Brain Ventricular System and Cerebrospinal Fluid Development and Function: Light at the End of the Tube: A Primer with Latest Insights.

The brain ventricular system is a series of connected cavities, filled with cerebrospinal fluid (CSF), that forms within the vertebrate central nervous system (CNS). The hollow neural tube is a hallmark of the chordate CNS, and a closed neural tube is essential for normal development. Development and function of the ventricular system is examined, emphasizing three interdigitating components that form a functional system: ventricle walls, CSF fluid properties, and activity of CSF constituent factors. The cellular lining of the ventricle both can produce and is responsive to CSF. Fluid properties and conserved CSF components contribute to normal CNS development. Anomalies of the CSF/ventricular system serve as diagnostics and may cause CNS disorders, further highlighting their importance. This review focuses on the evolution and development of the brain ventricular system, associated function, and connected pathologies. It is geared as an introduction for scholars with little background in the field.

Obtaining Xenopus Eggs and Embryos.

Collecting eggs from adult Xenopus laevis and Xenopus tropicalis to raise healthy embryos and tadpoles is relatively simple but requires careful handling of the frog. Eggs can be fertilized through natural matings or by in vitro fertilization and examined visually. Here we review how eggs are obtained and how to recognize healthy eggs that will develop into high-quality embryos.

The zic1 gene is an activator of Wnt signaling.

The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.

Validation of Protein Knockout in Mutant Zebrafish Lines Using In Vitro Translation Assays.

Advances in genome-editing technology have made creation of zebrafish mutant lines accessible to the community. Experimental validation of protein knockout is a critical step in verifying null mutants, but this can be a difficult task. Absence of protein can be confirmed by Western blotting; however, this approach requires target-specific antibodies that are generally not available for zebrafish proteins. We address this issue using in vitro translation assays, a fast and standard procedure that can be easily implemented.

Identification and characterisation of the posteriorly-expressed Xenopus neurotrophin receptor homolog genes fullback and fullback-like.

We have identified fullback and fullback-like, two Xenopus laevis neurotrophin receptor homolog (NRH1) genes. The sequences of Fullback and Fullback-like are very similar to that of the neurotrophin receptor p75NTR, in both their extracellular and their intracellular domains. As their names imply, fullback and fullback-like are expressed in essentially identical patterns in the posterior of the embryo from the early gastrula stage onward. At tailbud and tadpole stages transcripts are also present in dorsal somites and the head, in addition to the growing tailbud. This expression pattern differs from that of p75NTR, suggesting that fullback and fullback-like have different functions from p75NTR during early development.

Isolation of DNA from red blood cells in Xenopus.

The amphibian Xenopus laevis is an important model organism that is particularly valuable for studies of early vertebrate development. Genomic DNA constitutes the total genetic information of an organism and it is used for Southern blotting, for determining gene structure, and for detecting the presence or absence of genes of interest. Genomic DNA can be extracted from Xenopus red blood cells, which are unlike the mammalian equivalent in that they contain nuclei. This article describes a protocol for the isolation of genomic DNA from frog red blood cells.

Microinjection of Xenopus embryos.

This article describes the factors that should be considered when microinjecting Xenopus oocytes. Forward planning is particularly important for embryo injection because the frogs lay eggs only for a limited time, and once the eggs are fertilized there is a very short period during which the embryos are suitable for injection.

Isolation of Xenopus oocytes.

Xenopus oocytes are obtained from sexually mature females by surgically removing parts of the ovary. The operation is not fatal and can be performed on an anesthetized frog several times during its lifetime. However, a recovery period of 2 wk is recommended between operations. A careful record of all operations performed, including details of oocyte quality, should be kept. A frog that produces one good batch of oocytes; e.g., those that translate injected messenger RNAs (mRNAs) efficiently, should be recorded and used again, because oocyte quality is generally frog-dependent.

Defolliculation of Xenopus oocytes.

It is possible to microinject Xenopus oocytes that are still contained within their ovarian follicles, but most researchers find it more convenient to work with defolliculated oocytes. Defolliculation can be carried out enzymatically by treatment with collagenase, or it can be performed manually. Enzymatic treatment is recommended for preparation of large numbers of oocytes (more than 1000); however, this treatment causes complications because it often damages the quality of the oocytes. The manual procedure, which requires some practice, is recommended for experiments requiring only a few hundred oocytes. This protocol details both the enzymatic and manual procedures for defolliculation of Xenopus oocytes.

Microinjection of Xenopus oocytes.

