Goosecoid and HNF-3beta genetically interact to regulate neural tube patterning during mouse embryogenesis

The homeobox gene goosecoid (gsc) and the winged-helix gene Hepatic Nuclear Factor-3beta (HNF-3beta) are co-expressed in all three germ layers in the anterior primitive streak and at the rostral end of mouse embryos during gastrulation. In this paper, we have tested the possibility of functional synergism or redundancy between these two genes during embryogenesis by generating double-mutant mice for gsc and HNF-3beta. Double-mutant embryos of genotype gsc(-/-);HNF-3beta(+/-) show a new phenotype as early as embryonic days 8.75. Loss of Sonic hedgehog (Shh) and HNF-3beta expression was observed in the notochord and ventral neural tube of these embryos. These results indicate that gsc and HNF-3beta interact to regulate Shh expression and consequently dorsal-ventral patterning in the neural tube. In the forebrain of the mutant embryos, severe growth defects and absence of optic vesicles could involve loss of expression of fibroblast growth factor-8, in addition to Shh. Our results also suggest that interaction between gsc and HNF-3beta regulates other signalling molecules required for proper development of the foregut, branchial arches and heart

Glyphosate Induces Neurotoxicity in Zebrafish

Glyphosate based herbicides (GBH) like Roundup® are used extensively in agriculture as well as in urban and rural settings as a broad spectrum herbicide. Its mechanism of action was thought to be specific only to plants and thus considered safe and non-toxic. However, mounting evidence suggests that GBHs may not be as safe as once thought as initial studies in frogs suggest that GBHs may be teratogenic. Here we utilize the zebrafish vertebrate model system to study early effects of glyphosate exposure using technical grade glyphosate and the Roundup® Classic formulation. We find morphological abnormalities including cephalic and eye reductions and a loss of delineated brain ventricles. Concomitant with structural changes in the developing brain, using in situ hybridization analysis, we detect decreases in genes expressed in the eye, fore and midbrain regions of the brain including pax2, pax6, otx2 and ephA4. However, we do not detect changes in hindbrain expression domains of ephA4 nor exclusive hindbrain markers krox-20 and hoxb1a. Additionally, using a Retinoic Acid (RA) mediated reporter transgenic, we detect no alterations in the RA expression domains in the hindbrain and spinal cord, but do detect a loss of expression in the retina. We conclude that glyphosate and the Roundup® formulation is developmentally toxic to the forebrain and midbrain but does not affect the hindbrain after 24 hour exposure

Analysis of neural induction and patterning in amniote embryos

When grafted into a host embryo, Hensen's node, the chick gastrular organiser, is able to induce an ectopic second neural axis. The use of pan-neural and regionally specific neural genes, particularly forebrain markers such as BF-1 and GANF, shows that the chick node is able to induce a nervous system with complete anterior pattern, expressing all the markers tested. Evidence in mouse suggests that additional signalling information, established separately from the node and its derivatives, is required to generate complete anterior pattern. The ability of mouse node grafts to induce the same range of neural markers in chick hosts has therefore also been examined. In this assay, the mouse node is not able to induce expression of chick forebrain markers, and an anteriorly truncated second axis is formed. This work has also investigated the role of the foregut endoderm in patterning the anterior brain of the chick. This tissue is formed by cells that move through the node into the lower layer during gastrulation. Removal of the lower layer at stage 4 has no apparent effect. However, removal of foregut endoderm during the early head process stages (4+ to 5) causes reduction in forebrain pattern. By the 12 somite stage, most neuraxes lack telencephalon and eyes, and BF-1 and GANF expression domains are absent or severely reduced. This syndrome is preceded by a failure to establish normal FGF 8 expression in the anterior neural ridge signalling centre, at early somite stages. However, gene expression throughout axial mesoderm {BMP 7, chordin and shh) appears unaffected in all embryos. The homeobox gene hex and the chick Frzb homologue crescent are both expressed in the anterior definitive endoderm at the time when removal of this tissue results in forebrain defects. These results suggest that the definitive foregut endoderm contains information crucial for forebrain patterning in the chick embryo

Effects of High Salt-Exposure on the Development of Retina and Lens in 5.5-Day Chick Embryo

