Persistent expression of Hlxb9 in the pancreatic epithelium impairs pancreatic development.

The homebox gene Hlxb9, encoding Hb9, exhibits a dual expression profile during pancreatic development. The early expression in the dorsal and ventral pancreatic epithelium is transient and spans from embryonic day (e) 8 to e9-e10, whereas the later expression is confined to differentiating beta-cells as they appear. We previously showed that Hlxb9 is critically required for the initiation of the dorsal, but not the ventral, pancreatic program. Here, we demonstrate the requirement for a stringent temporal regulation of Hlxb9 expression during early stages of pancreatic development. In transgenic mice, where Hlxb9 expression, under control of the Ipf1/Pdx1 promoter, was extended beyond e9-e10, the development of the pancreas was drastically perturbed. Morphological analyses showed that the growth and morphogenesis of the pancreatic epithelium was impaired. Moreover, differentiation of pancreatic endocrine and exocrine cells was diminished and instead the pancreatic epithelium with its adjacent mesenchyme adopted an intestinal-like differentiation program. Together, these data point to a need for a tight temporal regulation of Hlxb9 expression. Thus, a total loss of Hlxb9 expression results in a block of the initiation of the dorsal pancreatic program, while a temporally extended expression of Hlxb9 results in a complete impairment of pancreatic development.     

The distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of Viviparus ater (Mollusca, Gastropoda)

The timing and spatial distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of the mollusc Viviparus ater are examined using immunohistochemistry. FMRFamide-like molecules emerge in the early stage E8 (8% of embryonic development) before the 5-HT immunoreactivity, and they are not only found during nervous system ontogeny. As the parts of the digestive tract differentiated, the pattern of the diffuse gut endocrine cells, present in adults, start to be established (E20-E30), and both open and closed cell types are immunoreactive to anti-FMRFamide antibody. From their appearance (E20), cells with a 5-HT-like phenotype are distributed in the central nervous ganglia and progressively assembled during embryonic development. The early occurrence of both these molecules in V. ater embryos reinforces the growing view that neurotransmitters play a regulatory role in embryogenic processes. In particular, the very early presence of FMRFamide-related factors suggests an involvement of these molecules in the regulation of basic, not only neuronal, cell behaviours, while 5-HT seems to be a more specific neural development signal.     

Developmental biology of the Psammomys obesus pancreas: cloning and expression of the Neurogenin-3 gene.

The desert gerbil Psammomys obesus, an established model of type 2 diabetes (T2D), has previously been shown to lack pancreatic and duodenal homeobox gene 1 (Pdx-1) expression. Pdx-1 deficiency leads to pancreas agenesis in both mice and humans. We have therefore further examined the pancreas of P. obesus during embryonic development. Using Pdx-1 antisera raised against evolutionary conserved epitopes, we failed to detect Pdx-1 immunoreactivity at any time points. However, at E14.5, Nkx6.1 immunoreactivity marks the nuclei of all epithelial cells of the ventral and dorsal pancreatic buds and the only endocrine cell types found at this time point are glucagon and PYY. At E18.5 the pancreas is well branched and both glucagon- and ghrelin-positive cells are scattered or found in clusters, whereas insulin-positive cells are not found. At E22.5, the acini of the exocrine pancreas are starting to mature, and amylase and carboxypeptidase A immunoreactivity is found scattered and not in all acini. Ghrelin-, glucagon-, PYY-, gastrin-, somatostatin (SS)-, pancreatic polypeptide (PP)-, and insulin-immunoreactive cells are found scattered or in small groups within or lining the developing ductal epithelium as marked by cytokeratin 19. Using degenerate PCR, the P. obesus Neurogenin-3 (Ngn-3) gene was cloned. Nucleotide and amino acid sequences show high homology with known Ngn-3 sequences. Using specific antiserum, we can observe that Ngn-3-immunoreactive cells are rare at E14.5 but readily detectable at E18.5 and E22.5. In conclusion, despite the lack of detection of Pdx-1, the P. obesus pancreas develops similarly to Muridae species, and the Ngn-3 sequence and expression pattern is highly conserved in P. obesus.     