Xenopus oocytes can be injected with messenger RNA (mRNA) or DNA constructs into the cytoplasm or nucleus, respectively. The cytoplasm can withstand the introduction of up to 50 nL of injected material, and the nucleus can tolerate up to 20 nL. This protocol describes microinjection of both defolliculated and folliculated Xenopus oocytes. Oocytes remain in good condition for a relatively long time, and injections can be continued, if necessary, over a period of days.

Calibration of the injection volume for microinjection of Xenopus oocytes and embryos.

Microinjection of Xenopus oocytes or embryos with messenger RNA (mRNA) or DNA is a powerful technique for studying development. Before microinjection can be performed, the injection volume must be calibrated carefully. This protocol describes the calibration procedure for a pressure injector.

Microinjection of RNA and preparation of secreted proteins from Xenopus oocytes.

Microinjection of Xenopus oocytes or embryos with messenger RNA (mRNA) is a powerful technique for studying development, including protein expression during development. This protocol describes a method for the preparation of secreted (35)S-labeled proteins following mRNA microinjection into Xenopus oocytes.

The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth.

The primary mouth forms from ectoderm and endoderm at the extreme anterior of the embryo, a conserved mesoderm-free region. In Xenopus, a very early step in primary mouth formation is loss of the basement membrane between the ectoderm and endoderm. In an unbiased microarray screen, we defined genes encoding the sFRPs Frzb-1 and Crescent as transiently and locally expressed in the primary mouth anlage. Using antisense oligonucleotides and ;face transplants', we show that frzb-1 and crescent expression is specifically required in the primary mouth region at the time this organ begins to form. Several assays indicate that Frzb-1 and Crescent modulate primary mouth formation by suppressing Wnt signaling, which is likely to be mediated by beta-catenin. First, a similar phenotype (no primary mouth) is seen after loss of Frzb-1/Crescent function to that seen after temporally and spatially restricted overexpression of Wnt-8. Second, overexpression of either Frzb-1 or Dkk-1 results in an enlarged primary mouth anlage. Third, overexpression of Dkk-1 can restore a primary mouth to embryos in which Frzb-1/Crescent expression has been inhibited. We show that Frzb-1/Crescent function locally promotes basement membrane dissolution in the primary mouth primordium. Consistently, Frzb-1 overexpression decreases RNA levels of the essential basement membrane genes fibronectin and laminin, whereas Wnt-8 overexpression increases the levels of these RNAs. These data are the first to connect Wnt signaling and basement membrane integrity during primary mouth development, and suggest a general paradigm for the regulation of basement membrane remodeling.

Dejellying Xenopus laevis Embryos.

INTRODUCTIONThis protocol presents a method for preparing Xenopus embryos for manipulation. Embryos are surrounded by a series of thick, protective jelly membranes. Removal of these membranes is the first step in most micromanipulation procedures. These membranes must be completely removed for embryo dissection. For microinjection, membranes can be completely or partially removed.

Removing the Vitelline Membrane from Xenopus laevis Embryos.

INTRODUCTIONThis protocol presents a method for the removal of the vitelline membrane from Xenopus embryos, which is essential for embryo dissection experiments. Removal of the vitelline membrane is a technique that becomes easier with practice. The easiest embryos on which to learn this technique are those with a large space between the membrane and the embryo, that is, late neurula stages.

Handling Xenopus laevis Adults.

INTRODUCTIONThis protocol describes the proper technique for handling adult Xenopus animals.

Inducing Ovulation in Xenopus laevis.

INTRODUCTIONThis protocol describes a method for inducing ovulation in Xenopus laevis for the purpose of in vitro fertilization. Ovulation is induced by injection of human chorionic gonadotropin (hCG) into the dorsal lymph sac of a female frog. Females can lay many hundreds of eggs. However, because the actual number of eggs laid and the efficiency of fertilization are unpredictable, ovulation should be induced in more than one female, even when only a few eggs are required.

Isolating Xenopus laevis Testes.

INTRODUCTIONThis protocol describes a method of isolating Xenopus laevis testes for use in in vitro fertilization. The testes from one male Xenopus contain sufficient sperm to fertilize several thousand eggs.

Xenopus laevis Egg Collection.

INTRODUCTIONThis protocol describes procedures for collecting Xenopus laevis eggs from females in which ovulation has been induced.

Xenopus laevis In Vitro Fertilization and Natural Mating Methods.

INTRODUCTIONThis protocol describes in vitro fertilization and natural mating methods for Xenopus laevis.