Advances in our understanding of the many levels of regulation of TGFβ and BMP signaling were reported at the recent FASEB Summer Conference entitled `The TGFβ Superfamily: Development and Disease', which was held in Carefree, Arizona, USA, on the northern edge of the Sonoran Desert. This conference was the fifth meeting in a biannual FASEB conference series and, as with the previous meetings, brought together biochemists, geneticists, developmental and tissue biologists interested in the inter-workings of TGFβ/BMP signaling pathways and in the consequences of these pathways going awry.

Transforming growth factor β (TGFβ) pathways are implicated in metazoan development, adult homeostasis and disease. TGFβ ligands signal via receptor serine/threonine kinases that phosphorylate, and activate, intracellular Smad effectors as well as other signaling proteins. Oligomeric Smad complexes associate with chromatin and regulate transcription, defining the biological response of a cell to TGFβ family members. Signaling is modulated by negative-feedback regulation via inhibitory Smads. We review here the mechanisms of TGFβ signal transduction in metazoans and emphasize events crucial for embryonic development.

In many cases, the level, positioning and timing of signaling through the bone morphogenetic protein (BMP) pathway are regulated by molecules that bind BMP ligands in the extracellular space. Whereas many BMP-binding proteins inhibit signaling by sequestering BMPs from their receptors, other BMP-binding proteins cause remarkably context-specific gains or losses in signaling. Here, we review recent findings and hypotheses on the complex mechanisms that lead to these effects, with data from developing systems, biochemical analyses and mathematical modeling.

In recent years, informatics studies have predicted several new ways in which the transforming growth factor β (TGFβ) signaling pathway can be post-translationally regulated. Subsequently, many of these predictions were experimentally validated. These approaches include phylogenetic predictions for the phosphorylation, sumoylation and ubiquitylation of pathway components, as well as kinetic models of endocytosis, phosphorylation and nucleo-cytoplasmic shuttling. We review these studies and provide a brief `how to' guide for phylogenetics. Our hope is to stimulate experimental tests of informatics-based predictions for TGFβ signaling, as well as for other signaling pathways, and to expand the number of developmental pathways that are being analyzed computationally.

The conducting airways (bronchi and bronchioles) and peripheral gas exchange (alveolar) regions of the mammalian lung are generated by a process of branching morphogenesis. Evidence suggests that during embryonic development, the undifferentiated epithelial progenitors are located at the distal tips of the branching epithelium. To test this hypothesis, we used an Id2-CreER^T2 knock-in mouse strain to lineage trace the distal epithelial tip cells during either the pseudoglandular or canalicular phases of development. During the pseudoglandular stage, the tip cells both self-renew and contribute descendents to all epithelial cell lineages, including neuroendocrine cells. In addition, individual Id2⁺ tip cells can self-renew and contribute descendents to both the bronchiolar and alveolar compartments. By contrast, during the later canalicular stage, the distal epithelial tip cells only contribute descendents to the alveoli. Taken together, this evidence supports a model in which the distal tip of the developing lung contains a multipotent epithelial population, the fate of which changes during development.

We report here experiments aimed at understanding the connections between cell competition and growth in the Drosophila wing disc. The principal assay has been to generate discs containing marked cells that proliferate at different rates and to study their interactions and their contribution to the final structure. It is known that single clones of fast-dividing cells within a compartment may occupy the larger part of the compartment without affecting its size. This has suggested the existence of interactions involving cell competition between fast- and slow-dividing cells directed to accommodate the contribution of each cell to the final compartment. Here we show that indeed fast-dividing cells can outcompete slow-dividing ones in their proximity. However, we argue that this elimination is of little consequence because preventing apoptosis, and therefore cell competition, in those compartments does not affect the size of the clones or the size of the compartments. Our experiments indicate that cells within a compartment proliferate autonomously at their own rate. The contribution of each cell to the compartment is exclusively determined by its division rate within the frame of a size control mechanism that stops growth once the compartment has reached the final arresting size. This is supported by a computer simulation of the contribution of individual fast clones growing within a population of slower dividing cells and without interacting with them. The values predicted by the simulation are very close to those obtained experimentally.

