D imaging method. Here, we show the detailed processes and mechanisms of intestinal development in rat embryos, including the development of the umbilicus, the formation of loops inside the umbilical coelom, and the subsequent shift of these loops into the abdominal cavity. Our 3D datasets of developing intestines will substantially advance the understanding of normal mammalian midgut embryology and offer new possibilities to reveal unknown mechanisms in the pathogenesis of congenital disorders.

Systematic identification of circular RNAs and corresponding regulatory networks unveil their potential roles in the midgut ofApis cerana ceranaworkers

BackgroundCircular RNAs (circRNAs) are newly discovered noncoding RNAs (ncRNAs) that play key roles in various biological functions, such as the regulation of gene expression and alternative splicing. CircRNAs have been identified in some species, including western honeybees. However, the understanding of honeybee circRNA is still very limited, and to date, no study on eastern honeybee circRNA has been conducted. Here, the circRNAs in the midguts ofApis cerana ceranaworkers were identified and validated, and the regulatory networks were constructed. Differentially expressed circRNAs (DEcircRNAs) and the corresponding competitively endogenous RNA (ceRNA) networks in the development of the worker’s midgut were further investigated.ResultsHere, 7- and 10-day-oldA. c. ceranaworkers’ midguts (Ac1 and Ac2) were sequenced using RNA-seq, and a total of 9589 circRNAs were predicted using bioinformatics. These circRNAs were approximately 201-800 nt in length and could be classified into six types; the annotated exonic circRNAs were the most abundant. Additionally, five novelA. c. ceranacircRNAs were confirmed by PCR amplification and Sanger sequencing, indicating the authenticity ofA. c. ceranacircRNAs. Interestingly, novel_circ_003723, novel_circ_002714, novel_circ_002451 and novel_circ_001980 were the most highly expressed circRNAs in both Ac1 and Ac2, which is indicative of their key roles in the development of the midgut. Moreover, 55 DEcircRNAs were identified in the Ac1 vs Ac2 comparison group, including 34 upregulated and 21 downregulated circRNAs. Further investigation showed that the source genes of circRNAs were classified into 34 GO terms and were involved in 141 KEGG pathways. In addition, the source genes of DEcircRNAs were categorized into 10 GO terms and 15 KEGG pathways, which demonstrated that the corresponding DEcircRNAs may affect the growth, development, and material and energy metabolisms of the worker’s midgut by regulating the expression of the related source genes. Additionally, the circRNA-miRNA regulatory networks were constructed and analyzed, and the results demonstrated that 1060 circRNAs can bind to 74 miRNAs and that 71.51% of circRNAs can be linked to only one miRNA. Furthermore, the DEcircRNA-miRNA-mRNA networks were constructed and explored, and the results indicate that the 13 downregulated circRNAs can bind to eight miRNAs and to 29 target genes. In addition, the results indicate that the 16 upregulated circRNAs can bind to 9 miRNAs and to 29 target genes, demonstrating that DEcircRNAs are likely involved in the regulation of midgut development via ceRNA mechanisms. Moreover, the regulatory networks of miR-6001-y-targeted DEcircRNAs were analyzed, and the results showed that eight DEcircRNAs may affect the development ofA. c. ceranaworkers’ midguts by targeting miR-6001-y. Finally, four randomly selected DEcircRNAs were verified via RT-qPCR, confirming the reliability of our sequencing data.ConclusionThis is the first systematic investigation of circRNAs and their corresponding regulatory networks in eastern honeybees. The identified circRNAs from theA. c. ceranaworker’s midgut will enrich the known reservoir of honeybee ncRNAs. DEcircRNAs may play a comprehensive role during the development of the worker’s midgut via the regulation of source genes and the interaction with miRNAs by acting as ceRNAs. The eight DEcircRNAs that targeted miR-6001-y were likely to be vital for the development of the worker’s midgut. Our results provide a valuable resource for the future studies ofA. c. ceranacircRNA and lay a foundation to reveal the molecular mechanisms underlying the regulatory networks of circRNAs responsible for the worker’s midgut development; in addition, these findings facilitate a functional study on the key circRNAs involved in the developmental process.Graphical Abstract

