RORβ Induces Barrel-like Neuronal Clusters in the Developing Neocortex

Neurons in layer IV of the rodent whisker somatosensory cortex are tangentially organized in periodic clusters called barrels, each of which is innervated by thalamocortical axons transmitting sensory information from a single principal whisker, together forming a somatotopic map of the whisker pad. Proper thalamocortical innervation is critical for barrel formation during development, but the molecular mechanisms controlling layer IV neuron clustering are unknown. Here, we investigate the role in this mapping of the nuclear orphan receptor RORβ, which is expressed in neurons in layer IV during corticogenesis. We find that RORβ protein expression specifically increases in the whisker barrel cortex during barrel formation and that in vivo overexpression of RORβ is sufficient to induce periodic barrel-like clustering of cortical neurons. Remarkably, this clustering can be induced as early as E18, prior to innervation by thalamocortical afferents and whisker derived-input. At later developmental stages, these ectopic neuronal clusters are specifically innervated by thalamocortical axons, demonstrated by anterograde labeling from the thalamus and by expression of thalamocortical-specific synaptic markers. Together, these data indicate that RORβ expression levels control cytoarchitectural patterning of neocortical neurons during development, a critical process for the topographical mapping of whisker input onto the cortical surfac

Pre-Steady-State Decoding of the Bicoid Morphogen Gradient

Morphogen gradients are established by the localized production and subsequent diffusion of signaling molecules. It is generally assumed that cell fates are induced only after morphogen profiles have reached their steady state. Yet, patterning processes during early development occur rapidly, and tissue patterning may precede the convergence of the gradient to its steady state. Here we consider the implications of pre-steady-state decoding of the Bicoid morphogen gradient for patterning of the anterior–posterior axis of the Drosophila embryo. Quantitative analysis of the shift in the expression domains of several Bicoid targets (gap genes) upon alteration of bcd dosage, as well as a temporal analysis of a reporter for Bicoid activity, suggest that a transient decoding mechanism is employed in this setting. We show that decoding the pre-steady-state morphogen profile can reduce patterning errors caused by fluctuations in the rate of morphogen production. This can explain the surprisingly small shifts in gap and pair-rule gene expression domains observed in response to alterations in bcd dosage

RORβ induces barrel-like neuronal clusters in the developing neocortex

Neurons in layer IV of the rodent whisker somatosensory cortex are tangentially organized in periodic clusters called barrels, each of which is innervated by thalamocortical axons transmitting sensory information from a single principal whisker, together forming a somatotopic map of the whisker pad. Proper thalamocortical innervation is critical for barrel formation during development, but the molecular mechanisms controlling layer IV neuron clustering are unknown. Here, we investigate the role in this mapping of the nuclear orphan receptor RORβ, which is expressed in neurons in layer IV during corticogenesis. We find that RORβ protein expression specifically increases in the whisker barrel cortex during barrel formation and that in vivo overexpression of RORβ is sufficient to induce periodic barrel-like clustering of cortical neurons. Remarkably, this clustering can be induced as early as E18, prior to innervation by thalamocortical afferents and whisker derived-input. At later developmental stages, these ectopic neuronal clusters are specifically innervated by thalamocortical axons, demonstrated by anterograde labeling from the thalamus and by expression of thalamocortical-specific synaptic markers. Together, these data indicate that RORβ expression levels control cytoarchitectural patterning of neocortical neurons during development, a critical process for the topographical mapping of whisker input onto the cortical surface

Lmo4 and Clim1 progressively delineate cortical projection neuron subtypes during development

Molecular controls over the development of the exceptional neuronal subtype diversity of the cerebral cortex are now beginning to be identified. The initial subtype fate decision early in the life of a neuron, and the malleability of this fate when the balance of key postmitotic signals is modified, reveals not only that a neuron is deterministically set on a general developmental path at its birth, but also that this program must be precisely executed during postmitotic differentiation. Here, we show that callosal projection neurons (CPN) and subcerebral projection neurons (subcerebral PN) in layer V of the neocortex share aspects of molecular identity after their birth that are progressively resolved during differentiation. The LIM-homeodomain-related genes Lmo4 and Clim1 are initially expressed by both CPN and subcerebral PN in layer V, and only during mid to late differentiation does expression of Lmo4 and Clim1 become largely segregated into distinct neuronal subtypes. This progressive postmitotic resolution of molecular identity reveals similarities and possibly shared evolutionary origin between layer V CPN and subcerebral PN, and provides insight into how and when these neuronal subtypes achieve their distinct identities during cortical development.Stem Cell and Regenerative Biolog

Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting

For precise motor control, distinct subpopulations of corticospinal neurons (CSN) must extend axons to distinct spinal segments, from proximal targets in the brainstem and cervical cord to distal targets in thoracic and lumbar spinal segments. We find that developing CSN subpopulations exhibit striking axon targeting specificity in spinal white matter, which establishes the foundation for durable specificity of adult corticospinal circuitry. Employing developmental retrograde and anterograde labeling, and their distinct neocortical locations, we purified developing CSN subpopulations using fluorescence-activated cell sorting to identify genes differentially expressed between bulbar-cervical and thoracolumbar-projecting CSN subpopulations at critical developmental times. These segmentally distinct CSN subpopulations are molecularly distinct from the earliest stages of axon extension, enabling prospective identification even before eventual axon targeting decisions are evident in the spinal cord. This molecular delineation extends beyond simple spatial separation of these subpopulations in the cortex. Together, these results identify candidate molecular controls over segmentally specific corticospinal axon projection targeting

Analysis of Bcd-Dependent <i>lac</i>Z Reporter Expression over Cleavage Cycles 11, 12, and 13

<div><p>The posterior boundary of the <i>lac</i>Z expression domain is shown as a function of the normalized nuclear density for each embryo (colored dots). Embryos fall into three classes of nuclear density corresponding to their cleavage cycle (11, red; 12, green; and 13, blue). Average nuclear density and domain boundary for each cycle are indicated by big circles, and whiskers denote standard deviations.</p> <p>(B) The distribution of the expression boundary is shown for the three cycles (bin size is 2% EL). Note the progression in time of the boundary.</p></div

Properties of the Pre-Steady-State Morphogen Distribution

<div><p>(A) The morphogen distribution <i>M</i>(<i>x</i>, <i>t</i>) is plotted as a function of position <i>x</i> for different times <i>t</i> (legend). The plots were obtained by solving the reaction diffusion <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-e001" target="_blank">Equation 1</a> in one dimension (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-sd001" target="_blank">Protocol S1</a>). The position <i>x</i> is in units of the decay length scale , while the time <i>t</i> is in units of the decay time <i>τ.</i></p> <p>(B) Same as in (A), except that each profile was rescaled such that it has unit concentration at <i>x</i> = 0 and decays to 1/<i>e</i> at <i>x =</i> 1. Note the logarithmic scale. At early times, the profile tail decays super-exponentially, while at later times the morphogen distribution is well approximated by an exponential.</p> <p>(C) Alteration of the steady-state morphogen concentration upon 2-fold reduction in morphogen production rate. The original profile corresponds to the solid line and the altered profile to the dotted line. Note that the indicated positional shifts Δ<i>x = |x − x′|</i> at different morphogen thresholds do not depend on the position <i>x</i>.</p> <p>(D) Same as in (C) but for the pre-steady-state profile.</p> <p>(E) The shift Δ<i>x</i> is shown as a function of <i>x</i> for different times <i>t</i> (see legend in [A]). For pre-steady-state profiles, Δ<i>x</i> decreases as a function of <i>x</i>.</p> <p>(F) The shift Δ<i>x</i> as a function of time <i>t</i> is shown for different positions <i>x</i>, as indicated. While at late times the shift is almost independent of the position, at early times the shift decreases with increasing distance from the source.</p> <p>(G) The exact solution for the pre-steady-state profile was fitted to the phenomenological approximation <i>M_p</i>(<i>x, t</i>) = <i>M</i>₀(<i>t</i>) exp[−(<i>x</i>/<i>λ</i>(<i>t</i>))<i>^p</i>^(<i>t</i>)]<i>.</i> The best-fitted exponent <i>p</i> is shown as a function of time (in units of the decay time <i>τ</i>).</p> <p>(H) To estimate the deviation of the time-dependent solution from an exponential, we compared the residual error obtained for the best-fit <i>p</i> approximation <i>(R_p)</i> to the residual error obtained when fitting to exponential with <i>p =</i>1 (<i>R</i>_lin). The ratio of these residual errors is shown as a function of time.</p> <p>(I) The best-fitted exponent <i>p</i> for quantitative Bcd profiles corresponding to wild-type embryos between cycles 10 and 14. Data were downloaded from the FlyEx database [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-b056" target="_blank">56</a>]. For embryos in cycles 10–12, the average <i>p</i> is significantly larger than 1, indicating superexponential decay, while Bcd profiles at cycles 13 and 14 are consistent with <i>p</i> = 1. Note, however, the large fluctuations.</p></div