Background/Aims: Excess maternal salt intake during pregnancy may alter fetal development. However, our knowledge on how an increased salt intake during pregnancy influences fetal eye development is limited. In this study, we investigated the effects of high-salt treatment on the developing eyes in chick embryos, especially focusing on the development of the retina and the lens. Methods: 5.5-day chick embryos were exposed to 280mosm/l (n=17), or 300mosm/l (n=16) NaCl. The treated embryos were then incubated for 96 hours before they were fixed with 4% paraformaldehyde for H&E staining, whole-mount embryo immunostaining and TUNEL staining. BrdU and PH3 incorporation experiments were performed on the chick embryos after high-salt treatment. RT-PCR analyses were conducted from chick retina tissues. Results: We demonstrated that high-salt treatment altered the size of eyes in chick embryos, induced malformation of the eyes and impaired the development of the lens and the retina. We found an impaired expression of Paired box 6 (PAX6) and neuronal cells in the developing retina as revealed by neurofilament immunofluorescent staining. There was a reduction in the number of BrdU-positive cells and PH3-positive cells in the retina, indicating an impaired cell proliferation with high-salt treatment. High-salt treatment also resulted in an increased number of TUNEL-positive cells in the retina, indicating a higher amount of cell death. RT-PCR data displayed that the expression of the pro-apoptotic molecule nerve growth factor (NGF) in chick retina was increased and CyclinD1 was reduced with high-salt treatment. The size of the lens was reduced and Pax6 expression in the lens was significantly inhibited. High salt-treatment was detrimental to the migration of neural crest cells. Conclusion: Taken together, our study demonstrated that high-salt exposure of 5.5-day chick embryos led to an impairment of retina and lens development, possibly through interfering with Pax6 expression

Opponent Activities of Shh and BMP Signaling during Floor Plate Induction In Vivo

AbstractWe performed in vivo experiments in chick embryos that examined whether application of an exogenous source of Shh protein mimics the ability of the notochord to induce ectopic floor plate cells in the neural tube. Shh cannot act alone to induce a floor plate. However, coapplication of Shh and chordin, a BMP antagonist normally coexpressed with Shh in the notochord, results in a marked switch from dorsal to ventral cell fate, including a dramatic and widespread induction of floor plate cells. These data provide in vivo evidence that notochord-derived BMP antagonists may normally generate a permissive environment for the Shh-mediated induction of floor plate. Further experiments performed to address the source of BMPs that are inhibited by the action of chordin suggest that they derive specifically from the surface ectoderm and dorsal-most neuroepithelium. These data indicate that, at neural groove stages, dorsally derived BMPs affect ventral-most regions of the neural plate, suggesting a novel long-range action of BMPs. Together, these studies suggest that the balance of dorsally derived signals and notochord-derived signals determines the extent of floor plate cell induction

Alterations In Ocular Surface System During The Pathogenesis Of Herpes Stromal Keratitis

