Control of Astrocyte Progenitor Specification, Migration and Maturation by Nkx6.1 Homeodomain Transcription Factor

Although astrocytes are the most abundant cell type in the central nervous system (CNS), little is known about their molecular specification and differentiation. It has previously been reported that transcription factor Nkx6.1 is expressed in neuroepithelial cells that give rise to astrocyte precursors in the ventral spinal cord. In the present study, we systematically investigated the function of Nkx6.1 in astrocyte development using both conventional and conditional Nkx6.1 mutant mice. At early postnatal stages, Nkx6.1 was expressed in a subpopulation of astrocytes in the ventral spinal cord. In the conventional Nkx6.1KO spinal cord, the initial specification of astrocyte progenitors was affected by the mutation, and subsequent migration and differentiation were disrupted in newborn mice. In addition, the development of VA2 subtype astrocytes was also inhibited in the white matter. Further studies with Nkx6.1 conditional mutants revealed significantly delayed differentiation and disorganized arrangement of fibrous astrocytes in the ventral white matter. Together, our studies indicate that Nkx6.1 plays a vital role in astrocyte specification and differentiation in the ventral spinal cord.


Introduction
Astrocytes and oligodendrocytes are macroglial cells found in all regions of the central nervous system (CNS). It is estimated that macroglial cells constitute as many as 90% of cells in some regions of the CNS, with astrocytes being the predominant cell type. Although both glial cell types act to support the activities of neurons, oligodendrocytes and astrocytes have clearly distinct functions. The primary function of an oligodendrocyte is to form myelin sheaths around multiple axons for rapid transmission of electrical pulses along axons. In contrast, astrocytes play many diverse roles, both supportive and active, in the functioning of the CNS. Among those are the regulation of ion and neurotransmitter concentrations, formation of the brain blood barrier, modulation of synapse formation and efficacy [1], and induction of neurogenesis in the adult brain [2]. Thus, it is unsurprising that abnormalities in astrocyte density and functioning have recently been implicated in several common neurological diseases including neuropathic pain, depression, and schizophrenia [3,4].
Spinal cord has served as an excellent model system to study the origin and molecular specification of neurogenesis and gliogenesis due to its relatively simple anatomy and structures. In the developing spinal cord, neuroepithelial cells in the ventricular zone (VZ) first give rise to neurons which subsequently migrate away from the ventricular zone by radial migration. At later stages, neuroepithelial cells switch to produce glial cells, i.e. astrocytes or oligodendrocytes. In the past decade, great progress has been made in our understanding of the origin and molecular control of oligodendrocyte development. During gliogenesis, early oligodendrocyte progenitor cells (OPCs) originate from the ventral motor neuron progenitor domain (pMN) of the ventral neuroepithelium [5,6], but a small number of OPCs are also generated from dorsal neural progenitor cells at later stage [7,8]. Recent molecular and genetic evidence suggests that astrocytes are produced from other domains of neural progenitor cells, particularly from the p1-p3 domains in the ventral spinal cord [9,10]. Although considerable progress has been made in our understanding of the molecular specification of oligodendrocytes, the molecular mechanisms that control the development of astrocytes have been lagging, partly due to the lack of well-defined stage-specific markers for astrocyte lineage.
Previous studies have demonstrated that the Nkx6.1 homeodomain transcription factor is widely expressed by neural progenitor cells within the ventral neural tube and it plays a prominent role in ventral neural patterning and neurogenesis [11][12][13]. During gliogenesis, Nkx6.1 expression is retained in neuroepithelial cells in the ventricular zone (VZ), including the progenitor cells that produce astrocyte precursors [14]. More recently, it has been shown that the identity of positionally distinct ventral astrocyte subtypes (VAs) is determined by Nkx6.1 [15]. In this study, we showed that Nkx6.1 is selectively expressed by ventral astrocytes after they migrate away from the VZ to the surrounding parenchyma. Nkx6.1 ablation leads to abnormal specification, delayed differentiation and disorganized morphology of ventral astrocytes, indicating an important role for Nkx6.1 in the development of astrocytes in the ventral spinal cord.

