Localization of cartilage linking protein 1 during primary neurulation in the chick embryo.

Primary neurulation is a form-shaping event during the early development of the vertebrate embryo in which the neural plate is rolled up into the neural tube, the rudiment of the central nervous system. In an effort to identify genes specifically expressed in tissues lateral to the chick neural plate--tissues known to generate extrinsic forces for primary neurulation--we designed a subtractive scheme and identified a positive clone as the gene encoding chick cartilage linking protein 1 (CRTL1). CRTL1 (also known as link protein) is a small glycoprotein of the extracellular matrix that was originally identified for its role in stabilizing aggregates of aggrecan and hyaluronan in cartilage. In addition to being expressed in cartilage, CRTL1 is also immunolocalized in several noncartilaginous tissues as assessed with the 4B6 monoclonal antibody. Using the 4B6 antibody and the G9 riboprobe derived from our subtraction, we report the detailed distribution of CRTL1 protein and crtl1 transcripts during primary neurulation in chick embryos. This report emphasizes and briefly discusses important differences between the RNA expression pattern and the domains of accumulation of the protein. CRTL1 prominently accumulates in the basal lamina of the epidermal ectoderm just lateral to the neural plate. Based on the crucial role of the interface between this tissue and the neuroepithelium in the formation of the neural folds, and because of the biophysical role of hyaluronan in tissue morphogenesis, we propose that crtl1 represents is an excellent candidate neurulation gene, worthy of further study.

Clonal analysis in mice underlines the importance of rhombomeric boundaries in cell movement restriction during hindbrain segmentation.

BACKGROUND: Boundaries that prevent cell movement allow groups of cells to maintain their identity and follow independent developmental trajectories without the need for ongoing instructive signals from surrounding tissues. This is the case of vertebrate rhombomeric boundaries. Analysis in the developing chick hindbrain provided the first evidence that rhombomeres are units of cell lineage. The appearance of morphologically visible rhombomeres requires the segment restricted expression of a series of transcription factors, which position the boundaries and prefigure where morphological boundaries will be established. When the boundaries are established, when the cells are committed to a particular rhombomere and how they are organized within the hindbrain are important questions to our understanding of developmental regionalization. METHODOLOGY/PRINCIPAL FINDINGS: Sophisticated experimental tools with high-resolution analysis have allowed us to explore cell lineage restriction within the hindbrain in mouse embryos. This novel strategy is based on knock-in alleles of ubiquitous expression and allows unrestricted clonal analysis of cell lineage from the two-cell stage to the adult mouse. Combining this analysis with statistical and mathematical tools we show that there is lineage compartmentalization along the anteroposterior axis from very early stages of mouse embryonic development. CONCLUSIONS: Our results show that the compartment border coincides with the morphological boundary in the mouse hindbrain. The restriction of the cells to cross rhombomeric boundaries seen in chick is also observed in mouse. We show that the rhombomeric boundaries themselves are involved in cell movement restriction, although an underlying pre-pattern during early embryonic development might influence the way that cell populations organize.

Live optical projection tomography.

Optical projection tomography (OPT) is a technology ideally suited for imaging embryonic organs. We emphasize here recent successes in translating this potential into the field of live imaging. Live OPT (also known as 4D OPT, or time-lapse OPT) is already in position to accumulate good quantitative data on the developmental dynamics of organogenesis, a prerequisite for building realistic computer models and tackling new biological problems. Yet, live OPT is being further developed by merging state-of-the-art mouse embryo culture with the OPT system. We discuss the technological challenges that this entails and the prospects for expansion of this molecular imaging technique into a wider range of applications.

Assessing the contributions of gene products to the form-shaping events of neurulation: a transgenic approach in chick.

Most of our current knowledge on the tissue and cellular basis of neurulation in amniotes has been gained using the chick embryo as an experimental model system. Gene manipulation during chick neurulation has been difficult, greatly limiting our ability to assess the contribution of gene products to the tissue and cellular behaviors of neurulation. Using electroporation, we have developed a simple and reliable method for expressing transgenes in the ectoderm of the neural folds of chick embryos developing in whole-embryo culture. Sense- or antisense-expressing plasmids are electroporated, resulting in gain or loss of gene function, respectively. The morphogenesis of transgenic tissues was compared to the morphogenesis of contralateral wildtype tissues as neurulation was taking place. As a proof of principle, we present a functional analysis of the chick gene encoding Cartilage Linking Protein 1 (CRTL1), identified as a candidate neurulation gene using subtractive hybridization. This experimental approach provides a much-needed innovation for studying the mechanisms by which genes influence neurulation and reveals here important contributions of CRTL1 to the formation of the neural folds.

Differential expression of two cell adhesion molecules, Ephrin-A5 and Integrin alpha6, during cranial neurulation in the chick embryo.

The formation of the neural tube (neurulation) depends on the physical properties of the cells and tissues both inside and outside the neural plate. One such important physical property is cell adhesion. Theoretical and biological evidence support a role for cell adhesion in neurulation, but few specific cell adhesion molecules have been identified during this process. Ephrin-A5 and Integrin alpha6 are two of the known genes encoding cell adhesion molecules that are likely to be directly involved in neurulation because neural tube defects result when they are knocked out in mice. Yet it remains unclear how they can act on the cell and tissue behaviors of neurulation, because their domains of expression in neurulating tissues have not been reported. We report here the detailed pattern of expression of these two cell adhesion molecules in the chick embryo throughout the stages of neurulation at the mRNA and protein level. We show that Ephrin-A5 and Integrin alpha6 are differentially expressed in the ectoderm, outside and inside the neural plate, respectively, and that they are both restricted to neurulation at cranial (brain) levels. We discuss the potential contribution of this differential expression to the cell adhesion mechanisms involved in cranial neurulation and anencephaly.