Quantitative Effects of Altered Maternal <i>bcd</i> Gene Dosage on Zygotic Target Gene Expression

<div><p>(A) Dorsal view of a representative cycle-14 wild-type <i>Drosophila</i> embryo stained for the Eve (green) and Kr (red) proteins. The contours of the embryo were determined from the transmitted light image (blue).</p> <p>(B) (C) Quantitative analysis of stripe positions was performed by semiautomated software as follows: the positions of the embryo poles and of the first and last Eve stripes were defined manually. Based on these definitions, a rectangular area (yellow dashed in [A]) corresponding in height to 10% EL was extracted automatically. Intensity profiles (solid lines in [C]) were obtained by averaging the fluorescence signal along the dorsal–ventral axis in this area and subsequent smoothing. Stripe positions (green dotted lines in [C]) and boundaries (red dotted lines in [C]) were defined based on local maxima of these profiles and their first derivative, respectively.</p> <p>(D) Expression domains of Eve (green) and the gap genes <i>Gt, Hb,</i> or <i>Kr</i> (red; as indicated) in embryos derived from females bearing one, two, or four copies of <i>bcd.</i> In each panel, the top part displays a representative confocal image, while quantitative results obtained from multiple embryos are shown at the bottom part. The widths of the stripes correspond to the standard errors (bright) and deviations (shaded). <i>n</i> denotes the number of embryos used in each analysis.</p> <p>(E) Observed shifts of target gene expression domains (center, left, and right boundaries, if applicable) in embryos with one functional <i>bcd</i> allele (1 × <i>bcd</i>) as a function of the wild-type (2 × <i>bcd</i>) position. Gray lines indicate theoretical predictions for different Bcd decay times (compare with <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-g001" target="_blank">Figure 1</a>G and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-g001" target="_blank">1</a>H).</p> <p>(F) Same as in (G) but for embryos with four copies of <i>bcd</i> (4 × <i>bcd</i>).</p></div

In Silico Simulation of the Gap Gene Network

<div><p>(A) A scheme of the gap gene interactions used: Bcd activates the expression of the gap genes in a concentration-dependent manner. Most gap genes mutually suppress each other (see Figure 9 in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-b045" target="_blank">45</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-b046" target="_blank">46</a>]). These interactions were modeled by a set of reaction diffusion equations, as specified in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050046#pbio-0050046-sd001" target="_blank">Protocol S1</a>.</p> <p>(B) Spatial distributions of the different network proteins (color-coded as in [A]), at a time when the Bcd gradient has fully evolved and is close to exponential are shown for different <i>bcd</i> dosages. The gap genes are expressed in adjacent stripes, consistent with their in vivo expression domains.</p> <p>(C) The position of the Hb expression boundary in wild-type embryos (with 2 copies of <i>bcd</i>) and in embryos bearing altered <i>bcd</i> dosage (one and four copies, as shown). The results in our simulation (black circles) are compared to the experimental measurements (blue bars). Also shown is the prediction based on a steady-state gradient (red bars).</p> <p>(D) The temporal change in the position of the Hb expression boundary (diamonds) and in the Bcd concentration at this position (circles) are shown. We considered the wild-type situation (2 × <i>bcd</i>) and show the behavior following the initialization of gap gene expression.</p> <p>(E) We performed the simulations for different values of the Bcd diffusion constant <i>D</i>. Shown here is the shift of the Hb expression boundary in embryos with one <i>bcd</i> allele (1 × <i>bcd</i>) with respect to the wild-type as a function of <i>D</i>.</p> <p>(F) Same as in (E) but for different values of the gap gene diffusion constant.</p> <p>(G) Shifts of gap gene expression domains (center, left and right boundary, if applicable) in embryos with one functional <i>bcd</i> allele (1 × <i>bcd</i>) as a function of the wild-type (2 × <i>bcd</i>) position.</p> <p>(H) Same as in (G) but simulating embryos with four copies of <i>bcd</i> (4 × <i>bcd</i>).</p></div