Animals
Mice used in this study were handled according to the protocols approved by Institutional Animal Care and Use Committee (IACUC), University of Louisville (IACUC: 12034). C57BL/6N mice were obtained from Jackson Laboratory. The Nkx6.1 homozygous null (KO) embryos were obtained by the interbreeding of double heterozygous animals. For generation of tissuespecific Nkx6.1 conditional mutant mice, Nkx6.1 conditional knockout hGFAP Cre/+ ;Nkx6.1 flox/flox mice were bred with the hGFAP Cre line to generate tissuespecific Nkx6.1-knockout mice.

Western blotting
Spinal cord tissues were lysed in tissue lysis buffer (Sigma) with protease inhibitor cocktail (Sigma). 30 ng protein from control and mutant tissues was loaded for SDS-PAGE electrophoresis and subsequently detected with anti-Olig2 (Abcam, 1:10000) anti-GFAP (Millipore Bioscience Research Reagents), anti-Nkx6.1 (DSHB Inc.), and mouse anti-GAPDH (Sigma, 1:5000) antibodies according to the standard protocol. The optical density of blots on films was assessed with the analysis tool in Quantity One software of BIO-RAD ChemiDoc XRS (Biorad, USA) and the relative densitometric values were used for statistical analyses on the expression level of target proteins.

Primary Astrocyte culture and Nkx6.1 knockdown in vitro
Cerebral cortices from P0 s.d. rats were dissected out, minced and digested in 0.25% trypsin at 37uC. The digestion was stopped by the addition of Dulbecco's modified Eagle's medium (DMEM)/ F12 containing 10% fetal bovine serum (FBS). The dissociated cells were plated in a 75 cm 2 tissue culture flask coated with 100 mg/ml poly-L-lysine, and the whole medium was changed next day. After 10 days' culture, the cells were rinsed three times with culture medium and pre-shaken for 1 hour at 200 rpm to remove microglia. Then flasks were sealed and shaken at 250 rpm at 37uC for 15-18 hours to remove oligodendrocyte precursor cells. The medium with the detached cells was collected and first plated on tissue culture dishes (non PDL coated) for 30-60 minutes at 37uC with a gentle swirling of the dishes to eliminate contaminating dead cells and residual microglia. The nonadherent cells were collected, centrifuged and replated in DMEM containing 10% FBS.

Statistical Analysis
Statistical differences were determined by Student's t test for two-group comparison or by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. The accepted level of significance was P,0.05. Data in the text and figures are presented as mean6SEM.

Nkx6.1 is selectively expressed in ventral gray and white matter astrocytes
Nkx6.1 is widely expressed by neural progenitor cells within the ventral neural tube throughout embryogenesis [11][12][13][14]. As the majority of ventral domains contribute to astrogliogenesis, it is likely that the Nkx6.1 may regulate astrocyte development in the spinal cord. Immunostaining in postnatal mouse spinal cords revealed scattered Nkx6.1-expressing cells in the ventral spinal cords of early postnatal stages. Double immunostaining in postnatal spinal cord revealed that Nkx6.1+ cells did not coexpress the oligodendrocytes markers Olig2 and Sox10, nor the microglia marker Iba1 ( Figure 1A, B, H). Instead, they coexpressed the astrocytic progenitor markers BLBP and Fgfr3 [22][23][24] (Figure 1C, F, G). Detailed analysis revealed that Nkx6.1+ nuclei were localized in GS+ protoplasmic astrocytes and in a few NeuN+ neurons in the ventral gray matter ( Figure 1E, I), and were intimately associated with GFAP+ fibrous astrocytic processes in the white matter ( Figure 1D). These expression studies demonstrate that during gliogenesis stage, Nkx6.1 is specifically expressed