Herpes stromal keratitis (HSK) is a chronic immuno-inflammatory ocular disease caused by Herpes simplex virus-1 (HSV-1) infection in the cornea. HSK is characterized by the development of corneal opacity and angiogenesis accompanied by the loss of corneal transparency. Despite extensive studies on inhibiting corneal angiogenesis and strategies to reduce HSK disease development, a key underlying mechanism to maintain an intact and a transparent corneal surface during HSK disease progression, remain obscure. Our study has addressed possible causes for the HSK disease severity by identifying the key factors such as the development of inflammatory hypoxia, reduced tear volume, Inflammation of extra-orbital lacrimal gland and the conjunctiva and the transdifferentiation of the corneal epithelium in the HSK developing eyes. Intriguingly, our study also emphasizes the protective role of IGFBP-3 in inhibiting the severe disease progression of HSK. During the pre-clinical and the clinical disease phase of HSK, the influx of neutrophils have been studied as the most prominent innate immune cell type. Development of corneal hem- and lymph-angiogenesis, opacity and epithelial defects hinders the clear vision and is known to be the cause of the increased neutrophil influx in HSV-1 infected cornea. Previous studies have indicated that the influx of neutrophils into mucosal tissue can re-model the development of inflammatory hypoxia. Therefore, the aim of this study was to investigate the development of hypoxia and hypoxia-associated gene expression during the progression of HSK disease. Our results showed an increased influx of neutrophils during the clinical disease of HSK that leads to the development of hypoxia, and the upregulation of hypoxia-associated genes. This study provided us novel insights on the prevalence of the glycolytic metabolism in the HSK developing eyes. Additionally, our data demonstrated that the blocking of Hypoxia-Inducible Factor (HIF) with Acriflavine alone might not be an efficacious drug in reducing HSK disease severity. Overall, these results pave way for using novel therapeutic targets that play a dual role to block HIF and control corneal inflammation during HSK. Maintaining a healthy corneal epithelium, retaining a steady tear volume and its secretion could be the other factors that may aid in alleviating HSK disease severity. Mounting evidence corroborates corneal nerves as a critical factor in the maintenance of corneal integrity and homeostasis of the ocular surface by mediating the reflex actions of blinking and tearing. In support, in a mouse model during HSV-1 ocular infection, corneal nerve damage has been demonstrated. Hence, corneal transparency and clear vision are critically dependent upon the integrity of corneal tissue maintained by the intricate network of corneal nerves, the presence of intact corneal epithelium and tear film that is controlled by a cross-talk between the components of the lacrimal functional unit. Our study showed a reduced measurement of tear volume in the HSK developing eyes correlating to the inflammation in the extra-orbital lacrimal gland (EoLG) and the conjunctiva. Atrophy of tear secreting acinar cells in the EoLG, the significant loss of goblet cells in the conjunctiva and the influx of leukocytes and CD4 T cells were the major events occurring during the HSV-1 infection. These events caused dysfunction to the lacrimal functional unit, thereby inducing severe HSK and ‘dry eye’ conditions. Nevertheless, topical treatment of lacritin (tear glycoprotein) delayed but did not prevent the reduction in tear volume nor reduced the severity of HSK. Existence of a healthy, smooth and lubricated corneal surface is critical for the maintenance of transparent cornea to aid clear vision. Corneal epithelium comprises of non-keratinized stratified squamous epithelium. Nevertheless, under pathological conditions non-keratinized epithelium transforms into a keratinized epithelium that closely matches the features of skin epithelium (epidermis). The keratin expression profile determines this switch in keratinization in the cornea. Cytokeratin-12 (K12) is one of the keratins specific to the corneal epithelium, which is replaced by epidermis-specific cytokeratin-10 (K10) expression under pathological conditions. Intriguingly, our data showed the loss of K12 expression with the emergence of K10 expression during the clinical stage of HSK disease. Our data suggested the occurrence of corneal epithelial transformation. Another prominent marker, PAX-6 (paired box -6 transcription factor) has a crucial role in the maintenance of corneal epithelial identity by regulation of its differentiation. Our data showed a dramatic loss of PAX-6 expression in the HSK developing eyes. ‘Squamous metaplasia’ is the pathological phenomenon characterized by abnormal differentiation of corneal epithelium into the skin like epithelium. Overall, our results signified the ‘trans-differentiation of corneal epithelial cells to the skin like epithelium, in HSV-1 infected mouse cornea. Corneal hem-angiogenesis is one of the hallmarks of HSK. Hence, it is essential to understand better for the molecules involved in regulating hem-angiogenesis, immune cell survival, and its effector function. Intriguingly, angiogenesis array data showed an increased expression of IGFBP-3 in the HSV-1 infected corneas of C57BL/6 (B6) mice. Therefore, we were inclined to study the role of IGFBP-3 molecule in HSV-1 infected B6 corneas. IGFBP-3 is one of the six highly conserved IGF-binding proteins. IGFBP-3 binds to 75% of the serum IGF-I and IGF-II in complexes and regulates IGF-1R signaling by sequestering IGF molecule. IGF binding to IGF-1R results in phosphorylation of tyrosine residues in IGF-1Rβ chain. IGF-1R signaling that has been reported to promote angiogenesis (hem- and lymph-angiogenesis). Our data indicated severe HSK disease with robust angiogenesis in IGFBP-3-/- mice associated with a significant increase in IGF-1R phosphorylation in the infiltrated leukocytes. These results suggest the protective role of IGFBP-3 in inhibiting IGF-1R phosphorylation that leads to the development of severe HSK disease progression. Altogether, this study broadens our knowledge on understanding several factors involved in the alterations of the ocular surface system during the pathogenesis of HSK. In summary, this study highlights on five crucial factors that may play an essential role in reducing HSK disease pathogenesis in the cornea; 1) regulating the development of inflammatory hypoxia 2) maintaining of the stable tear volume 3) reducing the lacrimal and conjunctival inflammation 4) maintaining the corneal epithelial integrity 3) alleviating corneal hem-angiogenesis (Figure 42)

Whole-mount in situ hybridization in the Rotifer Brachionus plicatilis representing a basal branch of lophotrochozoans

In order to broaden the comparative scope of evolutionary developmental biology and to refine our picture of animal macroevolution, it is necessary to establish new model organisms, especially from previously underrepresented groups, like the Lophotrochozoa. We have established the culture and protocols for molecular developmental biology in the rotifer species Brachionus plicatilis Müller (Rotifera, Monogononta). Rotifers are nonsegmented animals with enigmatic basal position within the lophotrochozoans and marked by several evolutionary novelties like the wheel organ (corona), the median eye, and the nonpaired posterior foot. The expression of Bp-Pax-6 is shown using whole-mount in situ hybridization. The inexpensive easy culture and experimental tractability of Brachionus as well as the range of interesting questions to which it holds the key make it a promising addition to the “zoo” of evo-devo model organisms