Neural progenitor cells often produce distinct types of neurons in a specific order, but the determinants that control the sequential generation of distinct neuronal subclasses in the vertebrate CNS remain poorly defined. We examined the sequential generation of visceral motor neurons and serotonergic neurons from a common pool of neural progenitors located in the ventral hindbrain. We found that the temporal specification of these neurons varies along the anterior-posterior axis of the hindbrain, and that the timing of their generation critically depends on the integrated activities of Nkx-and Hox-class homeodomain proteins. A primary function of these proteins is to coordinate the spatial and temporal activation of the homeodomain protein Phox2b, which in turn acts as a binary switch in the selection of motor neuron or serotonergic neuronal fate. These findings assign new roles for Nkx, Hox, and Phox2 proteins in the control of temporal neuronal fate determination, and link spatial and temporal patterning of CNS neuronal fates. Neuronal cell diversity is established by mechanisms that operate in space and over time during central nervous system (CNS) development. Insight has been obtained regarding the initial steps of spatial patterning of neurons along the dorsal-ventral (DV) and anterior-posterior (AP) axes of the neural tube Jessell 2000). Local inductive signals determine the spatial pattern of expression of transcription factors along both these axes, so that neural progenitors at different positions acquire distinct molecular identities. In the ventral neural tube, neuronal fate along the DV axis depends on the Shh-mediated patterning of Nkx-, Dbx-, Pax-, and Irx-class homeodomain (HD) proteins . Along the AP axis, the overlapping, or nested, expression pattern of Hox HD proteins provides positional values that influence the fate of neurons . Despite significant advances, however, DV and AP patterning have generally been analyzed independently, leaving open the issue as to what degree these orthogonal patterning mechanisms are integrated (Davenne et al. 1999;Gaufo et al. 2000). Compared to spatial patterning, little is known about the mechanisms that underlie how neural progenitors produce distinct types of neurons in a specific temporal order. Studies of the retina (Livesey and Cepko 2001) and developing neo-cortex (Monuki and Walsh 2001) suggest that the sequential production of different neuronal subtypes reflects temporal changes in neural progenitors, either in response to extrinsic cues or mechanisms intrinsic to neural progenitor cells. Recent data indicate that modulation of Notch signaling by the bHLH protein Mash1 and the HD proteins Dlx1/2 may control the sequential specification of progenitors in subcortical areas of the telencephalon (Yun et al. 2002). Apart from this, few molecular determinants that influence these temporal processes in the vertebrate CNS have been identified to date. ResultsTo address how spatial and temporal aspects of cell patterning are integrated during development, w...

gene regulation ͉ hydroxylation ͉ signal transduction R educed oxygen levels (hypoxia) lead to a set of cellular adaptations, including increased angiogenesis and erythropoiesis and a switch to glycolytic metabolism. The cellular machinery that senses hypoxia is composed of several proteins. A critical component is the transcription factor hypoxiainducible factor 1␣ (HIF-1␣) (1). The level and activity of HIF-1␣ are controlled by oxygen-dependent prolyl (PHD) and asparaginyl factor-inhibiting HIF-1␣ (FIH-1)] hydroxylases. PHDs hydroxylate two proline residues in the degradation domain of HIF-1␣ in normoxia, which makes HIF-1␣ a substrate for the von Hippel-Lindau E3 ubiquitin ligase and proteasomal degradation. After stabilization in hypoxia, HIF-1␣ interacts with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to hypoxia response elements (HREs)

Morphogens orchestrate tissue patterning in a concentration-dependent fashion during vertebrate embryogenesis, yet little is known of how positional information provided by such signals is translated into discrete transcriptional outputs. Here we have identified and characterized cis-regulatory modules (CRMs) of genes operating downstream of graded Shh signaling and bifunctional Gli proteins in neural patterning. Unexpectedly, we find that Gli activators have a noninstructive role in long-range patterning and cooperate with SoxB1 proteins to facilitate a largely concentration-independent mode of gene activation. Instead, the opposing Gli-repressor gradient is interpreted at transcriptional levels, and, together with CRM-specific repressive input of homeodomain proteins, comprises a repressive network that translates graded Shh signaling into regional gene expression patterns. Moreover, local and long-range interpretation of Shh signaling differs with respect to CRM context sensitivity and Gli-activator dependence, and we propose that these differences provide insight into how morphogen function may have mechanistically evolved from an initially binary inductive event.

