The present study described the full morphology of the duodenum of the Japanese quail during the embryonic stage from 3rd day of incubation till hatching using the light and electron (scanning and transmission) microscope. The specimens were collected, analyzed and described anatomically, morphometrically and microscopically. The first recognition of the prospective duodenum was at the 4th day of incubation and developed continuously by age progression. The prospective duodenum consisted of a flat pseudostratified epithelium, mesenchyme and covering mesothelium. On day 8th of incubation, the epithelium developed three evaginations lead to formation three previllous ridges protruding inside the duodenal lumen, which later at the 9th day differentiated into numbers of projections; villi. On the 9th day, the epithelium lined the villi transformed into a simple columnar type, the duodenal villi appeared as pyramidal-shaped projections, had wide base and narrow apex and by age progression, the duodenal villi went through changes in number, size and shape. On hatching day, the duodenal epithelium consisted of enterocytes interspersed with secretory goblet cells, which stained positive for both Periodic Acid Schiff (PAS) and Alcian blue AB and represented filled with metachromatic granules. The muscular wall started as mesenchymal condensation on the 6th day then differentiated into the circular smooth muscle layer on the 9th day of incubation. Giving detailed information about the morphological development of the duodenum during the incubation period of quail embryo helps in reaching a satisfactory explanation about how the duodenum plays a vital role in digestion, absorption and immunity. HIGHLIGHTS: Studying the quail duodenum can be used as a model for understanding the mammalian duodenum. Understanding the duodenal structure and its function is the best way to maximize the efficiency of the production of the livestock through giving the best type of diet. Duodenum plays a vital role in digestion through the digestible secretions, absorption of nutrients, and immunity against invaders. Duodenum is the spot where the food digestion mostly occurs.

Embryonic salivary gland mesenchyme (eSGM) secretes various growth factors (bioactives) that support the proper growth and differentiation of salivary gland epithelium. Therefore, eSGM cells can be used as feeder cells for in vitro-cultured artificial salivary gland if their survival and bioactivity are properly maintained. As eSGM is encapsulated in a hyaluronan (HA)-rich developmental milieu, we hypothesized that mimicking this environment in vitro via surface immobilization of HA might enhance survival and bioactivity of eSGM. In this study, various HA derivatives, conjugated with catechol (HA-CA), thiol (HA-SH), or amine (HA-EDA) moieties, respectively, were screened for their efficacy of culturing eSGM-derived feeder cells in vitro. Among these HA derivatives, HA-CA showed the highest surface coating efficiency and growth enhancement effect on the embryonic submandibular gland. In addition, the HA-CA coating enhanced the production of growth factors EGF and FGF7, but not FGF10. These effects were maintained when eSGM cells isolated from the embryonic salivary gland were re-seeded to develop the feeder layer cells. CD44s (a major HA receptor) in eSGM cells were clustered at the cell membrane, and enhanced EGF expression was detected only in CD44 cluster-positive cells, suggesting that membrane clustering of CD44 is the key mechanism for the increased expression of EGF.

UNASSIGNED: To investigate the role of the periotic mesenchyme (POM) in the development of sensory cells of developing auditory epithelium.
UNASSIGNED: Developing auditory epithelium with or without periotic mesenchyme was isolated from mice at embryonic days 11.5 (E11.5), E12.5 and E13.5, respectively, and cultured in vitro to an equivalent of E18.5\'s epithelium in vivo. Then, the explants were co-stained with antibodies targeting myosin VIIA, Sox2 and BrdU.
UNASSIGNED: More hair cells in E11.5 + 7 DIV, E12.5 + 6 DIV and E13.5 + 5 DIV auditory epithelia were found upon culture with POM (225.90 ± 62.44, 476.94 ± 100.81, and 1386.60 ± 202.38, respectively) compared with the non-POM group (68.17 ± 23.74, 205.00 ± 44.23, and 1266.80 ± 38.84, respectively). Moreover, regardless of developmental stage, the mesenchymal tissue increased the amount of cochlear sensory cells as well as the ratio of differentiated hair cells to total sensory cells.
UNASSIGNED: The periotic mesenchyme promotes the development of cochlear sensory cells, and its effect depends on the developmental stage of the auditory epithelium.

