Maternal factors control development prior to the activation of the embryonic genome. In vertebrates, little is known about the molecular mechanisms by which maternal factors regulate embryonic development. To understand the processes controlled by maternal factors and identify key genes involved, we embarked on a maternal-effect mutant screen in the zebrafish. We identified 68 maternal-effect mutants. Here we describe 15 mutations in genes controlling processes prior to the midblastula transition, including egg development, blastodisc formation, embryonic polarity, initiation of cell cleavage, and cell division. These mutants exhibit phenotypes not previously observed in zygotic mutant screens. This collection of maternal-effect mutants provides the basis for a molecular genetic analysis of the maternal control of embryogenesis in vertebrates.

Many maternal factors in the oocyte persist in the embryo. They are required to initiate zygotic transcription but also function beyond this stage, where they interact with zygotic gene products during embryonic development. In a four-generation screen in the zebrafish, we identified 47 maternal-effect and five paternal-effect mutants that manifest their phenotypes at the time of, or after, zygotic genome activation. We propagated a subset of 13 mutations that cause developmental arrest at the midblastula transition, defects in cell viability, embryonic morphogenesis, and establishment of the embryonic body plan. This diverse group of mutants, many not previously observed in vertebrates, demonstrates a substantial maternal contribution to the "zygotic" period of embryogenesis and a surprising degree of paternal control. These mutants provide powerful tools to dissect the maternal and paternal control of vertebrate embryogenesis.

The short-term survival of highly purified embryonic spinal motor neurons (SMNs) in culture can be promoted by many peptide trophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF). We have asked whether these peptides are sufficient to promote the long-term survival of purified E15 SMNs. Contrary to previous reports, we find that when SMNs are cultured in serum-free medium containing a single peptide trophic factor only approximately one-third of the cells survive for 3 d in culture. When multiple factors are combined, additive effects on survival are observed transiently, but by 7 d of culture the majority of SMNs has died. Surprisingly, when cAMP levels are elevated, the majority of SMNs extend processes and survive for 1 week in culture in the absence of peptide trophic factors, even in low-density cultures. A combination of five peptide trophic factors, together with cAMP elevation, promotes the long-term survival of most of the SMNs in serum-free culture for 3 weeks. These findings provide useful culture conditions for studying the properties of SMNs and have implications for the treatment of motor neuron diseases.

Whereas PNS neurons in culture are intrinsically responsive to peptide trophic factors, retinal ganglion cells (RGCs) are not unless they are depolarized, or their intracellular levels of cyclic AMP (cAMP) are elevated. We show here that depolarization increases cAMP in cultured RGCs sufficiently to enhance their responsiveness and that the trophic responsiveness of developing RGCs in intact retinas depends on physiological levels of activity and cAMP elevation. Responsiveness is lost after axotomy but is restored by cAMP elevation. The death of axotomized RGCs can be prevented if they are simultaneously stimulated by several trophic factors together with cAMP elevation. Thus, the death of RGCs after axotomy is not caused solely by the loss of retrograde trophic stimuli but also by a profound loss of trophic responsiveness.

Adenosine 3:5-cyclic monophosphate (cAMP) is a key second messenger in signaling pathways governing many cellular processes. To define the subcellular localization and relative abundance of cAMP, we developed a novel immunochemical approach based on acrolein fixation to visualize cAMP within cells. We describe here the fixation and immobilization of cAMP within cells and the production of specific, high titer polyclonal antibodies that recognize cAMP. Relative levels of cAMP immunofluorescence were quantitated in glial cells (oligodendrocytes, astrocytes, Schwann cells, and glioma cells) that were either untreated or treated with activators of endogenous adenylyl cyclase to raise cAMP levels. In treated cells, cAMP immunofluorescence is strongly localized in the perinuclear cytoplasm.Many signals that regulate the growth, development, and metabolism in cells use cAMP as a second messenger (1, 2). Neurotransmitters, growth factors, and hormones signal to appropriate receptors, causing either the stimulation or inhibition of adenylyl cyclase through intermediate guanyl nucleotide regulatory proteins. Adenylyl cyclase catalyzes the conversion of ATP to cAMP, which acts by binding to protein kinase A (PKA 1 ) and causing the release of activated catalytic subunits, or by binding and activating cAMP-gated ion channels (3-6). Activated PKA regulates the function of pre-existing cellular proteins by selective phosphorylation and also regulates the synthesis of new cellular proteins by phosphorylating and altering the activity of transcription factors (7-9).In the oligodendrocyte, the myelinating cell of the central nervous system, cAMP is thought to play independent roles in the regulation of cell growth and differentiation. Precursor cells, which are mobile and proliferative, exit the cell cycle upon exposure to cAMP analogs (10). In oligodendrocyte-lineage cells that are differentiating, exposure to cAMP analogs or activators of adenylyl cyclase increases the rate of differentiation (11,12). Whereas experimentally induced cAMP can have potent regulatory effects in these processes, the role of cAMP in normal oligodendrocyte development is not well understood. Cyclic AMP has similar effects in the peripheral myelinating glia, Schwann cells (13), and it is thought that axonal contact causes an elevation in cAMP, inducing proliferation and differentiation (14).Many questions remain about how cAMP relays signals, due in part to technical limitations that have prevented determination of the subcellular distribution of cAMP or comparison of cAMP levels between single cells. Homogeneous populations of cells may vary in their cAMP content from cell to cell, and conventional assays of cAMP extracted from homogenates of such a population generate a simple average level for the set (15). These assay procedures also mask any useful information about cell-specific changes in cAMP levels in the heterogeneous environments of tissue or cell culture. Finally, there have been no methods for accurately measuring free cAMP levels within ...