Dynamic morphological changes in mitochondria depend on the balance of fusion and fission in various eukaryotes, and are crucial for mitochondrial activity. Mitochondrial dysfunction has emerged as a common theme that underlies numerous neurological disorders, including neurodegeneration. However, how this abnormal mitochondrial activity leads to neurodegenerative disorders is still largely unknown. Here, we show that the Drosophila mitochondrial protein Preli-like (Prel), a member of the conserved PRELI/MSF1 family, contributes to the integrity of mitochondrial structures, the activity of respiratory chain complex IV and the cellular ATP level. When Prel function was impaired in neurons in vivo, the cellular ATP level decreased and mitochondria became fragmented and sparsely distributed in dendrites and axons. Notably, the dendritic arbors were simplified and downsized, probably as a result of breakage of proximal dendrites and progressive retraction of terminal branches. By contrast, abrogation of the mitochondria transport machinery per se had a much less profound effect on the arbor morphogenesis. Interestingly, overexpression of Drob-1 (Debcl), a Drosophila Bax-like Bcl-2 family protein, in the wild-type background produced dendrite phenotypes that were reminiscent of the prel phenotype. Moreover, expression of the Drob-1 antagonist Buffy in prel mutant neurons substantially restored the dendritic phenotype. Our observations suggest that Prel-dependent regulation of mitochondrial activity is important for both growth and prevention of breakage of dendritic branches.

The characterisation of interspecies differences in gene regulation is crucial to understanding the molecular basis of phenotypic diversity and evolution. The atonal homologue Atoh7 participates in the ontogenesis of the vertebrate retina. Our study reveals how evolutionarily conserved, non-coding DNA sequences mediate both the conserved and the species-specific transcriptional features of the Atoh7 gene. In the mouse and chick retina, species-related variations in the chromatin-binding profiles of bHLH transcription factors correlate with distinct features of the Atoh7 promoters and underlie variations in the transcriptional rates of the Atoh7 genes. The different expression kinetics of the Atoh7 genes generate differences in the expression patterns of a set of genes that are regulated by Atoh7 in a dose-dependent manner, including those involved in neurite outgrowth and growth cone migration. In summary, we show how highly conserved regulatory elements are put to use in mediating non-conserved functions and creating interspecies neuronal diversity.

Normal patterning of tissues and organs requires the tight restriction of signaling molecules to well-defined organizing centers. In the limb bud, one of the main signaling centers is the zone of polarizing activity (ZPA) that controls growth and patterning through the production of sonic hedgehog (SHH). The appropriate temporal and spatial expression of Shh is crucial for normal limb bud patterning, because modifications, even if subtle, have important phenotypic consequences. However, although there is a lot of information about the factors that activate and maintain Shh expression, much less is known about the mechanisms that restrict its expression to the ZPA. In this study, we show that BMP activity negatively regulates Shh transcription and that a BMP-Shh negative-feedback loop serves to confine Shh expression. BMP-dependent downregulation of Shh is achieved by interfering with the FGF and Wnt signaling activities that maintain Shh expression. We also show that FGF induction of Shh requires protein synthesis and is mediated by the ERK1/2 MAPK transduction pathway. BMP gene expression in the posterior limb bud mesoderm is positively regulated by FGF signaling and finely regulated by an auto-regulatory loop. Our study emphasizes the intricacy of the crosstalk between the major signaling pathways in the posterior limb bud.

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. We demonstrate that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. We show that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance our understanding of a key gene regulatory network that, when disrupted, causes HPE.

Wnt signaling through Frizzled proteins guides posterior cells and axons in C. elegans into different spatial domains. Here we demonstrate an essential role for Wnt signaling through Ror tyrosine kinase homologs in the most prominent anterior neuropil, the nerve ring. A genetic screen uncovered cwn-2, the C. elegans homolog of Wnt5, as a regulator of nerve ring placement. In cwn-2 mutants, all neuronal structures in and around the nerve ring are shifted to an abnormal anterior position. cwn-2 is required at the time of nerve ring formation; it is expressed by cells posterior of the nerve ring, but its precise site of expression is not critical for its function. In nerve ring development, cwn-2 acts primarily through the Wnt receptor CAM-1 (Ror), together with the Frizzled protein MIG-1, with parallel roles for the Frizzled protein CFZ-2. The identification of CAM-1 as a CWN-2 receptor contrasts with CAM-1 action as a non-receptor in other C. elegans Wnt pathways. Cell-specific rescue of cam-1 and cell ablation experiments reveal a crucial role for the SIA and SIB neurons in positioning the nerve ring, linking Wnt signaling to specific cells that organize the anterior nervous system.

