Chao Yun Irene Yan

Rho Signaling Pathway and Apical Constriction in the Early Lens Placode

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho‐ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan‐Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368–379, 2011.

Samba, a Xenopus hnRNP expressed in neural and neural crest tissues.

RNA binding proteins regulate gene expression at the posttranscriptional level and play important roles in embryonic development. Here, we report the cloning and expression of Samba, a Xenopus hnRNP that is maternally expressed and persists at least until tail bud stages. During gastrula stages, Samba is enriched in the dorsal regions. Subsequently, its expression is elevated only in neural and neural crest tissues. In the latter, Samba expression overlaps with that of Slug in migratory neural crest cells. Thereafter, Samba is maintained in the neural crest derivatives, as well as other neural tissues, including the anterior and posterior neural tube and the eyes. Overexpression of Samba in the animal pole leads to defects in neural crest migration and cranial cartilage development. Thus, Samba encodes a Xenopus hnRNP that is expressed early in neural and neural crest derivatives and may regulate crest cells migratory behavior. Developmental Dynamics 238:204–209, 2009.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

The transcription factor chicken Scratch2 is expressed in a subset of early postmitotic neural progenitors

Scratch proteins are members of the Snail superfamily which have been shown to regulate invertebrate neural development. However, in vertebrates, little is known about the function of Scratch or its relationship to other neural transcription factors. We report the cloning of chicken Scratch2 (cScrt2) and describe its expression pattern in the chick embryo from HH15 through HH29. cScrt2 was detected in cranial ganglia, the nasal placode and neural tube. At all stages examined, cScrt2 expression is only detected within a subregion of the intermediate zone of the neural tube. cScrt2 is also expressed in the developing dorsal root ganglia from HH22-23 onwards and becomes limited to its dorsal medial domain at HH29. phospho-Histone H3 and BrdU-labeling revealed that the cScrt2 expression domain is located immediately external to the proliferative region. In contrast, cScrt2 domain overlapped almost completely with that of the postmitotic neural transcription factor NeuroM/Ath3/NEUROD4. Together, these data define cScrt2-positive cells as a subset of immediately postmitotic neural progenitors. Previous data has shown that Scrt2 is a repressor of E-box-driven transcription whereas NeuroM is an E-box-transactivator. In light of these data, the co-localization detected here suggests that Scrt2 and NeuroM may have opposing roles during definition of neural subtypes.

Rho GTPases Control Ciliary Epithelium Cells Proliferation and Progenitor Profile Induction In Vivo

PURPOSE Rho GTPases play a central role in actin-based cytoskeleton reorganization and regulate multiple signaling pathways that control gene transcription, cell survival, and proliferation. We investigated the effect of Rho GTPases on cell cycle regulation and progenitor genes expression on mouse ciliary epithelium (CE), a potential source of progenitor/stem cells in the adult retina. METHODS Rho GTPases were activated by intraocular injection of lysophosphatidic acid and inactivated by Clostridium difficile Toxin A (general Rho GTPase inhibitor), NSC23766 (Rac1 activation inhibitor), or Y27632 (Rho-associated protein kinase [ROCK] inhibitor). Thereafter, we assayed for RhoA, RhoB, and Rac1 protein localization in CE cells. Proliferation was examined by the expression levels of cell cycle regulators p27(kip), p16(INK4a), and Ki67 and the effects on progenitors by determining the changes in Pax6 and Chx10 progenitor markers expression. RESULTS All GTPases investigated were expressed in mouse CE cells. Activation increased the coexpression of Pax6 and Chx10, but had no significant effect on the proliferation of CE cells. In contrast, Rho GTPases inactivation increased cell proliferation and potentiated the proliferative effect of growth factors. Specific inactivation of Rac1 or ROCK increased the levels of Ki67 and decreased the expression of the cell cycle inhibitors p27(kip) and p16(INK4a). CONCLUSIONS This study reports that Rho GTPase modulation (activation and inactivation) controls the expression of retinal progenitor genes and proliferation, respectively, in the adult ciliary epithelial progenitor/stem cells of rodent eyes. The modulation of these two different mechanisms (proliferation and reprogramming) may provide a potential new approach in retinal repair.

