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In many organisms, early germline development takes place within cysts of interconnected cells that form by incomplete cytokinesis and later undergo programmed breakdown. We recently identified similar cell clusters within the fetal mouse ovary, but the fate and functional significance of these germ cell cysts remained unclear. Here, we show that mouse cysts undergo programmed breakdown between 20.5-22.5 dpc, during which approximately 33% of the oocytes survive to form primordial follicles. This process accounts for most of the perinatal reduction in germ cell numbers and germ cell apoptosis reported by previous authors, and suggests that perinatal germ cell loss is a developmentally regulated process that is distinct from the follicular atresia that occurs during adult life. Our observations also suggest a novel function for a transient cyst stage of germ cell development. Prior to breakdown, mitochondria and ER reorganize into perinuclear aggregates, and can be seen within the ring canals joining adjacent germ cells. Cysts may ensure that oocytes destined to form primordial follicles acquire populations of functional mitochondria, through an active process that has been evolutionarily conserved.

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Germ cells in many vertebrate and invertebrate species initiate gametogenesis by forming groups of interconnected cells known as germline cysts. Recent studies using Xenopus, mouse and Drosophila are beginning to uncover the cellular and molecular mechanisms that control germline cyst formation and, in conjunction with morphological evidence, suggest that the process is highly conserved during evolution. This article discusses these recent findings and argues that cysts play an important and general role in germ line development.

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Pair-rule genes serve two important functions during Drosophila development: they first initiate periodic patterns, and subsequently interact with each other to refine these patterns to the precision required for definition of segmental compartments. Previously, we described a pair-rule input region of the runt gene. Here we further characterize this region through the use of reporter gene constructs and by comparison with corresponding sequences from Drosophila virilis. We find that many but not all regulatory properties of this '7-stripe region' are functionally conserved. Moreover, the similarity between these homologous sequences is surprisingly low. When compared to similar data for gap gene input element, our data suggest that pair-rule target sequences are less constrained during evolution, and that functional elements mediating pair-rule interactions can be dispersed over many kilobases.

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Oocytes from many invertebrates initiate development within distinctive cysts of interconnected cells, which are formed through synchronous divisions of a progenitor cell. Recently, processes underlying cyst formation have been extensively characterized at the molecular level in Drosophila. Defects in this process cause sterility in female flies. Early female mouse germ cells are organized as cell clusters as well, but it is uncertain whether these groups are similar to the cysts of invertebrates. We find that mouse germ cells are connected by intercellular bridges in the ovaries of 11.5 to 17.5 days postcoitum embryos; microtubules and organelles have been observed within these bridges. Confocal microscopy shows that cells within mouse clusters divide synchronously and frequently correspond in number to powers of two. Thus, female mouse germ cell clusters exhibit key characteristics of invertebrate germline cysts indicating that the process of germline cyst formation is conserved in the mouse.

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The Runt domain gene AML1 is essential for definitive hematopoiesis during murine embryogenesis. We have isolated Xaml, a Xenopus AML1 homologue in order to investigate the patterning mechanisms responsible for the generation of hematopoietic precursors. Xaml is expressed early in the developing ventral blood island in a pattern that anticipates that of later globin. Analysis of globin and Xaml expression in explants, in embryos with perturbed dorsal ventral patterning, and by lineage tracing indicates that the formation of the ventral blood island is more complex than previously thought and involves contributions from both dorsal and ventral tissues. A truncated Xaml protein interferes with primitive hematopoiesis. Based on these results, we propose that Runt domain proteins function in the specification of hematopoietic stem cells in vertebrate embryos.

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The Drosophila runt gene is the founding member of the Runt domain family of transcriptional regulators. Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicated that flies were likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, we identified two Drosophila genes, referred to as Brother and Big-brother, that have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirmed the functional homology of the Brother, Big-brother, and PEBP2/CBF beta proteins and demonstrated that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins in the presence and absence of their partners were also examined. Our results show that Runt domain proteins bend DNA and that this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos revealed that Brother and Big-brother are likely to interact with runt in vivo and further suggested that the activity of these proteins is not restricted to their interaction with Runt.

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A phylogenetic approach was used to identify conserved regions of the transcriptional regulator Runt. Alignment of the deduced protein sequences from Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virilis revealed eight blocks of high sequence homology separated by regions with little or no homology. The largest conserved block contains the Runt domain, a DNA and protein binding domain conserved in a small family of mammalian transcription factors. The functional properties of the Runt domain from the D. melanogaster gene and the human AML1 (acute myeloid leukemia 1) gene were compared in vitro and in vivo. Electrophoretic mobility-shift assays with Runt/AML1 chimeras demonstrated that the different DNA binding properties of Runt and AML1 are due to differences within their respective Runt domains. Ectopic expression experiments indicated that proteins containing the AML1 Runt domain function in Drosophila embryos and that sequences outside of this domain are important in vivo.

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Retroviruses and related genetic elements generate terminally redundant RNA products by differential polyadenylylation within a long terminal repeat. Expression of the white-apricot (wa) allele of Drosophila melanogaster, which carries an insertion of the 5.1-kilobase retrovirus-like transposable element copia in a small intron, is influenced by signals within copia. By using this indicator, we have isolated a 518-base-pair deletion, 312 base pairs upstream of the copia polyadenylylation site, that is phenotypically like much larger deletions and eliminates RNA species polyadenylylated in copia. This requirement of distant upstream sequences for copia polyadenylylation has implications for the expression of many genetic elements bearing long terminal repeats.

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