Formation of the vertebrate embryo is known to depend on the activity of organizing centers. The dorsal Spemann organizer is the source of growth factor antagonists that participate in the creation of signaling gradients. In various species, the existence of head, trunk and trunk-tail inducers has been proposed to explain the formation of different parts of the embryo along the anteroposterior (A/P) axis. In zebrafish, two organizing centers have been described, the dorsal and tail organizers, located at the dorsal and ventral gastrula margins, respectively. Here, we report that organizer functions are executed not only by the dorsal and ventral margins, but also by all parts of the blastula-gastrula margin. The position of different marginal territories along the dorsoventral axis defines the A/P nature of the structures they are able to organize. At the molecular level, we show that this organizing activity results from the simultaneous activation of BMP and Nodal signaling pathways. Furthermore, the A/P character of the organized structures is not defined by absolute levels but instead by the ratio of BMP and Nodal activities. Rather than resulting from the activity of discrete centers, organization of the zebrafish embryo depends on the activity of the entire margin acting as a continuous and global organizer that is established by a gradual ventral-to-dorsal modulation of the ratio of marginal BMP to Nodal activity.

The establishment of sexual identity is a crucial step of germ cell development in sexually reproducing organisms. Sex determination in the germline is controlled differently than in the soma, and often depends on communication from the soma. To investigate how sexual identity is established in the Drosophila germline, we first conducted a molecular screen for genes expressed in a sex-specific manner in embryonic germ cells. Sex-specific expression of these genes is initiated at the time of gonad formation (stage 15), indicating that sexual identity in the germline is established by this time. Experiments where the sex of the soma was altered relative to that of the germline (by manipulating transformer) reveal a dominant role for the soma in regulating initial germline sexual identity. Germ cells largely take on the sex of the surrounding soma, although the sex chromosome constitution of the germ cells still plays some role at this time. The male soma signals to the germline through the JAK/STAT pathway, while the nature of the signal from the female soma remains unknown. We also find that the genes ovo and ovarian tumor (otu) are expressed in a female-specific manner in embryonic germ cells, consistent with their role in promoting female germline identity. However, removing the function of ovo and otu, or reducing germline function of Sex lethal, had little effect on establishment of germline sexual identity. This is consistent with our findings that signals from the soma are dominant over germline autonomous cues at the initial stage of germline sex determination.

Cell fate determination is governed by complex signaling molecules at appropriate concentrations that regulate the cell decision-making process. In vertebrates, however, concentration and kinetic parameters are practically unknown, and therefore the mechanism by which these molecules interact is obscure. In myogenesis, for example, multipotent cells differentiate into skeletal muscle as a result of appropriate interplay between several signaling molecules, which is not sufficiently characterized. Here we demonstrate that treatment of biochemical events with SAT (satisfiability) formalism, which has been primarily applied for solving decision-making problems, can provide a simple conceptual tool for describing the relationship between causes and effects in biological phenomena. Specifically, we applied the Lukasiewicz logic to a diffusible protein system that leads to myogenesis. The creation of an automaton that describes the myogenesis SAT problem has led to a comprehensive overview of this non-trivial phenomenon and also to a hypothesis that was subsequently verified experimentally. This example demonstrates the power of applying Lukasiewicz logic in describing and predicting any decision-making problem in general, and developmental processes in particular.

Coordination of voluntary motor activity depends on the generation of the appropriate neuronal subtypes in the basal ganglia and their integration into functional neuronal circuits. The largest nucleus of the basal ganglia, the striatum, contains two classes of neurons: the principal population of medium-sized dense spiny neurons (MSNs; 97-98% of all striatal neurons in rodents), which project to the globus pallidus and the substantia nigra, and the locally projecting striatal interneurons (SINs; 2-3% in rodents). SINs are further subdivided into two non-overlapping groups: those producing acetylcholine (cholinergic) and those producing -amino butyric acid (GABAergic). Despite the pivotal role of SINs in integrating the output of striatal circuits and the function of neuronal networks in the ventral forebrain, the lineage relationship of SIN subtypes and the molecular mechanisms that control their differentiation are currently unclear. Using genetic fate mapping, we demonstrate here that the majority of cholinergic and GABAergic SINs are derived from common precursors generated in the medial ganglionic eminence during embryogenesis. These precursors express the LIM homeodomain protein Lhx6 and have characteristics of proto-GABAergic neurons. By combining gene expression analysis with loss-of-function and misexpression experiments, we provide evidence that the differentiation of the common precursor into mature SIN subtypes is regulated by the combinatorial activity of the LIM homeodomain proteins Lhx6, Lhx7 (Lhx8) and Isl1. These studies suggest that a LIM homeodomain transcriptional code confers cell-fate specification and neurotransmitter identity in neuronal subpopulations of the ventral forebrain.