A detailed description of an economical setup for electroporation of chick embryos in ovo

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

Role of myosin Va in neuritogenesis of chick dorsal root ganglia nociceptive neurons

Myosin‐Va, widely distributed throughout the developing nervous system, is involved in the transport of vesicles and other intracellular components with its globular tail domain (GTD) implicated in cargo recognition/interaction. Inactivation of myosin‐Va in dorsal root ganglia (DRG) neurons of chick embryos, in vitro, decreases the rate of filopodial extension. MYO5A mutant mice have severe neurological defects. We have found that the overexpression of GTD in DRG cultures reduces the number of neurons with long neurites (above fourfold cell body length) and increased the number of neurons with short or no neurites. However, if transfection occurred after the onset of neuritogenesis, this was not seen. In embryo, we characterized the expression pattern of myosin‐Va during neuritogenesis of TrkA‐positive cells at different stages of chick DRG development. Myosin‐Va expression was detected starting from HH25. At this stage, it was present in cells both with and without neurites. The presence of myosin‐Va in DRG neurites persisted throughout the last stage analysed (HH34). The data suggest that Myosin Va can participate in embryonic DRG neuritogenesis.

Functional analysis of Scratch2 domains: implications in the evolution of Snail transcriptional repressors

The Snail superfamily of transcription factors have a modular organization and their similarities and divergences are the basis for subdividing the superfamily into the Snail1/2 and Scratch families. As it is generally accepted that the Snail and Scratch families originated through gene duplication, understanding the functional contribution of each module could provide us with further insight about the molecular and functional evolution of the Snail superfamily. Thus, in this work, we investigated the function of the SNAG and SCRATCH domains in chicken Scratch2. Through evolutionary comparison analysis we identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. Similar to members of the Snail1/2 families, Scratch2- mediated transcriptional repression requires SNAG and nuclear localization requires the zinc-finger domain. We also identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. HINGE is highly conserved in amniotes. Single mutations of the conserved Tyrosine and Serine residues of HINGE downregulated Scratch2-mediated transcriptional repression. This effect depended on the presence of the SCRATCH domain.

Role of Scratch2 in mouse embryonic stem cells cortical differentiation

The decision to replicate is controlled by checkpoints located in the G1 phase and leads to cell type-specific cell cycle length. For example, in culture, the population doubling time for mouse embryonic fibroblasts (MEF) is ~30 hrs, while that of mouse embryonic stem cells (mESC) is ~12 hrs. Alterations in cell cycle time has been attributed to altered control of regulators of G1, including cyclins, cyclin dependant kinases (CDKs), CDK inhibitors, Retinoblastoma (Rb), Rb-related pocket proteins and other accessory factors. Reprogramming of MEFs to an induced pluripotent state offers an excellent system to study stage-specific cell cycle control. During the reprogramming process, two important early programs are altered. First, there is a dismantling of the controls that regulate the somatic cell cycle and a conversion to a shortened cell cycle with reduced Gap phases, resulting in a rapid proliferation rate. Second, is a cell fate change involving the Mesenchymal to Epithelial transition (MET). In order to study the mechanisms that effect these changes, we designed an inducible reprogramming switch using a 4-hydroxy Tamoxifen (OHT)-dependant Oct-3/4. Using this inducible system we find that Oct-3/4 and Klf-4 can trigger the initial changes that lead to the speeding up of the cell cycle, a crucial step in the initial phase of reprogramming. We also show that Oct-3/4 and Klf-4 acting in concert initiate MET by upregulating E-Cadherin and downregulating N-Cadherin, important epithelial and mesenchymal markers. These results suggest that Oct-3/4 and Klf-4 are crucial for the initial phase of reprogramming and may also play a key role in maintaining the shortened cell cycle in mESCs.

Par3 in chick lens placode development: MELO et al.

The lens originates from a simple cuboidal epithelium, which, on its basal side, contacts the optic vesicle, whilst facing the extraembryonic environment on its apical side. As this epithelium changes into the pseudostratified lens placode, its cells elongate and become narrower at their apical ends. This is due to the formation of an apical actin network, whose appearance is restricted to cells of the placodal region, as a result of region‐specific signaling mechanisms that remain largely unknown. Here, we investigated the role of the polarity protein PAR3 and the phosphorylation state of its Threonine 833 (T833) aPKC‐binding site in the recruitment of aPKC and in the establishment of actin network in the chick lens placode. Overexpression of wild type PAR3 recruited aPKC and punctate actin clusters to the basolateral membranes of the placodal cells. Recruitment of aPKC depended on the charge of the residue that replaced the T833 residue. In contrast, induction of the ectopic actin spots was independent on the charge of this residue.