The tenacious prevalence of human pancreatic diseases such as diabetes mellitus and adenocarcinoma has prompted huge research interest in better understanding of pancreatic organogenesis. The plethora of signaling pathways involved in pancreas development is activated in a highly coordinated manner to assure unmitigated development and morphogenesis in vertebrates. Therefore, a complex mesenchymal-epithelial signaling network has been implicated to play a pivotal role in organogenesis through its interactions with other germ layers, specifically the endoderm. The Fibroblast Growth Factor Receptor FGFR2-IIIb splicing isoform (FGFR2b) and its high affinity ligand Fibroblast Growth Factor 10 (FGF10) are expressed in the epithelium and mesenchyme, respectively, and therefore are well positioned to transmit mesenchymal to epithelial signaling. FGF10 is a typical paracrine FGF and chiefly mediates biological responses by activating FGFR2b with heparin/heparan sulfate (HS) as cofactor. A substantial number of studies using genetically engineered mouse models have demonstrated an essential role of FGF10 in the development of many organs and tissues including the pancreas. During mouse embryonic development, FGF10 signaling is crucial for epithelial cell proliferation, maintenance of progenitor cell fate and branching morphogenesis in the pancreas. FGF10 is also implicated in pancreatic cancer, and that overexpression of FGFR2b is associated with metastatic invasion. A thorough understanding of FGF10 signaling machinery and its crosstalk with other pathways in development and pathological states may provide novel opportunities for pancreatic cancer targeted therapy and regenerative medicine.

Metamorphosis takes place within the life cycle of most marine invertebrates. The marine ascidian is a classical model to study complex cellular processes and underlying molecular mechanisms involved in its larval metamorphosis. The detailed molecular signaling pathways remain elusive, though extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinase (JNK) have been revealed to regulate cell migration, differentiation, and apoptosis in ascidian larval organ regression and juvenile organ development. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level. Large numbers of miRNAs have been demonstrated to be involved in many developmental and metamorphic processes. However, the identification of miRNAs in ascidian larval metamorphosis has not yet been investigated.
Totally, 106 known and 59 novel miRNAs were screened out through RNA-sequencing of three small RNA libraries from 18 to 21-h post-fertilization (hpf) tailbud embryos as well as from 42 hpf larvae (after tail regression) in Ciona savignyi. Expression profiling of miRNAs was confirmed by quantitative real-time PCR, showing that the expression levels of csa-miR-4040, csa-miR-4086, csa-miR-4055, csa-miR-4060, csa-miR-216a, csa-miR-216b, csa-miR-217, csa-miR-183, and csa-miR-92c were significantly higher in 42 hpf larvae, whereas those of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were higher in 18 and 21 hpf embryos; then, their expression in 42 hpf larvae became significantly low. For these 12 miRNAs, whose expression levels significantly changed, we predicted their target genes through the combination of miRanda and TargetScan. This prediction analysis revealed 332 miRNA-target gene pairs that were associated with the ERK, JNK, and transforming growth factor beta signaling pathways, suggesting that the identified miRNAs are involved in the regulation of C. savignyi larval metamorphosis via controlling the expression of their target genes. Furthermore, we validated the expression of five selected miRNAs by northern blotting. Among the selected miRNAs, the expression patterns of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were further examined by whole-mount in situ hybridization. The results showed that all three miRNAs were specifically expressed in a cell population resembling mesenchymal cells at the head and trunk part in swimming larvae but not in metamorphic larvae. Utilizing the luciferase assay, we also confirmed that miR-4000f targeted Mapk1, suggesting that the csa-miR-4018a/csa-miR-4018b/csa-miR-4000f cluster regulates larval metamorphosis through the Mapk1-mediated signaling pathway.
Totally, 165 miRNAs, including 59 novel ones, were identified from the embryos and larvae of C. savignyi. Twelve of them showed significant changes in expression before and during metamorphosis. In situ hybridization and northern blotting results revealed that three miRNAs are potentially involved in the signaling regulatory network for the migration and differentiation of mesenchymal cells in larval metamorphosis. Furthermore, the luciferase reporter assay revealed that Mapk1 is a target of csa-miR-4000f. Our results not only present a list and profile of miRNAs involved in Ciona metamorphosis but also provide informative cues to further understand their function in ascidian larval metamorphosis.

