Clifton W. Ragsdale

Cell-type homologies and the origins of the neocortex

The six-layered neocortex is a uniquely mammalian structure with evolutionary origins that remain in dispute. One long-standing hypothesis, based on similarities in neuronal connectivity, proposes that homologs of the layer 4 input and layer 5 output neurons of neocortex are present in the avian forebrain, where they contribute to specific nuclei rather than to layers. We devised a molecular test of this hypothesis based on layer-specific gene expression that is shared across rodent and carnivore neocortex. Our findings establish that the layer 4 input and the layer 5 output cell types are conserved across the amniotes, but are organized into very different architectures, forming nuclei in birds, cortical areas in reptiles, and cortical layers in mammals.

The octopus genome and the evolution of cephalopod neural and morphological novelties

Coleoid cephalopods (octopus, squid and cuttlefish) are active, resourceful predators with a rich behavioural repertoire. They have the largest nervous systems among the invertebrates and present other striking morphological innovations including camera-like eyes, prehensile arms, a highly derived early embryogenesis and a remarkably sophisticated adaptive colouration system. To investigate the molecular bases of cephalopod brain and body innovations, we sequenced the genome and multiple transcriptomes of the California two-spot octopus, Octopus bimaculoides. We found no evidence for hypothesized whole-genome duplications in the octopus lineage. The core developmental and neuronal gene repertoire of the octopus is broadly similar to that found across invertebrate bilaterians, except for massive expansions in two gene families previously thought to be uniquely enlarged in vertebrates: the protocadherins, which regulate neuronal development, and the C2H2 superfamily of zinc-finger transcription factors. Extensive messenger RNA editing generates transcript and protein diversity in genes involved in neural excitability, as previously described, as well as in genes participating in a broad range of other cellular functions. We identified hundreds of cephalopod-specific genes, many of which showed elevated expression levels in such specialized structures as the skin, the suckers and the nervous system. Finally, we found evidence for large-scale genomic rearrangements that are closely associated with transposable element expansions. Our analysis suggests that substantial expansion of a handful of gene families, along with extensive remodelling of genome linkage and repetitive content, played a critical role in the evolution of cephalopod morphological innovations, including their large and complex nervous systems.

Patterning the mammalian cerebral cortex.

When and how is the area map of the cerebral cortex set up during development? Recent studies indicate that regional pattern emerges early in cortical neurogenesis, and that this pattern does not require cues from extrinsic innervation. Studies of mutant mice indicate a role for embryonic signaling centers and for specific transcription factors in regionalizing the cortex. Thus, it is increasingly probable that the cortex is partitioned using the same types of mechanisms--and in some cases, the same gene families--that are used in patterning other parts of the embryo. This emerging model is likely to be the basis for many future studies. However, new evidence also confirms the special nature of the cerebral cortex, in that cues from developing connections appear to modify and refine the final area map.

A role for midbrain arcs in nucleogenesis

Nuclei are fundamental units of vertebrate brain organization, but the mechanisms by which they are generated in development remain unclear. One possibility is that the early patterning of brain tissue into reiterated territories such as neuromeres and columns serves to allocate neurons to distinct nuclear fates. We tested this possibility in chick embryonic ventral midbrain, where a periodic pattern of molecularly distinct stripes (midbrain arcs) precedes the appearance of midbrain nuclei. We found that midbrain arc patterning has a direct relationship to the formation of nuclei. Both differential homeobox gene expression and diagnostic axon tracing studies established that the most medial arc contains primordia for two major midbrain nuclei: the oculomotor complex and the red nucleus. We tested the relationship of the medial arc to oculomotor complex and red nucleus development by perturbing arc pattern formation in Sonic Hedgehog and FGF8 misexpression experiments. We found that Sonic Hedgehog manipulations that induce ectopic arcs or expand the normal arc pattern elicit precisely parallel inductions or expansions of the red nucleus and oculomotor complex primordia. We further found that FGF8 manipulations that push the medial arc rostrally coordinately move both the red nucleus and oculomotor complex anlagen. Taken together, these findings suggest that arcs represent a patterning mechanism by which midbrain progenitor cells are allocated to specific nuclear fates.

AGGRECAN IS EXPRESSED BY EMBRYONIC BRAIN GLIA AND REGULATES ASTROCYTE DEVELOPMENT

Determination of the molecules that regulate astrocyte development has been hindered by the paucity of markers that identify astrocytic precursors in vivo. Here we report that the chondroitin sulfate proteoglycan aggrecan both regulates astrocyte development and is expressed by embryonic glial precursors. During chick brain development, the onset of aggrecan expression precedes that of the astrocytic marker GFAP and is concomitant with detection of the early glial markers GLAST and glutamine synthetase. In co-expression studies, we established that aggrecan-rich cells contain the radial glial markers nestin, BLBP and GLAST and later in embryogenesis, the astroglial marker GFAP. Parallel in vitro studies showed that ventricular zone cultures, enriched in aggrecan-expressing cells, could be directed to a GFAP-positive fate in G5-supplemented differentiation media. Analysis of the chick aggrecan mutant nanomelia revealed marked increases in the expression of the astrocyte differentiation genes GFAP, GLAST and GS in the absence of extracellular aggrecan. These increases in astrocytic marker gene expression could not be accounted for by changes in precursor proliferation or cell death, suggesting that aggrecan regulates the rate of astrocyte differentiation. Taken together, these results indicate a major role for aggrecan in the control of glial cell maturation during brain development.

