Assessing the mechanisms directing cell fate in the dorsal spinal cord

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Project Summary The somatosensory system permits us to perceive and react to the environment through modalities that include touch, nociception, thermosensation and proprioception. Somatosensory information is received peripherally and then relayed centrally by different populations of dorsal interneurons (dIs; dI1-dI6) in the spinal cord. Our research objectives are to understand the mechanisms that establish dIs during development and then apply these principles towards designing differentiation protocols to direct the formation of specific populations of dIs from pluripotent stem cells. These cells have the potential to repair damaged sensory circuits and act as substrates for drug screening platforms. We have focused on dissecting the role of the bone morphogenetic protein (BMP) family in dI fate specification. BMPs were widely assumed to act as morphogens, patterning the dorsal spinal cord in a concentration-dependent mechanism similar to the manner in which sonic hedgehog (Shh) patterns the ventral spinal cord. However, our recent studies using mouse, chicken, and mouse embryonic stem cell (mESC) models have found that no evidence that BMPs act as morphogens. Rather, BMPs have signal-specific activities, with differential abilities to direct dorsal progenitor (dP) patterning and/or differentiation through specific type I BMP receptors. Our recent in vivo and in vitro studies have also suggested that dI fates are established in a series of nested choice points. In our model, spinal progenitors are dorsalized by retinoic acid (RA), subdivided into multipotential dP subgroups by BMP signaling, and then resolve into specific dI fates. Since little is known about this patterning process, we will assess in Aim 1 how multipotential dP fates are first established by RA and BMP signaling and identify the mechanisms directing multipotential dPs into specific dI identities. In Aim 2, we will determine the nature of the intracellular response that permits specific BMPs to drive dPs towards different dI identities, addressing two unresolved questions: [1] are canonical receptor regulated (R)-Smads activated in a BMP-specific manner to result in distinct patterning activities? And [2] what factors do the R-Smads in turn regulate to promote dI fates? Together, these studies will investigate a long-standing problem in developmental biology, i.e., understanding how specific outcomes arise from a common signal, and shed light on the specification of dIs, cell types needed to permit paralyzed patients to again interpret their sensory environment. Our specific aims are as follows: Aim 1: Identify the mechanism(s) that establish multipotential dPs and assign them into individual dI fates. Hypothesis: RA±BMP4 direct the formation of multipotential dP subgroups, which resolve into specific dI fates by the asymmetric activation of additional endogenous signaling pathways, such as the Wnt pathway. Aim 2: Assess the role of the Smad and Id families in BMP-induced dI fate specification. Hypothesis: BMPs differentially activate Smad1 and/or Smad5, to regulate the activity of Id family members.