Nkx6.1 is required for the migration of astrocyte progenitors
To investigate the function of Nkx6.1 in regulating astrogliogenesis in the ventral spinal cord, we first examined the effect of Nkx6.1 null mutation on the expression of S100b, an early astroglial marker expressed in the VZ [25]. At embryonic day 15.5 (E15.5), S100b was predominantly expressed in the VZ (Fig 2A). In Nkx6.1 mutant, ventricular expression of S100b was dramatically reduced, but its ectopic expression was detected within the floor plate (Fig 2B). At E18.5, ventricular S100b expression was still present in the wild-type tissue, but completely vanished in the mutant spinal cord (Fig 2C-D), suggesting that Nkx6.1 mutation led to an abnormal fate specification of ventricular neural progenitor cells.
We next examined the expression of two other astrocytic progenitor markers, Fgfr3 and Glast [22,26,27] in the ventral spinal parenchyma by RNA in situ hybridization (ISH). At E12.5, expression of Fgfr3 and Glast was restricted to the ventricular zone of the wild-type and Nkx6.1 mutant embryos ( Figure 3A-B9). At E14.5, numerous Fgfr3+ and Glast+ astrocyte progenitors had migrated out into white matter in control embryos, but few were seen in the mutants ( Figure 3C-D9, I). At later stages, the number of astrocyte progenitors in the white matter of Nkx6.1 mutants increased rapidly ( Figure 3E-F9). By E18.5, no apparent difference was found in the wild-type and mutant animals ( Figure 3G-H9). These results suggested that Nkx6.1 is required for the timingly generation and migration of astrocyte progenitors in the ventral spinal cord.

Differentiation of fibrous astrocytes in ventral white matter is retarded in Nkx6.1 mutants
We next assessed whether the differentiation of astrocytes is also influenced by Nkx6.1 mutation in the ventral spinal cord. GS and GFAP are lineage-specific markers that label protoplasmic in the gray matter and fibrous astrocytes in the white matter, respectively [28,29]. At E18.5, GFAP is largely expressed by fibrous astrocytes in the ventral white matter in control animals ( Figure 4A, E). However, a marked decrease of GFAP expression was observed in the ventral white matter of Nkx6.1 mutant mice at both the mRNA ( Figure 4B) and protein levels ( Figure 4F). As expected, expression of GFAP in dorsal spinal cord was not affected by the mutation (Figure 4C, D). At the same stage, strong GS immunoreactivity was observed in the entire gray matter in both the wildtype and mutant spinal cords, and no significant difference was detected between these two genotypes (Fig 4G, H).
Since Nkx6.1 mutant animals die immediately after birth due to defective motor neuron development, it is not clear whether Nkx6.1 function is required for postnatal astrocyte development. Thus, we generated the hGFAPCre;Nkx6.1 flox/flox conditional mice (Cko) in which Nkx6.1 expression was selectively ablated in the ventricular progenitor cells after neurogenesis [30] (Figure 5A9-D9). Consistent with the results observed in the Nkx6.1 conventional knockouts, GFAP was significantly reduced in the ventral white matter of Cko mutants during embryogenesis ( Figure 5A, A9). However, at postanatal stages, expression of GFAP in Cko mutants increased with time in the white matter ( Figure 5B9, C9, D9), despite the persistently lower level of its expression than that in the wild-type animal at all stages examined ( Figure 5B, C, D). Western blotting results revealed a similar reduction of GFAP protein expression in Cko mutant spinal cords from E18.5 up to postnatal P5 ( Figure 5E, F). Together, these observations suggest that Nkx6.1 ablation results in delayed expression of GFAP in the ventral spinal cord, indicating that Nkx6.1 activity is vital for the timely differentiation of GFAP+ astrocytes in this region.

Nkx6.1 knockdown suppress astrocyte differentiation in culture
To address the cell-autonomous effects of Nkx6.1 deficiency in astroglia lineage development, cultured astrocytes were treated with siRNA to suppress Nkx6.1 expression. Consistent with the in vivo data, the treatment significantly reduced GFAP expression in primary astrocyte culture ( Figure 6A), suggesting an autonomous role for Nkx6.1 in the regulation of astrocyte differentiation. Meanwhile, the expression of Olig2 and STAT3 was also reduced in the astrocytes treated with siRNA-Nkx6.1 ( Figure 6B). Since STAT3 is involved in astrocyte differentiation [31], it has raised the possibility that Nkx6.1 may regulate astrocyte differentiation through the JaK/STAT signaling pathway.

