SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals.

The reiterative deployment of a small cadre of morphogen signals underlies patterning and growth of most tissues during embyogenesis, but how such inductive events result in tissue-specific responses remains poorly understood. By characterizing cis-regulatory modules (CRMs) associated with genes regulated by Sonic hedgehog (Shh), retinoids, or bone morphogenetic proteins in the CNS, we provide evidence that the neural-specific interpretation of morphogen signaling reflects a direct integration of these pathways with SoxB1 proteins at the CRM level. Moreover, expression of SoxB1 proteins in the limb bud confers on mesodermal cells the potential to activate neural-specific target genes upon Shh, retinoid, or bone morphogenetic protein signaling, and the collocation of binding sites for SoxB1 and morphogen-mediatory transcription factors in CRMs faithfully predicts neural-specific gene activity. Thus, an unexpectedly simple transcriptional paradigm appears to conceptually explain the neural-specific interpretation of pleiotropic signaling during vertebrate development. Importantly, genes induced in a SoxB1-dependent manner appear to constitute repressive gene regulatory networks that are directly interlinked at the CRM level to constrain the regional expression of patterning genes. Accordingly, not only does the topology of SoxB1-driven gene regulatory networks provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within the developing neural tube.

Mechanistic differences in the transcriptional interpretation of local and long-range Shh morphogen signaling.

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.

Transcription factor-induced lineage selection of stem-cell-derived neural progenitor cells.

The generation of specific types of neurons from stem cells offers important opportunities in regenerative medicine. However, future applications and proper verification of cell identities will require stringent ways to generate homogeneous neuronal cultures. Here we show that transcription factors like Lmx1a, Phox2b, Nkx2.2, and Olig2 can induce desired neuronal lineages from most expressing neural progenitor cells by a mechanism resembling developmental binary cell-fate switching. Such efficient selection of cell fate resulted in remarkable cellular enrichment that enabled global gene-expression validation of generated neurons and identification of previously unrecognized features in the studied cell lineages. Several sources of stem cells have a limited competence to differentiate into specific neuronal cell types; e.g., dopamine neurons. However, we show that the combination of factors that normally promote either regional or dedicated neuronal specification can overcome limitations in cellular competence and also promote efficient reprogramming in more remote neural contexts, including human neural progenitor cells.

A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning.

The 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.

Rmst is a novel marker for the mouse ventral mesencephalic floor plate and the anterior dorsal midline cells.

The availability of specific markers expressed in different regions of the developing nervous system provides a useful tool to illuminate their development, regulation and function. We have identified by expression profiling a putative non-coding RNA, Rmst, that exhibits prominent expression in the midbrain floor plate region, the isthmus and the roof plate of the anterior neural tube. At the developmental stage when the ventral dopaminergic neuron territory is being established, Rmst expression appears to be restricted to the presumptive dopaminergic neurons of the ventral tegmental area that lies close to the ventral midline. Thus this study presents Rmst as a novel marker for the developing dopaminergic neurons in the mesencephalic floor plate as well as a marker for the dorsal midline cells of the anterior neural tube and the isthmic organizer.