Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at an enormous rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in vertebrate model organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches1-3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes3,4, this number falls significantly short of all >22,000 mouse protein-coding genes5. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning6, insertional mutagenesis7-9, antisense morpholino oligonucleotides10, targeted re-sequencing11-13 and zinc finger and TAL endonucleases14-17 have made significant contributions to our understanding of the biological activity of vertebrate genes, but the number of genes studied again falls well short of the >26,000 zebrafish protein-coding genes18. Importantly, for both mice and zebrafish, none of these strategies is particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Enabled by a well-annotated zebrafish reference genome sequence18,19, high-throughput sequencing and efficient chemical mutagenesis, we describe an active project that aims to identify and phenotype disruptive mutations in every zebrafish protein-coding gene. Thus far we have identified potentially disruptive mutations in more than 38% of all known protein coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

Transcription is an essential component of basic cellular and developmental processes. However, early embryonic development occurs in the absence of transcription and instead relies upon maternal mRNAs and proteins deposited in the egg during oocyte maturation. Although the early zebrafish embryo is competent to transcribe exogenous DNA, factors present in the embryo maintain genomic DNA in a state that is incompatible with transcription. The cell cycles of the early embryo titrate out these factors, leading to zygotic transcription initiation, presumably in response to a change in genomic DNA chromatin structure to a state that supports transcription. To understand the molecular mechanisms controlling this maternal to zygotic transition, it is important to distinguish between the maternal and zygotic transcriptomes during this period. Here we use exome sequencing and RNA-seq to achieve such discrimination and in doing so have identified the first zygotic genes to be expressed in the embryo. Our work revealed different profiles of maternal mRNA post-transcriptional regulation prior to zygotic transcription initiation. Finally, we demonstrate that maternal mRNAs are required for different modes of zygotic transcription initiation, which is not simply dependent on the titration of factors that maintain genomic DNA in a transcriptionally incompetent state.

The initial phases of embryonic development occur in the absence of de novo transcription and are instead controlled by maternally inherited mRNAs and proteins. During this initial period, cell cycles are synchronous and lack gap phases. Following this period of transcriptional silence, zygotic transcription begins, the maternal influence on development starts to decrease, and dramatic changes to the cell cycle take place. Here, we discuss recent work that is shedding light on the maternal to zygotic transition and the interrelated but distinct mechanisms regulating the onset of zygotic transcription and changes to the cell cycle during early embryonic development.

During embryonic development, signalling molecules known as morphogens act in a concentration-dependent manner to provide positional information to responding tissues. In the early zebrafish embryo, graded signalling by members of the nodal family induces the formation of mesoderm and endoderm, thereby patterning the embryo into three germ layers. Nodal signalling has also been implicated in the establishment of the dorso-ventral axis of the embryo. Although one can infer the existence of nodal gradients by comparing gene expression patterns in wild-type embryos and embryos in which nodal signalling is diminished or augmented, real understanding can only come from directly observing the gradients. One approach is to determine local ligand concentrations in the embryo, but this is technically challenging, and the presence of inhibitors might cause the effective concentration of a ligand to differ from its actual concentration. We have therefore taken two approaches to visualise a direct response to nodal signalling. In the first, we have used transgenic embryos to study the nuclear accumulation of a Smad2-Venus fusion protein, and in the second we have used bimolecular fluorescence complementation to visualise the formation of a complex between Smad2 and Smad4. This has allowed us to visualise, in living embryos, the formation of a graded distribution of nodal signalling activity. We have quantified the formation of the gradient in time and space, and our results not only confirm that nodal signalling patterns the embryo into three germ layers, but also shed light on its role in patterning the dorso-ventral axis and highlight unexpected complexities of mesodermal patterning.

Chicken growth hormone has been isolated from adenohypophysial tissue from which the glycoprotein hormones had been removed. The procedure entailed alkali extraction, ammonium sulphate precipitation and ion-exchange chromatography on DEAE-cellulose. The resulting fraction was homogeneous, active in the rat tibia bioassay and had a similar isoelectric point, molecular weight and amino acid composition to mammalian growth hormone. A specific homologous radioimmunoassay has been developed using the avian growth hormone.

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SUMMARYDuring early zebrafish development the nodal signalling pathway patterns the embryo into three germ layers, in part by inducing the expression of no tail (ntl), which is essential for correct mesoderm formation. When nodal signalling is inhibited ntl fails to be expressed in the dorsal margin, but ventral ntl expression is unaffected. These observations indicate that ntl transcription is under both nodal-dependent and nodal-independent regulation. Consistent with these observations and with a role for ntl in mesoderm formation, some somites form within the tail region of embryos lacking nodal signalling. In an effort to understand how ntl is regulated and thus how mesoderm forms, we have mapped the elements responsible for nodaldependent and nodal-independent expression of ntl in the margin of the embryo. Our work demonstrates that expression of ntl in the margin is the consequence of two separate enhancers, which act to mediate different mechanisms of mesoderm formation. One of these enhancers responds to nodal signalling, and the other to Wnt and BMP signalling. We demonstrate that the nodal-independent regulation of ntl is essential for tail formation. Misexpression of Wnt and BMP ligands can induce the formation of an ectopic tail, which contains somites, in embryos devoid of nodal signalling, and this tail formation is dependent on ntl function. Similarly, nodal-independent tail somite formation requires ntl. At later stages in development ntl is required for notochord formation, and our analysis has also led to the identification of the enhancer required for ntl expression in the developing notochord.

The concentrations of prolactin, LH, progesterone and GH were measured in the blood of broody bantam hens. The concentration of prolactin was at its highest when the birds began to incubate their eggs and in six out of nine hens it tended to remain raised until the eggs hatched. The increase in the concentration of prolactin was small: in incubating hens it was only 23% higher than in hens caring for their young and 14% higher than in laying hens (P less than 0.05 for both comparisons). The concentration of GH tended to be depressed in hens caring for young but otherwise was not related to reproductive activity. The concentrations of LH and progesterone decreased at the onset of incubation and remained depressed while the hens sat on their eggs (P less than 0.001) for both comparisons). After the chicks hatched, the level of LH began to increase slowly whereas the level of progesterone remained low. The hens stopped showing broody behaviour between 4 and 10 weeks after the chicks had hatched; this corresponded to the time when the concentration of LH had increased to values found in laying hens. These observations provide some evidence that prolactin secretion increases at the onset of incubation and support the view that the hormone is not secreted at an increased rate while hens are caring for their young.