Mothers against dpp encodes a conserved cytoplasmic protein required in DPP/TGF-beta responsive cells

The proteins necessary for signal transduction in cells responding to ligands of the TGF-beta family are largely unknown. We have previously identified Mad (Mothers against dpp), a gene that interacts with the TGF-beta family member encoded by decapentaplegic (dpp) in Drosophila. Assay of Mad's role in the DPP-dependent events of embryonic midgut development demonstrates that Mad is required for any response of the visceral mesoderm or endoderm to DPP signals from the visceral mesoderm. Replacement of the normal DPP promoter with a heterologous (hsp70) promoter fails to restore DPP-dependent responses in Mad mutant midguts. Experiments utilizing Mad transgenes regulated by tissue-specific promoters show that MAD is required specifically in cells responding to DPP. Immunohistochemical studies localize MAD to the cytoplasm in all tissues examined. Experiments in Xenopus embryos demonstrate that Drosophila MAD can function in the signaling pathway of BMP-4, a vertebrate homolog of dpp. Based on these results, we propose that Mad is a highly conserved and essential element of the DPP signal transduction pathway.

Genomic regions required for morphogenesis of the Drosophila embryonic midgut.

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.

Role of the teashirt gene in Drosophila midgut morphogenesis: secreted proteins mediate the action of homeotic genes

Homeotic genes control the development of embryonic structure by coordinating the activities of downstream ‘target’ genes. The identities and functions of target genes must be understood in order to learn how homeotic genes control morphogenesis. Drosophila midgut development is regulated by homeotic genes expressed in the visceral mesoderm, where two of their target genes have been identified. Both encode secreted proteins. The Ultrabithorax (Ubx) homeotic gene activates transcription of the decapentaplegic (dpp) gene, which encodes a TGF beta class protein, while in adjacent mesoderm cells the abdominal-A (abd-A) homeotic gene activates transcription of the wingless (wg) gene, which encodes a Wnt class protein. The homeotic genes Antennapedia (Antp) and Sex combs reduced (Scr) act in more anterior midgut regions. Here we report the identification of another homeotic gene target in the midgut mesoderm, the teashirt (tsh) gene, which encodes a protein with zinc finger motifs. tsh is necessary for proper formation of anterior and central midgut structures. Antp activates tsh in anterior midgut mesoderm. In the central midgut mesoderm Ubx, abd-A, dpp, and wg are required for proper tsh expression. The control of tsh by Ubx and abd-A, and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals as well as localized transcription regulators, the tsh transcription factor is produced in a spatial pattern distinct from any of the homeotic genes.

A role for the mesoderm in endodermal migration and morphogenesis in Drosophila

The endodermal midgut arises from two primordia, the anterior midgut (AMG) primordium and the posterior midgut (PMG) primordium, which are separated by almost the entire length of the Drosophila embryo. To form the midgut, these two parts have to extend towards each other and to fuse laterally on both sides of the yolk. Shortly before and during that movement, AMG and PMG are arranged as mesenchymal cell masses, but later the midgut cells form an epithelium. We show that these two aspects of midgut development, migration of AMG and PMG and transition to an epithelium, depend on the mesoderm. The extension of the midgut primordia is achieved by cell migration along the visceral mesoderm which forms a continuous layer of cells within the germ band. In mutant embryos lacking the entire mesoderm or failing to differentiate the visceral mesoderm, AMG and PMG are formed but do not migrate properly. In addition, they fail to form an epithelium and instead either remain as compact cell masses anterior and posterior to the yolk (in twist and snail mutant embryos) or only occasionally wrap around the yolk before embryogenesis is completed (in tinman-deficient embryos). We conclude that the visceral mesoderm serves as a substratum for the migrating endodermal cells and that the contact between visceral mesoderm and endoderm is required for the latter to become an epithelium.