Research
We are currently investigating cellular and molecular mechanisms controlling the development of the nervous and skeletal systems during embryogenesis. We are using the Chicken (Gallus gallus) and Quail (Coturnix coturnix japonica) embryos as model organisms.
3 Day-Old Chick Embryo
Developmental Neurobiology: Cellular and molecular mechanisms that control the specification, delamination and migration of neural crest cells.
The neural crest (NC) is a transient group of vertebrate progenitors. Its component cells yield an extensive variety of derivatives such as melanocytes, neurons of many kinds, glial, ectomesenchymal and endocrine cells. Initially, NC cells are epithelial and then undergo a time and axial level-specific conversion into mesenchyme that generates cell migration. NC cells then advance
through stereotyped pathways, reach their homing sites and differentiate (Le Douarin and Kalcheim, 1999). The molecular network underlying the generation of cellular movement remains incompletely understood (Kalcheim and Burstyn-Cohen, 2005). This process involves an epithelial-to-mesenchymal transition (EMT) of the premigratory NC cells residing in the dorsal neural tube (NT) followed by delamination. Work from our lab has shown that a balance between BMP and its inhibitor noggin underlies the emigration of trunk-level NC independently of earlier cell specification (Sela-Donenfeld and Kalcheim, 1999). A decreasing rostrocaudal gradient of BMP4 activity is established along the NT by a reciprocal gradient of noggin. Noggin downregulation is in turn triggered by the developing somites which thus determine the timing of NC emigration (Sela-Donenfeld and Kalcheim, 1999; Sela-Donenfeld and Kalcheim, 2000; Sela-Donenfeld and Kalcheim, 2002). BMP then induces EMT of NC by triggering Wnt1 transcription. The latter promotes G1/S transition which is a necessary step for delamination of trunk NC (Burstyn-Cohen and Kalcheim, 2002; Burstyn-Cohen et al., 2004).
Acting downstream of BMP is the activity of effector genes that act on re-organisation of the actin cytoskeleton, alterations in adhesive properties and consequent loss of epithelial polarity. In this context, N-cadherin was found to be a component of the BMP-dependent network leading to NC EMT. N-cadherin inhibits the onset of NC delamination both by a cell adhesion-dependent mechanism as well as by repressing canonical Wnt signaling. Relief from N-cadherin-mediated inhibition is attained in the dorsal NT during the onset of cell emigration. This is accounted for by an ADAM10-dependent cleavage of the full-length molecule into a soluble domain with pro-delamination properties, a process triggered by BMP (Shoval et al., 2007). Rho GTPases act as molecular switches to control a variety of signal transduction pathways. Their possible effects on NC delamination remained virtually unexplored. We reported that RhoA and RhoB, through Rock, act downstream of BMP and of G1/S transition to maintain NC cells in an epithelial conformation. They exert these effects both by stabilizing assembly of the actin cytoskeleton and by enhancing N-cadherin-mediated intercellular adhesion (Groysman et al, 2008).
During the course of the above studies, we observed that in the trunk, NC cells continuously delaminate from the NT for over two days, raising the fundamental question of the source and mechanisms accounting for the production of successive waves of NC progenitors. We found that the first NC to delaminate reside in the dorsal midline of the NT and generate sympathetic ganglia, and successive waves translocate ventrodorsally in the NT to replenish the dorsal midline and then delaminate. Hence, the dorsal midline is a dynamic region traversed sequentially by progenitors that colonize NC derivatives in a ventral to dorsal order (chromaffin cells, sympathetic ganglia, then Schwann cells, DRG and finally melanocytes). Based on our data invoking a dynamic behavior of premigratory NC cells, we hypothesize the existence of a dynamic spatiotemporal fate map of derivatives already within the NT. Preliminary data suggest the existence of such a map as well as of a fate restriction of premigratory cells to generate discrete derivatives (Krispin et al, 2010). Then, we will investigate the putative molecular code underlying specification to various fates. We will also examine the relationship between cell specification and migration by asking whether restriction to a certain fate conveys the cell with knowledge of the migratory pathway to follow and consequently, of its homing site, or alternatively, whether these are separable and distinctly regulated events. The regulation of epithelial-mesenchymal conversion in association with cell fate are among the most puzzling problems raised by morphogenesis, which go beyond developmental biology to concern the stability of the histiotypic state and its disruption in disease.