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Abstract-Independent component analysis (ICA) has recently been proposed as a tool to unmix hyperspectral data. ICA is founded on two assumptions: 1) the observed spectrum vector is a linear mixture of the constituent spectra (endmember spectra) weighted by the correspondent abundance fractions (sources); 2) sources are statistically independent. Independent factor analysis (IFA) extends ICA to linear mixtures of independent sources immersed in noise. Concerning hyperspectral data, the first assumption is valid whenever the multiple scattering among the distinct constituent substances (endmembers) is negligible, and the surface is partitioned according to the fractional abundances. The second assumption, however, is violated, since the sum of abundance fractions associated to each pixel is constant due to physical constraints in the data acquisition process. Thus, sources cannot be statistically independent, this compromising the performance of ICA/IFA algorithms in hyperspectral unmixing. This paper studies the impact of hyperspectral source statistical dependence on ICA and IFA performances. We conclude that the accuracy of these methods tends to improve with the increase of the signature variability, of the number of endmembers, and of the signal-to-noise ratio. In any case, there are always endmembers incorrectly unmixed. We arrive to this conclusion by minimizing the mutual information of simulated and real hyperspectral mixtures. The computation of mutual information is based on fitting mixtures of Gaussians to the observed data. A method to sort ICA and IFA estimates in terms of the likelihood of being correctly unmixed is proposed.Index Terms-Independent component analysis (ICA), independent factor analysis (IFA), mixture of Gaussians, unmixing hyperspectral data.

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.

How the sequential specification of neurons and progressive loss of potency associated with aging neural progenitors are regulated in vertebrate brain development is poorly understood. By examining a temporal differentiation lineage in the hindbrain, we here identify Tgfβ as a switch signal that executes the transition between early and late phases of neurogenesis and concurrently constrains progenitor potency. Young progenitors have inherent competence to produce late-born neurons, but implementation of late-differentiation programs requires suppression of early identity genes achieved through temporally programmed activation of Tgfβ downstream of Shh signaling. Unexpectedly, we find that sequentially occurring fate-switch decisions are temporally coupled, and onset of Tgfβ signaling appears thereby to impact on the overall lifespan of the temporal lineage. Our study establishes Tgfβ as a regulator of temporal identity and potency of neural stem cells, and provides proof of concept that Tgfβ can be applied to modulate temporal specification of neurons in stem cell engineering.

SUMMARYThe deployment of morphogen gradients is a core strategy to establish cell diversity in developing tissues, but little is known about how small differences in the concentration of extracellular signals are translated into robust patterning output in responding cells. We have examined the activity of homeodomain proteins, which are presumed to operate downstream of graded Shh signaling in neural patterning, and describe a feedback circuit between the Shh pathway and homeodomain transcription factors that establishes non-graded regulation of Shh signaling activity. Nkx2 proteins intrinsically strengthen Shh responses in a feed-forward amplification and are required for ventral floor plate and p3 progenitor fates. Conversely, Pax6 has an opposing function to antagonize Shh signaling, which provides intrinsic resistance to Shh responses and is important to constrain the inductive capacity of the Shh gradient over time. Our data further suggest that patterning of floor plate cells and p3 progenitors is gated by a temporal switch in neuronal potential, rather than by different Shh concentrations. These data establish that dynamic, non-graded changes in responding cells are essential for Shh morphogen interpretation, and provide a rationale to explain mechanistically the phenomenon of cellular memory of morphogen exposure.