Seema Agarwala

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.

Bone morphogenetic proteins regulate neural tube closure by interacting with the apicobasal polarity pathway

During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.

Diversification of the expression patterns and developmental functions of the Dishevelled gene family during chordate evolution

Dishevelled (Dvl) proteins are key transducers of Wnt signaling encoded by members of a multi‐gene family in vertebrates. We report here the divergent, tissue‐specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression patterns in mice. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, an outgroup to chordates, we find that the one Dvl gene is dynamically expressed in a tissue‐specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation in Xenopus. Most strikingly, we report a novel role for Dvl3 in the maintenance of gene expression in muscle and in the development of the Xenopus sclerotome. These data demonstrate that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution. Developmental Dynamics 238:2044–2057, 2009.

Ventral specification and perturbed boundary formation in the mouse midbrain in the absence of Hedgehog signaling

Although Hedgehog (HH) signaling plays a critical role in patterning the ventral midbrain, its role in early midbrain specification is not known. We examined the midbrains of sonic hedgehog (Shh) and smoothened (Smo) mutant mice where HH signaling is respectively attenuated and eliminated. We show that some ventral (Evx1+) cell fates are specified in the Shh−/− mouse in a Ptc1‐ and Gli1‐independent manner. HH‐independent ventral midbrain induction was further confirmed by the presence of a Pax7‐negative ventral midbrain territory in both Shh−/− and Smo−/− mice at and before embryonic day (E) 8.5. Midbrain signaling centers are severely disrupted in the Shh−/− mutant. Interestingly, dorsal markers are up‐regulated (Wnt1, Gdf7, Pax7), down‐regulated (Lfng), or otherwise altered (Zic1) in the Shh−/− midbrain. Together with the increased cell death seen specifically in Shh−/− dorsal midbrains (E8.5–E9), our results suggest specific regulation of dorsal patterning by SHH, rather than a simple deregulation due to its absence. Developmental Dynamics 237:1359‐1372, 2008.

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.

Bone morphogenetic proteins regulate hinge point formation during neural tube closure by dynamic modulation of apicobasal polarity

BACKGROUND A critical event in neural tube closure is the formation of median hinge points (MHPs) and dorsolateral hinge points (DLHPs). Together, they buckle the ventral midline and elevate and juxtapose the neural folds for proper neural tube closure. Dynamic cell behaviors occur at hinge points (HPs), but their molecular regulation is largely unexplored. Bone morphogenetic proteins (BMPs) have been implicated in a variety of neural tube closure defects, although the underlying mechanisms are poorly understood. METHODS In this study, we used in vivo electroporations, high-resolution microscopy, and biochemical analyses to explore the role of BMP signaling in chick midbrain neural tube closure. RESULTS We identified a cell-cycle-dependent BMP gradient in the midbrain neural plate, which results in low-level BMP activity at the MHP. We show that although BMP signaling does not have a role in midbrain cell-fate specification, its attenuation is necessary and sufficient for MHP formation and midbrain closure. BMP blockade induces MHP formation by regulating apical constriction and basal nuclear migration. Furthermore, BMP signaling is critically important for maintaining epithelial organization by biochemically interacting with apicobasal polarity proteins (e.g., PAR3). As a result, prolonged BMP blockade disrupts apical junctions, desegregating the apical (PAR3(+), ZO1(+)) and basolateral (LGL(+)) compartments. Direct apical LGL-GFP misexpression in turn is sufficient to induce ectopic HPs. CONCLUSIONS BMPs have a critical role in maintaining epithelial organization, a role that is conserved across species and tissue types. Its cell-cycle-dependent modulation in the neural plate dynamically regulates apicobasal polarity and helps to bend, shape, and close the neural tube.