Cdc25 and the importance of G2 control

While cell proliferation is an essential part of embryonic development, cells within an embryo cannot proliferate freely. Instead, they must balance proliferation and other cellular events such as differentiation and morphogenesis throughout embryonic growth. Although the G₁ phase has been a major focus of study in cell cycle control, it is becoming increasingly clear that G₂ regulation also plays an essential role during embryonic development. Here we discuss the role of Cdc25, a key regulator of mitotic entry, with a focus on several recent examples that show how the precise control of Cdc25 activity and the G₂/M transition are critical for different aspects of embryogenesis. We finish by discussing a promising technology that allows easy visualization of embryonic and adult cells potentially regulated at mitotic entry, permitting the rapid identification of other instances where the exit from G₂ plays an essential role in development and tissue homeostasis.     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem- or progenitor- cells for islet transplantation.     

Dynamics of embryonic pancreas development using real-time imaging

Current knowledge about developmental processes in complex organisms has relied almost exclusively on analyses of fixed specimens. However, organ growth is highly dynamic, and visualization of such dynamic processes, e.g., real-time tracking of cell movement and tissue morphogenesis, is becoming increasingly important. Here, we use live imaging to investigate expansion of the embryonic pancreatic epithelium in mouse. Using time-lapse imaging of tissue explants in culture, fluorescently labeled pancreatic epithelium was found to undergo significant expansion accompanied by branching. Quantification of the real-time imaging data revealed lateral branching as the predominant mode of morphogenesis during epithelial expansion. Live imaging also allowed documentation of dynamic beta-cell formation and migration. During in vitro growth, appearance of newly formed beta-cells was visualized using pancreatic explants from MIP-GFP transgenic animals. Migration and clustering of beta-cells were recorded for the first time using live imaging. Total beta-cell mass and concordant aggregation increased during the time of imaging, demonstrating that cells were clustering to form "pre-islets". Finally, inhibition of Hedgehog signaling in explant cultures led to a dramatic increase in total beta-cell mass, demonstrating application of the system in investigating roles of critical embryonic signaling pathways in pancreas development including beta-cell expansion. Thus, pancreas growth in vitro can be documented by live imaging, allowing visualization of the developing pancreas in real-time.     

Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation

The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic β-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically only to those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.     

Early embryonic mouse mandibular morphogenesis and cytodifferentiation in serumless, chemically defined medium: a model for studies of autocrine and/or paracrine regulatory factors

During craniofacial and mandibular development at least three interdependent processes become integrated: 1) regulation of time-dependent differential gene expression; 2) positional information resulting in pattern formations; and 3) morphogenesis. The present studies were designed to test the hypothesis that intrinsic and/or paracrine factors regulate the developmental program for embryonic mouse mandibular morphogenesis, histogenesis, and cytodifferentiation. Either E11 or E12 C57B110 (B10.A) strain mouse mandibular processes were cultured in serumless, chemically defined medium for periods up to 9 days in vitro. At selected stages of development 3H-thymidine incorporation into DNA was used to evaluate the mitotic labeling for selected tissue compartments. Macroscopic observations demonstrated that morphogenesis (shape/form) in vitro was comparable to that for in vivo controls. Histological results demonstrated that chondrogenesis, osteogenesis, tooth formation, tongue formation, lip formation, and epithelial differentiation with keratinization were expressed according to sequence, time, and positions comparable to those observed in controls. This experimental approach provided datasets to support the hypothesis that exogenous long-range factors are not required for embryonic mouse mandibular morphogenesis and further suggested that autocrine and/or paracrine factors mediate the timing and position of mandibular development.     

Immunocytochemical detection of neuronal nitric oxide synthase (nNOS)-IR in embryonic rat stomach between days 13 and 21 of gestation.

