Fusion protein Isl1–Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs

Abstract

Combinatorial transcription codes generate the myriad of cell types during development and thus likely provide crucial insights into directed differentiation of stem cells to a specific cell type. The LIM complex composed of Isl1 and Lhx3 directs the specification of spinal motor neurons (MNs) in embryos. Here, we report that Isl1-Lhx3, a LIM-complex mimicking fusion, induces a signature of MN transcriptome and concomitantly suppresses interneuron differentiation programs, thereby serving as a potent and specific inducer of MNs in stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the LIM domain of Lhx3 are crucial for generating MNs without up-regulating interneuron genes. These led us to design Isl1-Lhx3, which maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM domain of Lhx3. Isl1-Lhx3 drives MN differentiation with high specificity and efficiency in the spinal cord and embryonic stem cells, bypassing the need for sonic hedgehog (Shh). RNA-seq analysis revealed that Isl1-Lhx3 induces the expression of a battery of MN genes that control various functional aspects of MNs, while suppressing key interneuron genes. Our studies uncover a highly efficient method for directed MN generation and MN gene networks. Our results also demonstrate a general strategy of using embryonic transcription complexes for producing specific cell types from stem cells. D eveloping central nervous system (CNS) produces a vast number of neuronal types, but adult CNS has only limited capacity to regenerate neurons. This has prompted great interest in identifying methods to produce specific neuronal types from stem cells. Production of differentiated cell types from pluripotent stem cells, such as embryonic stem cells (ESCs), should enable a continuous supply of diseased cell types for drug screening and cell replacement therapy and provide valuable insights into the pathophysiology of human diseases. One important challenge in this effort is to steer stem cells into specific cell types. Recapitulation of normal developmental processes using embryonic inductive signals has been used to drive differentiation of pluripotent stem cells into specific cell types (1). However, this strategy tends to trigger formation of mixed cell types rather than a targeted cell type, because each inductive signal is used in multiple developmental pathways. This shortcoming might be circumvented by using more specific, downstream transcription factors of inductive signals. In this regard, it should be noted that many transcription factors function in combination to determine cell fates during development, suggesting that coexpression of multiple transcription factors could be a more effective method to generate a particular cell type from pluripotent stem cells.Motor neurons (MNs) in the spinal cord project axons to muscles and control their contraction. The developmental pathways to generate MNs have been relatively well studied. In the developing spinal cord, sonic hedgehog (Shh) signal triggers the expression of two LIM homeodomain...

Identification of STAM1 as a novel effector of ventral projection of spinal motor neurons

“…STAM1) and the ISL1-LHX3 complex, our results support the previous prediction made from several genome-wide studies (Lee et al, 2012(Lee et al, , 2013Mazzoni et al, 2013) that the ISL1-LHX3 complex functions as a terminal selector of MN fate by regulating a battery of terminal differentiation genes that establish unique MN properties, such as neurogenesis, axonogenesis, axon guidance, synapse function, and cholinergic neurotransmitter characteristics (Lee et al, 2012(Lee et al, , 2013Cho et al, 2014). Of note, the Stam2 gene was not associated with any ISL1-LHX3 complex-binding peak (data not shown).…”

“…In support of this idea, expression of STAM1 was induced as MNs were derived from embryonic stem cells under MN differentiation conditions (Lee et al, 2012;Wichterle et al, 2002) (Fig. S1).…”

“…These studies, combined with MN transcriptome analyses (Lee et al, 2012;Mazzoni et al, 2013), revealed that the ISL1-LHX3 complex directly regulates the expression of a wide range of downstream effector genes that function not only in cell fate specification but also in subsequent maturation steps of MN differentiation, such as cell migration, axon pathfinding and synapse formation. As a result, the roles of individual target genes of the ISL1-LHX3 complex in MN specification and differentiation/maturation have finally begun to be dissected (Cho et al, 2014;Lee et al, 2012Lee et al, , 2013Mazzoni et al, 2013). Interestingly, v-MNs in embryos deficient in both LHX3 and LHX4 reorients their axonal projections dorsally, whereas the misexpression of LHX3 in d-MNs reorients their axonal projections ventrally (Sharma et al, 1998).…”

“…To understand better the role of the ISL1-LHX3 complex in spinal MN specification and differentiation, a genome-wide mapping of ISL1-LHX3 complexbinding sites has been recently performed using chromatin immunoprecipitation sequencing (ChIP-seq) (Lee et al, 2013;Mazzoni et al, 2013). These studies, combined with MN transcriptome analyses (Lee et al, 2012;Mazzoni et al, 2013), revealed that the ISL1-LHX3 complex directly regulates the expression of a wide range of downstream effector genes that function not only in cell fate specification but also in subsequent maturation steps of MN differentiation, such as cell migration, axon pathfinding and synapse formation. As a result, the roles of individual target genes of the ISL1-LHX3 complex in MN specification and differentiation/maturation have finally begun to be dissected (Cho et al, 2014;Lee et al, 2012Lee et al, , 2013Mazzoni et al, 2013).…”

The Transcription Factors Islet and Lim3 Combinatorially Regulate Ion Channel Gene Expression

“…The mammalian homologs, Isl1 and LhX3, have been shown to form a hexameric complex, together with the self-dimerizing cofactor NL1, to promote motoneuron fate (Lee et al, 2012). Although the LhX3/Isl1 complex and Isl1 or LhX3 response elements share A/T-rich sequences, each site is unique (Lee et al, 2008).…”