Imaging Neural Development in Embryonic and Larval Sea Urchins

Abstract

Imaging is a critical tool in neuroscience, and our understanding of the structure and function of sea urchin nervous systems owes much to this approach. In particular, studies of neural development have been facilitated by methods that enable the accurate identification of specific types of neurons. Here we describe methods that have been successfully employed to study neural development in sea urchin embryos. Altering gene expression in part of an embryo is facilitated by injection of reagents into individual blastomeres, which enables studies of cell autonomous effects and single embryo rescue experiments. The simultaneous localization of an in situ RNA hybridization probe and a cell type specific antigen has enabled studies of gene expression in specific types of neurons. Fixatives and antibodies can be capricious; thus, we provide data on preservation of antigens with commonly used fixatives and buffers.

Eph and Ephrin function in dispersal and epithelial insertion of pigmented immunocytes in sea urchin embryos

“…Sp-Efn is expressed in the dorsal ectoderm and ciliary band (Krupke and Burke, 2014). A more detailed examination of the ligand indicates an apparent correlation between the relative fluorescence, or immunoreactivity of ectoderm expressing Sp-Efn and the distribution of pigmented immunocytes.…”

“…In hatched blastulae, cells in the vegetal plate that are immunoreactive with the immunocyte-specific antibody Sp1 (Gibson and Burke, 1985), are also immunoreactive with anti-Sp-Eph (Krupke and Burke, 2014) (Figure 3A–C). Sp-Efn is present at the same time on the basal surfaces of ectodermal cells (Figure 3D).…”

“…Suppressing expression of Sp-Eph produces embryos that gastrulate and have defects in the ciliary band (Krupke and Burke, 2014) (Figure 4A). Immunocyte precursors migrate to the ectoderm and at 40 hr there is no difference in the number of Sp1 immunoreactive cells between control and experimental treatments (Figure 4A,E).…”

“…Sperm was diluted 1:100 in filtered seawater prior to fertilization and embryos were grown at 12–14°C. Eggs were prepared for microinjection as described previously (Krupke et al, 2014). Injection solutions contained water, 125 mM KCl and either RNA or morpholinos.…”

“…2–4 pL of a 0.2 μM injection solution was injected into each freshly fertilized egg as previously described (Krupke and Burke, 2014). Single blastomere injections followed Krupke et al (2014). Eph inhibitor, NVP BHG 712 (NVP) was used at 1.75 μM (Cat.…”