In this study, the localization and appearance of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry techniques and light microscopy in embryonic rat stomach. The stomachs of Wistar rat embryos aged 13-21 days were used. The first nerve cells containing nNOS-IR were seen on embryonic Day 14. The occurrence of mesenchymal cell condensation near nNOS-IR neuroblasts on embryonic Day 15 may reflect an active nerve element-specific mesenchymal cell induction causing the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after nNOS-IR neuroblasts, on embryonic Day 18, suggests that the epithelial differentiation may depend on inputs coming from nNOS-IR neuroblasts, as well as other factors. Observation of nNOS-IR nerve fibers on embryonic Day 21 demonstrates that at this stage they contribute to nonadrenergic noncholinergic relaxation. In conclusion, depending on this study's results, it can be said that cells and tissues might be affected by NO secreted by nNOS-IR nerve cells during the development and differentiation of embryonic rat stomach.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Transient Calretinin Expression during Intervertebral Disc Formation of the Chick Embryo

Calretinin immunoreactivity was localized during chick embryonic cervical spine development from day 4 until day 16. A transitory expression of calretinin could be seen from embryonic day 5 to embryonic day 15 in the mesenchymal cells forming the intervertebral disc. Calretinin was most abundant at embryonic day 8 when a maximal proliferation of cells occurred.
At embryonic day 12, calretinin positive fibroblasts were located along the fibers forming the annulus fibrosus. At embryonic day 16, calretinin immunoreactivity could no more be detected in the cervical column. In conclusion, an intracellular calcium binding proteins belonging to the calmodulin/troponin C superfamily, appeared to be a marker for the disc formation.     

Hedgehog signaling regulates expansion of pancreatic epithelial cells

Embryonic Hedgehog signaling is essential for proper tissue morphogenesis and organ formation along the developing gastrointestinal tract. Hedgehog ligands are expressed throughout the endodermal epithelium at early embryonic stages but excluded from the region that will form the pancreas. Ectopic activation of Hedgehog signaling at the onset of pancreas development has been shown to inhibit organ morphogenesis. In contrast, Hedgehog signaling components are found within pancreatic tissue during subsequent stages of development as well as in the mature organ, indicating that a certain level of pathway activation is required for normal organ development and function. Here, we ectopically activate the Hedgehog pathway midway through pancreas development via expression of either Sonic (Shh) or Indian Hedgehog (Ihh) under control of the human Pax4-promoter. Similar pancreatic defects are observed in both Pax4-Shh and Pax4-Ihh transgenic lines, suggesting that regulation of the overall level of Hedgehog activity is critical for proper pancreas development. We also show that Hedgehog signaling controls mesenchymal vs. epithelial tissue differentiation and that pathway activation impairs formation of epithelial progenitors. Thus, tight control of Hedgehog pathway activity throughout embryonic development ensures proper pancreas organogenesis.     

Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear

The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.     

Developmental changes in the subcellular localization of R-cadherin in chick retinal pigment epithelium

 It has been reported that in the chick embryonic retina, N-cadherin first appears at the very early stages and is subsequently substituted by R-cadherin at the middle to late stages of development. To examine the role of R-cadherin in the morphogenesis of chick retinal pigment epithelium (RPE), the distribution of this adhesion molecule was studied by immunofluorescence cytochemistry and immunoelectron microscopy from embryonic day (E) 6 to hatching. R-cadherin immunoreactivity was detected at E6, and was strongest at E12–13. During these stages, R-cadherin was expressed uniformly on the lateral plasma membranes of RPE cells in contact with each other. Thereafter, R-cadherin immunoreactivity was markedly decreased, with intense immunoreactivity restricted to zonulae adherentes in latero-apical regions at E16. R-cadherin immunoreactivity was no longer detectable in the newly hatched chick RPE, even though morphologically well developed zonulae adherentes were present in latero-apical regions. No immunoreactivity was detected on the apical side facing the neural retina or on the basal side facing the basal lamina at any stage of development. These findings indicate that R-cadherin plays an important role as a major cadherin subtype in the morphogenesis of chick embryo RPE, and is involved initially in non-specific cell-cell adhesions, and subsequently in the formation and maintenance of developing zonulae adherentes. Accepted: 11 April 1997     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem-or progenitor-cells for islet transplantation.Key Words: islets, stem-cells, development, epithelium, mesenchyme, pancreas, stomach, chick-quail, 3-dimensional, endocrine     

