377754 Precision HOX Patterning in a Human Pluripotent Stem Cell Model of Posterior Neural Development
Colinear activation of HOX genes is a fundamental process that conveys positional information within many developing tissue structures, including the posterior central nervous system (CNS) consisting of the hindbrain and spinal cord. HOX expression patterns direct many significant events in posterior CNS development, including neural circuit organization, cell fate choices, and motor neuron innervation choreography. As such, the ability to precisely regulate HOXexpression in human pluripotent stem cell (hPSC)-derived neural cells would be a significant advancement in the fields of disease modeling and regenerative medicine.
The predominant theory for HOX patterning in the developing posterior CNS dictates that, in the presence of continuous Wnt/β-catenin signaling, counteracting gradients of retinoic acid (RA) and fibroblast growth factor (FGF) plus growth differentiation factor (GDF) regulate rostral versus caudal HOX domains. However, the application of these principles to PSC differentiation has only resulted in neural cells possessing coarse, heterogeneous HOX expression. To address this issue, we utilized a completely defined hPSC neural differentiation scheme to interrogate the explicit roles of morphogen combinations in regulating HOX expression. We discovered that combined activation of Wnt/β-catenin and FGF signaling was sufficient to establish a highly pure Sox2+/Brachyury+ neuromesodermal state that mimics the axial progenitors which form posterior neuroectoderm in vivo, and activation of these two signaling pathways was required for colinear activation of HOX1-9 paralogs, which constitute the hindbrain, cervical, and thoracic spinal cord. Activation of GDF signaling was then required to induce expression of HOX10-12 lumbosacral paralogs. At any point during neuromesodermal propagation, the addition of RA was necessary and sufficient to transition to highly pure (>83%) Sox2+/Pax6+ neuroectoderm. Moreover, RA acts as the ‘stop’ signal for HOX progression, thus producing neuroectoderm with deterministic HOX expression profiles characteristic of the cervical, thoracic, and lumbar spinal cord. We also observed cross-repressive interactions between Hox proteins via immunocytochemistry and mass spectrometry that were representative of these regional spinal cord domains.
Collectively, these results represent the first example of deterministic HOX patterning in an hPSC differentiation system. Our findings argue against the classical gradient model of HOX patterning in the developing neural tube and instead support a temporal exposure mechanism where different sets of morphogens activate or halt HOX progression to generate fixed spatial domains. Overall, this HOX patterning methodology will serve as the basis for generating a spectrum of neural cells possessing diverse and defined positional identities for disease modeling and potential regenerative therapies.