Forced Expression of LIM Homeodomain Transcription Factor 1b Enhances Differentiation of Mouse Embryonic Stem Cells into Serotonergic Neurons

Abstract

The LIM homeodomain transcription factor 1b (Lmx1b) is a key factor in the specification of the serotonergic neurotransmitter phenotype. Here, we explored the capacity of Lmx1b to direct differentiation of mouse embryonic stem (mES) cells into serotonergic neurons. mES cells stably expressing human Lmx1b were generated by lentiviral vector infection. Clones expressing Lmx1b at a low level showed increased neurogenesis and elevated production of neurons expressing serotonin, serotonin transporter, tryptophan hydroxylase 2, and transcription factor Pet1, the landmarks of serotonergic differentiation. To explore the role of Lmx1b in the specification of the serotonin neurotransmission phenotype further, a conditional system making use of a floxed inducible vector targeted into the ROSA26 locus and a hormone-dependent Cre recombinase was engineered. This novel strategy was tested with the reporter gene encoding human placental alkaline phosphatase, and demonstrated its capacity to drive transgene expression in nestin þ neural progenitors (NPs) and in Tuj1 þ neurons. When it was applied to inducible expression of human Lmx1b, it resulted in elevated expression of serotonergic markers. Treatment of neural precursors with the floor plate signal Sonic hedgehog further enhanced differentiation of Lmx1b-overexpressing NPs into neurons expressing 5-HT, serotonin transporter, tryptophan hydroxylase 2, and Pet1, when compared with Lmx1b-nonexpressing progenitors. Together, our results demonstrate the capacity of Lmx1b to specify a serotonin neurotransmitter phenotype when overexpressed in mES cell-derived NPs.

Conditional Induction of Math1 Specifies Embryonic Stem Cells to Cerebellar Granule Neuron Lineage and Promotes Differentiation into Mature Granule Neurons

“…We have shown here that combining a temporally controlled expression of Math1 along with extrinsic signals leads to 73% of neurons acquiring a granule fate. This approach has recently brought encouraging results when trying to derive difficult to get neuronal subtypes such as was shown with serotonin [23][24][25].…”

Regulatory Mechanisms Controlling Maturation of Serotonin Neuron Identity and Function

“…Several recent studies have tested exogenous factors and multiple combinations of transcription factors known to have a role in driving 5-HT neuron specification (Dolmazon et al, 2011; Vadodaria et al, 2016; Xu et al, 2016). An interesting first test showed the capability of Lmx1b to induce mouse embryonic stem cells to differentiate into 5-HT neurons (Dolmazon et al, 2011). In the presence of Shh, an inducible Lmx1b construct was able to activate expression of Tph2, Sert, and Pet-1, however 5-HT levels were not determined under those conditions.…”

Association of Transcription Factor Gene LMX1B with Autism

“…However, during development, Lmx1b precedes pet1 , and Lmx1b knock-out mice showed loss of Pet1 expression [10]. In vivo , Pet1 expression was increased in neurons overexpressing Lmx1b [11]. Thus, Lmx1b has been proposed as an essential link between Nkx2.2 and Pet1 in the genetic cascade that controls the early specification and terminal differentiation of 5HTergic neurons in the hindbrain.…”

“…Lmx1b knock-out mice were found to be lacking the entire central 5HTergic neurons [9], [10]. Further, it was shown that overexpression of Lmx1b enhances differentiation of mouse embryonic stem cells into 5HT neurons [11]. In addition to its role in the development of central 5HTeregic neurons, Lmx1b is also required for the normal biosynthesis of 5HT in adult brain, and possibly for the regulation of normal functions of 5HTergic neurons [12].…”