Understanding the role of growth factors in embryonic development: insights from the lens

Growth factors play key roles in influencing cell fate and behaviour during development. The epithelial cells and fibre cells that arise from the lens vesicle during lens morphogenesis are bathed by aqueous and vitreous, respectively. Vitreous has been shown to generate a high level of fibroblast growth factor (FGF) signalling that is required for secondary lens fibre differentiation. However, studies also show that FGF signalling is not sufficient and roles have been identified for transforming growth factor-β and Wnt/Frizzled families in regulating aspects of fibre differentiation. In the case of the epithelium, key roles for Wnt/β-catenin and Notch signalling have been demonstrated in embryonic development, but it is not known if other factors are required for its formation and maintenance. This review provides an overview of current knowledge about growth factor regulation of differentiation and maintenance of lens cells. It also highlights areas that warrant future study.     

The C. elegans LIM homeobox gene lin-11 specifies multiple cell fates during vulval development

LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lin-11 have previously been shown to alter the cell division pattern of a subset of the 2 degrees lineage vulval cells. We demonstrate multiple functions of lin-11 during vulval development. We examined the fate of vulval cells in lin-11 mutant animals using five cellular markers and found that lin-11 is necessary for the patterning of both 1 degrees and 2 degrees lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, we show that lin-11 is initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2 degrees lineage cells. Using a conditional RNAi approach, we show that lin-11 regulates vulval morphogenesis. Finally, we show that LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning.     

Conditional ablation of Notch signaling in pancreatic development

The role of the Notch signaling members Notch1, Notch2 and Rbpj in exocrine pancreatic development is not well defined. We therefore analyzed conditional pancreas-specific Rbpj and combined Notch1/Notch2 knockout mice using Ptf1a(+/Cre(ex1)) mice crossed with floxed Rbpj or Notch1/Notch2 mice. Mice were analyzed at different embryonic stages for pancreatic exocrine and endocrine development. The absence of Rbpj in pancreatic progenitor cells impaired exocrine pancreas development up to embryonic day 18.5 and led to premature differentiation of pancreatic progenitors into endocrine cells. In Rbpj-deficient pancreata, amylase-expressing acini and islets formed during late embryonic and postnatal development, suggesting an essential role of Rbpj in early but not late development. Contrary to this severe phenotype, the concomitant inactivation of Notch1 and Notch2 only moderately disturbed the proliferation of pancreatic epithelial cells during early embryonic development, and did not inhibit pancreatic development. Our results show that, in contrast to Rbpj, Notch1 and Notch2 are not essential for pancreatogenesis. These data favor a Notch-independent role of Rbpj in the development of the exocrine pancreas. Furthermore, our findings suggest that in late stages of pancreatic development exocrine cell differentiation and maintenance are independent of Rbpj.     

Distribution of cells containing FMRFamide-related molecules in the embryonic development of Ophryotrocha labronica (Polychaeta: Dorvilleidae)

Abstract. The timing and spatial distribution of cells containing FMRFamide-related molecules in the embryogenesis of the polychaete Ophryotrocha labronica were studied immunocytochemically. FMRFamide-like molecules emerge early during embryonic development. They are found at the one-cell stage, are asymmetrically distributed in the first phases of cleavage, associated with gastrular movements, and label the central nervous system morphogenesis. Moreover, during embryogenesis, the pattern of gut cells with the FMRFamide-like phenotype that is present in adults is already established. The early occurrence of FMRFamide-like molecules in O. labronica suggests that these molecules are involved as pre-nervous growth signals in the regulation of basic neuronal cell behaviors.