(A-A'') Confocal scanning of a somite (dorsal view) electroporated with GFP, and co-stained for the muscle marker Desmin. (B) A transverse section showing the presence of dermis (D) and the myotome containing both differentiated myocytes (desmin, green) and Pax7+ muscle progenitors (red). (C) A transverse section stained for the epithelial marker ZO-1, showing expression in the apical domain of both the neural tube and the somite. (D) A section through an embryo electroporated with GFP (green) and co-stained for the Neural Crest marker HNK-1 (pink).
The transition between Neural Crest and definitive roof plate
In a developing embryo, the multicellular patterns formed are remarkably precise. Through cell-cell communication, progenitors coordinate their activities, sequentially generating distinct tissues. During spinal cord formation, Neural Crest (NC) cells that generate the peripheral nervous system, and definitive Roof Plate (RP) cells of the central nervous system are sequentially formed in the same anlagen. We will investigate how these peripheral and central neural branches segregate from common stem cells, a vital yet unexplored process and then create experimental models of neural tube defects (Nitzan, E. et al. Dynamics of BMP and Hes1/Hairy1 signaling in the dorsal neural tube underlies the transition from neural crest to definitive roof plate. BMC Biol14, 23, doi:10.1186/s12915-016-0245-6, 2016). By transcriptome analysis, we recently discovered genes differentially expressed in NC and RP. Together with knowledge on upstream signals, we will elucidate the molecular network and the logic underlying this transition.
Development of the skeletal system: The molecular basis of intrasomitic diversification in relation to vertebral development and to segmentation of the peripheral nervous system. The multiwave nature of myotome formation.
The somites are epithelial structures arising in a metameric pattern from the paraxial mesoderm. In the course of development, somites undergo successive phases of de-epithelialization concomitant with the acquisition of diverse cell fates. Initially, the ventral somite dissociates to generate the sclerotome, which eventually forms the vertebrae, ribs and tendons. The remaining dorsal part, known as the dermomyotome (DM), contributes cells to the myotome, the precursor of skeletal muscles, and upon dissociation, also generates the dorsal dermis.
Initially, we mapped the origin of muscle and dermis from the DM. Whereas the initial myotome is established by a population of early specified pioneer myoblasts resident in the medial epithelial somite (Kahane et al., 2007; Kahane et al., 1998b), subsequent myofibers form from all four lips of the DM (Cinnamon et al., 2006; Cinnamon et al., 1999, Kahane et al., 1998a; Kahane et al., 2002) and their proper patterning is determined by the initial scaffold of pioneer fibers (Kahane et al., 2007). Further to the formation of unit length myofibers, the DM produces progenitors that remain mitotically active within the myotome (Ben-Yair and Kalcheim, 2005; Kahane et al., 2001). These are generated from the extreme lips of the DM (Kahane et al., 2001) and from the dissociating DM sheet that also produces dermis (Ben-Yair et al., 2003; Ben-Yair and Kalcheim, 2005). Both mitotic myotomal precursors and dermis originate from single cells residing in the central DM sheet. The diversification of these two lineages is accompanied by a striking shift in the plane of cell division in the epithelium that becomes perpendicular to the mediolateral aspect of the DM. This shift is coupled to the asymmetric segregation of N-Cadherin to the apical daughter cells that will become muscle but not to the basal cells that will give rise to dermis (Ben-Yair and Kalcheim, 2005; Cinnamon et al., 2006). We are currently investigating the role of the orientation of cell divisions in the generation of distinct DM-derived fates.
-Electroporation strategies used to transfect distinct somite domains -
To better understand the mechanisms responsible for the segregation of the DM epithelium into its derivatives; we turned our attention to the generation of two additional DM-derived lineages, endothelial and mural (vascular smooth muscle and pericytes) cells. Using discrete lineage analysis in ovo, we found that the lateral portion of the DM is the most productive source for both endothelial and mural cells. In addition, both lineages have already diverged in the epithelial somite, in contrast to myotomal and dermal cells that derive from single progenitors in the central DM sheet.
Importantly, differentiation of these lineages is driven by distinct signaling systems. Notch is necessary for smooth muscle production while inhibiting striated muscle differentiation, yet it does not affect initial development of endothelial cells. On the other hand, BMP signaling is required for endothelial cell differentiation and/or migration but inhibits striated muscle differentiation, and fails to impact smooth muscle cell production. Hence, while different mechanisms are responsible for the generation of smooth muscle and endothelium, the choice to become smooth versus striated muscle depends upon a single signaling system. Altogether, these findings underscore the spatial and temporal complexity of lineage diversification in an apparently homogeneous epithelium (Ben-Yair and Kalcheim, 2008). Current work in the lab addresses the mechanisms that mediate the activity of Notch on the acquisition of the smooth muscle traits.
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