Cephalopod Genomics: A Plan of Strategies and Organization

The Cephalopod Sequencing Consortium (CephSeq Consortium) was established at a NESCent Catalysis Group Meeting, “Paths to Cephalopod Genomics-Strategies, Choices, Organization,” held in Durham, North Carolina, USA on May 24–27, 2012. Twenty-eight participants representing nine countries (Austria, Australia, China, Denmark, France, Italy, Japan, Spain and the USA) met to address the pressing need for genome sequencing of cephalopod mollusks. This group, drawn from cephalopod biologists, neuroscientists, developmental and evolutionary biologists, materials scientists, bioinformaticians and researchers active in sequencing, assembling and annotating genomes, agreed on a set of cephalopod species of particular importance for initial sequencing and developed strategies and an organization (CephSeq Consortium) to promote this sequencing. The conclusions and recommendations of this meeting are described in this white paper.

Molecular analysis of neocortical layer structure in the ferret

Molecular markers that distinguish specific layers of rodent neocortex are increasingly employed to study cortical development and the physiology of cortical circuits. The extent to which these markers represent general features of neocortical cell type identity across mammals, however, is unknown. To assess the conservation of layer markers more broadly, we isolated orthologs for 15 layer‐enriched genes in the ferret, a carnivore with a large, gyrencephalic brain, and analyzed their patterns of neocortical gene expression. Our major findings are: 1) Many but not all layer markers tested show similar patterns of layer‐specific gene expression between mouse and ferret cortex, supporting the view that layer‐specific cell type identity is conserved at a molecular level across mammalian superorders; 2) Our panel of deep layer markers (ER81/ETV1, SULF2, PCP4, FEZF2/ZNF312, CACNA1H, KCNN2/SK2, SYT6, FOXP2, CTGF) provides molecular evidence that the specific stratifications of layers 5 and 6 into 5a, 5b, 6a, and 6b are also conserved between rodents and carnivores; 3) Variations in layer‐specific gene expression are more pronounced across areas of ferret cortex than between homologous areas of mouse and ferret cortex; 4) This variation of area gene expression was clearest with the superficial layer markers studied (SERPINE2, MDGA1, CUX1, UNC5D, RORB/NR1F2, EAG2/KCNH5). Most dramatically, the layer 4 markers RORB and EAG2 disclosed a molecular sublamination to ferret visual cortex and demonstrated a molecular dissociation among the so‐called agranular areas of the neocortex. Our findings establish molecular markers as a powerful complement to cytoarchitecture for neocortical layer and cell‐type comparisons across mammals. J. Comp. Neurol. 518:3272–3289, 2010.

Levels of Homology and the Problem of Neocortex

The neocortex is found only in mammals, and the fossil record is silent on how this soft tissue evolved. Understanding neocortex evolution thus devolves to a search for candidate homologous neocortex traits in the extant nonmammalian amniotes. The difficulty is that homology is based on similarity, and the six-layered neocortex structure could hardly be more dissimilar in appearance from the nuclear organization that is so conspicuous in the dorsal telencephalon of birds and other reptiles. Recent molecular data have, however, provided new support for one prominent hypothesis, based on neuronal circuits, that proposes the principal neocortical input and output cell types are a conserved feature of amniote dorsal telencephalon. Many puzzles remain, the greatest being understanding the selective pressures and molecular mechanisms that underlie such tremendous morphological variation in telencephalon structure.

PHOX2A regulation of oculomotor complex nucleogenesis.

Brain nuclei are spatially organized collections of neurons that share functional properties. Despite being central to vertebrate brain circuitry, little is known about how nuclei are generated during development. We have chosen the chick midbrain oculomotor complex (OMC) as a model with which to study the developmental mechanisms of nucleogenesis. The chick OMC comprises two distinct cell groups: a dorsal Edinger-Westphal nucleus of visceral oculomotor neurons and a ventral nucleus of somatic oculomotor neurons. Genetic studies in mice and humans have established that the homeobox transcription factor gene PHOX2A is required for midbrain motoneuron development. We probed, in forced expression experiments, the capacity of PHOX2A to generate a spatially organized midbrain OMC. We found that exogenous Phox2a delivery to embryonic chick midbrain can drive a complete OMC molecular program, including the production of visceral and somatic motoneurons. Phox2a overexpression was also able to generate ectopic motor nerves. The exit points of such auxiliary nerves were invested with ectopic boundary cap cells and, in four examples, the ectopic nerves were seen to innervate extraocular muscle directly. Finally, Phox2a delivery was able to direct ectopic visceral and somatic motoneurons to their correct native spatial positions, with visceral motoneurons settling close to the ventricular surface and somatic motoneurons migrating deeper into the midbrain. These findings establish that in midbrain, a single transcription factor can both specify motoneuron cell fates and orchestrate the construction of a spatially organized motoneuron nuclear complex.

Retinoids and their targets in vertebrate development

The elaboration of the effect of retinoic acid on limb morphogenesis has prompted renewed investigation into the teratology of retinoic acid treatment, with the hope that such analysis might give insight into mechanisms of vertebrate patterning. Retinoids, their nuclear receptors and their cytoplasmic binding proteins are now known to be deployed throughout development, but the extent to which they are natural agents of morphogenesis remains obscure. The study of retinoic acid receptors may offer molecular insight into gene regulation underlying vertebrate pattern formation.