Doublesex and mab-3–related transcription factor 5 promotes midbrain dopaminergic identity in pluripotent stem cells by enforcing a ventral-medial progenitor fate

Abstract

Understanding the control of cell-fate choices during embryonic stem cell (ESC) differentiation is crucial for harnessing strategies for efficient production of desired cell types for pharmaceutical drug screening and cell transplantation. Here we report the identification of the zinc finger-like doublesex and mab-3-related transcription factor 5 (Dmrt5) as a marker for mammalian ventralmedial mesencephalic neuroepithelium that give rise to dopamine neurons. Gain-and loss-of-function studies in ESC demonstrate that Dmrt5 is critically involved in the specification of ventralmedial neural progenitor cell fate and the subsequent generation of dopamine neurons expressing essential midbrain characteristics. Genome-wide analysis of Dmrt5-mediated transcriptome changes and expression profiling of ventral-medial and ventral-lateral mesencephalic neuroepithelium revealed suppressive and inductive regulatory roles for Dmrt5 in the transcription program associated with the ventral-medial neural progenitor fates. Together, these data identify Dmrt5 as an important player in ventral mesencephalic neural fate specification.A major goal of embryonic stem cell (ESC) research is to direct the cells' differentiation toward specific cell types, especially those targeted by devastating degenerative diseases. The advent of induced pluripotent stem cell technology, with its promise for disease modeling, drug screening, and cell therapy, places further demand on a better understanding on the control of lineage/cell-fate specification from pluripotent stem cells. One neuronal cell type in particular, the midbrain dopaminergic (mDA) neuron, is a prime target in applied stem cell research because of its association with Parkinson's disease.The mDA neurons are generated in the floor plate (FP) region of the ventral midbrain and are uniquely identified by their coexpression of tyrosine hydroxylase (TH) with the mDA-specific homeobox protein Pitx3 (1, 2). During development, local inductive signals-Shh, FGF8, and Wnt1-induce distinct cell-fate potentials through initiation of transcriptional cascades that govern the subsequent differentiation, migration, and maturation of the ventral-most progenitors into mDA neurons (3-5). The distinct cell-fate potentials of ventral midbrain progenitors are defined by domain-identifiable expression of transcription factors. For example, the Lmx1a + Foxa2 + FP exclusively gives rise to mDA neurons, whereas the ventral-lateral domains marked by Meis2, Mab21l2, Helt, and Lhx1 produce glutamatergic or GABAergic neurons (6-9). Perturbation of such a transcription "code" seen in genetic studies often led to misspecification of progenitor identity and subsequently to neural transmitter phenotypes (10-12). These studies demonstrate the mechanism of cell-fate determination to be a balance of the activation of "specification" programs and the repression of alternative fates, as observed in the spinal cord (13). However, how transcription factors coordinate distinct fate choice in the ventral midbrain remains poorl...

Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

“…Transcriptional profiling of this cell fraction identified a range of genes encoding for transcription factors and other transcriptional regulators, components of signaling pathways, and secreted molecules. Several of these genes have been identified and verified in previous gene expression studies (Dmrta1 and Dlk1) (14,15) or have well-established roles in mDA neurogenesis and differentiation (TH, Lmx1a, Nurr1, En1, and Foxa2) (reviewed in refs. 16 and 17).…”

The doublesex-related Dmrta2 safeguards neural progenitor maintenance involving transcriptional regulation of Hes1

“…To investigate a role for Dmrta2 in telencephalic NPC behavior, we first examined the effect of Dmrta2 gain of function in neuronal differentiation of ESC-derived NPCs using a tetracycline-inducible Dmrta2 transgenic mESC model (Dmrta2-ESCs) reported previously (9). These cells harbor the reverse tetracycline-controlled transactivator (rtTA) and produce significant levels of Dmrta2 protein in response to the addition of doxycycline to the culture medium.…”

“…Six mouse ESC lines were used: a Dmrta2 flox/flox control line and two Dmrta2 −/− lines derived from the control line (SI Materials and Methods), two independent doxycycline-inducible Dmrta2-overexpressing ESC lines, and their parental lines harboring rtTA, as reported previously (9). All ESCs were maintained under standard conditions as described previously (17).…”

“…Another site of expression and function of Dmrta2 has been found in the embryonic brain, however (9,10). Dmrta2 loss of function in zebrafish leads to significant reductions in cortical size, coupled with reduced neuronal numbers (10,11).…”

Activin A directs striatal projection neuron differentiation of human pluripotent stem cells

“…In the absence of exogenous factors, these forebrain progenitors comprise a mix of dorsal and ventral entities, as demonstrated by the co-existence of cells expressing cortical (DMRT5; also known as DMRTA2) and ventral forebrain (GSX2) transcription factors (Fig. 1B,E,F) (Gennet et al, 2011).…”