Gene expression analysis of the hedgehog signaling cascade in the chick midbrain and spinal cord

The signaling molecule Sonic Hedgehog (SHH) plays a critical role in patterning the ventral midbrain of vertebrates. Our recent studies have established that the requirement for Hedgehog (HH) signaling in the chick midbrain is modulated spatially and temporally in a complex manner across the midbrain anlage. Unfortunately, the patterns of expression of downstream regulators that might modulate the HH signal in the midbrain are not currently known. To fill this gap, we have examined across time, the expression pattern of 14 genes that function in the HH signaling cascade in the midbrain and spinal cord. Our results suggest that SHH expression in the axial mesendoderm begins before the expression of known HH receptors/HH‐binding proteins (e.g., PTC1, PTC2, HHIP, BOC, MEGALIN). In the midbrain, PTC and GLI genes are expressed and then eliminated very early from the ventral midline. However, they exhibit high and persistent expression in the midbrain region circumscribing the SHH source. Intriguingly, multiple HH‐binding proteins (BOC, MEGALIN) and HH effectors (GLI1‐3, SMO, SUFU, DZIP) are expressed in the dorsal midbrain and the midbrain–hindbrain boundary. Finally, we report for the first time that IHH is expressed in intermediate regions of the spinal cord, where its expression does not overlap with that of SHH. Developmental Dynamics 236:1363–1373, 2007.

A novel role for FOXA2 and SHH in organizing midbrain signaling centers

The floor plate (FP) is a midline signaling center, known to direct ventral cell fates and axon guidance in the neural tube. The recent identification of midbrain FP as a source of dopaminergic neurons has renewed interest in its specification and organization, which remain poorly understood. In this study, we have examined the chick midbrain and spinal FP and show that both can be partitioned into medial (MFP) and lateral (LFP) subdivisions. Although Hedgehog (HH) signaling is necessary and sufficient for LFP specification, it is not sufficient for MFP induction. By contrast, the transcription factor FOXA2 can execute the full midbrain and spinal cord FP program via HH-independent and dependent mechanisms. Interestingly, although HH-independent FOXA2 activity is necessary and sufficient for inducing MFP-specific gene expression (e.g., LMX1B, BMP7), it cannot confer ventral identity to midline cells without also turning on Sonic hedgehog (SHH). We also note that the signaling centers of the midbrain, the FP, roof plate (RP) and the midbrain-hindbrain boundary (MHB) are physically contiguous, with each expressing LMX1B and BMP7. Possibly as a result, SHH or FOXA2 misexpression can transform the MHB into FP and also suppress RP induction. Conversely, HH or FOXA2 knockdown expands the endogenous RP and transforms the MFP into a RP and/or MHB fate. Finally, combined HH blockade and FOXA2 misexpression in ventral midbrain induces LMX1B expression, which triggers the specification of the RP, rather than the MFP. Thus we identify HH-independent and dependent roles for FOXA2 in specifying the FP. In addition, we elucidate for the first time, a novel role for SHH in determining whether a midbrain signaling center will become the FP, MHB or RP.

Apicobasal polarity and neural tube closure

During development, a flat neural plate rolls up and closes to form a neural tube. This process, called neural tube closure, is complex and requires morphogenetic events to occur along multiple axes of the neural plate. Recent studies suggest that cell and tissue polarity play a major role in neural tube morphogenesis. While the planar cell polarity pathway is known to be involved in this process, a role for the apicobasal polarity pathway has only recently begun to be elucidated. These studies show that bone morphogenetic proteins can regulate the apicobasal polarity pathway in the neural plate in a cell cycle dependent manner. This dynamically modulates apical junctions in the neural plate, resulting in cell and tissue shape changes that help bend, shape and close the neural tube.

A simple technique for early in vivo electroporation of E1 chick embryos

Background: The amenability of the chick embryo to a variety of manipulations has made it an ideal experimental model organism for over 100 years. The ability to manipulate gene function via in ovo electroporations has further revolutionized its value as an experimental model in the last 15 years. Although in ovo electroporations are simple to conduct in embryos ≥ E2, in ovo electroporations at early E1 stages have proven to be technically challenging due to the tissue damage and embryonic lethality such electroporations produce. Results and Conclusions: Here we report our success with in vivo microelectroporations of E1 embryos as young as Hamburger‐Hamilton Stage 4 (HH4). We provide evidence that such electroporations can be varied in size and can be spatially targeted. They cause minimal disruption of tissue‐size, 3‐dimensional morphology, cell survival, proliferation, and cell‐fate specification. Our paradigm is easily adapted to a variety of experimental conditions since it does not depend upon the presence of a lumen to enclose the DNA solution during electroporation. It is thus compatible with the in vivo examination of E1 morphogenetic events (e.g., neural tube closure) where preservation of 3‐dimensional morphology is critical. Developmental Dynamics 241:545–552, 2012.