Lens placode modulates extracellular matrix formation during early eye development.

The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm and is regulated by transcription factor Pax6 and secreted BMP4. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2, a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.

Characterization of embryonic surface ectoderm cell protrusions.

BACKGROUND: During embryonic development, complex changes in cell behavior generate the final form of the tissues. Extension of cell protrusions have been described as an important component in this process. Cellular protrusions have been associated with generation of traction, intercellular communication or establishment of signaling gradients. Here, we describe and compare in detail from live imaging data the dynamics of protrusions in the surface ectoderm of chick and mouse embryos. In particular, we explore the differences between cells surrounding the lens placode and other regions of the head. RESULTS: Our results showed that protrusions from the eye region in mouse embryos are longer than those in chick embryos. In addition, protrusions from regions where there are no significant changes in tissue shape are longer and more stable than protrusions that surround the invaginating lens placode. We did not find a clear directionality to the protrusions in any region. Finally, we observed intercellular trafficking of membrane puncta in the protrusions of both embryos in all the regions analyzed. CONCLUSIONS: In summary, the results presented here suggest that the dynamics of these protrusions adapt to their surroundings and possibly contribute to intercellular communication in embryonic cephalic epithelia.

The Rst-Neph family of cell adhesion molecules in Gallus gallus.

The Rst-Neph family comprises an evolutionarily conserved group of single-pass transmembrane glycoproteins that belong to the immunoglobulin superfamily and participate in a wide range of cell adhesion and recognition events in both vertebrates and invertebrates. In mammals and fish, three Rst-Neph members, named Neph1-3, are present. Besides being widely expressed in the embryo, particularly in the developing nervous system, they also contribute to the formation and integrity of the urine filtration apparatus in the slit diaphragm of kidney glomerular podocytes, where they form homodimers, as well as heterodimers with Nephrin, another immunoglobulin-like cell adhesion molecule. In mice, absence of Neph1 causes severe proteinuria, podocyte effacement and perinatal death, while in humans, a mutated form of Nephrin leads to congenital nephrotic syndrome of the Finnish type. Intriguingly, neither Nephrin nor Neph3 are present in birds, which nevertheless have typical vertebrate kidneys with mammalian-like slit diaphragms. These characteristics make, in principle, avian systems very helpful for understanding the evolution and functional significance of the complex interactions displayed by Rst-Neph proteins. To this end we have started a systematic study of chicken Neph embryonic and post-embryonic expression, both at mRNA and protein level. RT-qPCR mRNA quantification of the two Neph paralogues in adult tissues showed that both are expressed in heart, brain, and retina. Neph1 is additionally present in kidney, liver, pancreas, lungs, and testicles, while Neph2 mRNA is barely detected in kidney, testicles, pancreas and absent in liver and lungs. In embryos, mRNA from both genes can already be detected at as early as stage HH14, and remain expressed until at least HH28. Finally, we used a specific antibody to examine the spatial dynamics and subcellular distribution of ggNeph2 between stages HH20-28, particularly in the mesonephros, dermomyotomes, developing heart, and retina.

Lens Placode Modulates Extracellular Matrix Formation During Early Eye Development.

The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2 , a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.

In Embryo Gene Reporter Assays for Evaluation of Cis-Regulatory Regions.

Gene expression reporter assays measure the relevance of cis-regulatory elements and DNA-binding proteins in modulating transcriptional activity. Commonly, they are performed in cell lines. However, regulation of transcriptional activity during development is complex and dynamic, and not many cell lines reproduce the embryonic conditions. Thus, conclusions derived from cell line data provide limited information about embryonic development. On the other hand, one of the major hurdles for embryonic assays is delivering reporter plasmids in a tissue-specific manner. In this sense, the chick embryo is a good model system to perform these assays. Electroporation of chick embryos provides temporal and spatially controlled plasmid delivery. Further, it is a well-established, easy, and an economical procedure. Here, we describe in detail how to measure in the chick neural tube (1) enhancer activity with GFP, (2) enhancer activity with luciferase, and (3) 3'UTR activity with luciferase.

Morphogenesis of the lens placode.

For over 100 years, the vertebrate eye has been an important model system to understand cell induction, cell shape change, and morphogenesis during development. In the past, most of the studies examined histological changes to detect the presence of induction mechanisms, but the advancement of molecular biology techniques has made exploring the genetic mechanisms behind lens development possible. Despite the particular emphasis given to the induction of the lens placode, there are still many aspects of the cell biology of lens morphogenesis to be explored. Here, we will revisit the classical detailed description of early lens morphological changes, correlating it with the cell biology mechanisms and with the molecules and signaling pathways identified up to now in chick and mouse embryos. A detailed description of lens development stages helps better understand the timeline of the events involved in early lens morphogenesis. We then point to some key questions that are still open.

RNA-Seq analysis of differential gene expression in electroporated chick embryonic spinal cord.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.