Organ development requires complex signaling by cells in different tissues. Epithelium and mesenchyme interactions are crucial for the development of skin, hair follicles, kidney, lungs, prostate, major glands, and teeth. Despite myriad literature on cell-cell interactions and ligand-receptor binding, the roles of extracellular vesicles in epithelium-mesenchyme interactions during organogenesis are poorly understood. Here, we discovered that ∼100 nm exosomes were secreted by the epithelium and mesenchyme of a developing tooth organ and diffused through the basement membrane. Exosomes were entocytosed by epithelium or mesenchyme cells with preference by reciprocal cells rather than self-uptake. Exosomes reciprocally evoked cell differentiation and matrix synthesis: epithelium exosomes induce mesenchyme cells to produce dentin sialoprotein and undergo mineralization, whereas mesenchyme exosomes induce epithelium cells to produce basement membrane components, ameloblastin and amelogenenin. Attenuated exosomal secretion by Rab27a/b knockdown or GW4869 disrupted the basement membrane and reduced enamel and dentin production in organ culture and reduced matrix synthesis and the size of the cervical loop, which harbors epithelium stem cells, in Rab27aash/ash mutant mice. We then profiled exosomal constituents including miRNAs and peptides and further crossed all epithelium exosomal miRNAs with literature-known miRNA Wnt regulators. Epithelium exosome-derived miR135a activated Wnt/β-catenin signaling and escalated mesenchymal production of dentin matrix proteins, partially reversible by Antago-miR135a attenuation. Our results suggest that exosomes may mediate epithelium-mesenchyme crosstalk in organ development, suggesting that these vesicles and/or the molecular contents they are transporting may be interventional targets for treatment of diseases or regeneration of tissues.

Mandibular patterning information initially resides in the epithelium during development. However, how transcriptional regulation of epithelium-derived signaling controls morphogenesis of the mandible remains elusive. Using ShhCre to target the mandibular epithelium, we ablated transcription factor Islet1, resulting in a distally truncated mandible via unbalanced cell apoptosis and decreased cell proliferation in the distal mesenchyme. Loss of Islet1 caused a lack of cartilage at the distal tip, leading the fusion of two growing mandibular elements surrounding the rostral process of Meckel\'s cartilage. Loss of Islet1 results in dysregulation of mesenchymal genes important for morphogenesis of the mandibular arch. We revealed that Islet1 is required for the activation of epithelial β-catenin signaling via repression of Wnt antagonists. Reactivation of β-catenin in the epithelium of the Islet1 mutant rescued mandibular morphogenesis through sonic hedgehog (SHH) signaling to the mesenchyme. Furthermore, overexpression of a transgenic hedgehog ligand in the epithelium also partially restored outgrowth of the mandible. These data reveal functional roles for an ISLET1-dependent network integrating β-catenin/SHH signals in mesenchymal cell survival and outgrowth of the mandible during development.

TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. We have previously shown that, in the absence of TWIST1, cells within the cranial mesoderm adopt an abnormal epithelial configuration via a process reminiscent of a mesenchymal to epithelial transition (MET). Here, we show by gene expression analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. ChIP-seq was used to identify TWIST1-binding sites in an in vitro model of a TWIST1-dependent mesenchymal cell state, and the data were combined with the transcriptome data to identify potential target genes. Three direct transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated by ChIP-PCR using mouse embryonic tissues and by luciferase assays. Our findings reveal that the mesenchymal properties of the cranial mesoderm are likely to be regulated by a network of TWIST1 targets that influences the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.