Nkx6.1 is required for the generation of VA2 white matter astrocytes
It has been shown that there are at least three subtypes of white matter astrocytes in the ventral spinal cord, ventral astrocyte subtypes 1, 2 and 3 (VA1, VA2 and VA3) that are produced from p1, p2 and p3 progenitor domains, respectively [15]. It appears that Nkx6.1 is sufficient for the emergence of Slit1+ VA2 and VA3 astrocytes; however, it is not clear if Nkx6.1 is required for VA2/3 astrocytes. Thus, we examined the Slit1 expression in the spinal cord of Nkx6.1KO mice at various embryonic stages. Prior to gliogenic stages, Slit1 was only expressed in ventricular neural progenitor cells ( Figure 7A, B). At E14.5, many Slit1+ cells had migrated out into the white matter from p3 domain in the wildtype embryo; however, fewer Slit1+ cells were detected in the ventral white matter in the mutants ( Figure 7C-F). By E18.5, Slit1 expression was observed in both VA2 and VA3 astrocytes in wildtype spinal cord, but only in VA3 astrocytes in Nkx6.1 mutants ( Figure 7G, H). These observations suggested that Nkx6.1 is essential for the development of VA2 white matter astrocytes.

Discussion
The Nkx6.1 homeobox gene is expressed in the ventral neural progenitor cells that give rise to motor (MN), V2, and V3 neurons [12,13] and ventral oligodendrocytes [19,30]. Mice carrying Nkx6.1 null mutation exhibit a ventral-to-dorsal switch in the identity of progenitor cells with a ventral expansion of V1 interneurons at the expense of MN and V2 neurons [16]. During gliogenesis, it causes a significant delay in OPC specification and differentiation [32,7]. In this study, we found that Nkx6.1 also plays a vital role in the development of astrocyte lineage in the spinal cord. Nkx6.1 is continuously expressed in astrocyte precursor cells after they migrate from the VZ into the parenchyma (Figure 1). Similarly, S100B is also expressed by astroglial progenitor cells in the VZ and immature astrocytes that are dispersed within the mantle zone (Fig 2) [33,34]. Disruption of Nkx6.1 lead to a loss or severe reduction of S100B expression in the VZ at E15.5 and E18.5 stages (Figure 2). The lack of S100B expression in the mutants strongly suggests that Nkx6.1 plays an essential role in the fate specification or lineage maintenance of astrocyte precursors in the ventral VZ. Consistently, we noticed that the expression of VA2 Slit1 was lost from E16.5 ventral spinal cord in Nkx6.1 null mice. This finding also indicated that Nkx6.1 is required for the development of VA2 but not VA3 white matter astrocytes (Figure 7). It has been proposed that co-expression of Pax6 and Nkx6.1 specifies a VA2 phenotype (Reelin+, Slit1+) [15]. The normal Reelin expression in Nkx6.1 mutant spinal cord ( Figure S1) suggests that VA2 astrocytes may acquire the VA1 phenotype (Reelin+/Slit1-), consistent with the finding that expression of Nkx2.2 and Pax6 in the VZ were not affected in the Nkx6.1 mutant [16].
Intriguingly, ectopic expression of S100B was observed in the floor plate of E15.5 spinal cords ( Figure 2B), which may be caused by the abnormal migration of mis-specified ventricular cells. Concommitantly, migration of Glast+ and Fgfr3+ astrocyte progenitors from the ventral ventricular zone into the ventral parenchyma was also affected and delayed (Figure 3). Expression of mature astrocyte marker GFAP was also retarded in the ventral white matter (Figure 4). Moreover, the processes of GFAP+ fibrous astrocytes in the white matter become irregular (Figure 3, 4). In support of this notion, siRNA-Nkx6.1 treatment reduces GFAP expression in primary astrocyte culture in vitro ( Figure 6), suggesting an autonomous role for Nkx6.1 in the regulation of astrocyte differentiation. Collectively, these observations demonstrate that Nkx6.1 function is required for the normal differentiation and morphogenesis of ventral fibrous astrocytes.
The mechanisms underlying the retarded differention and maturation of astrocyte progenitor cells are currently unknown. Previous studies demonstrated that Olig2 function is important for white matter astrocyte formation in the spinal cord at postnatal stages [16]. We found that Nkx6.1 mutation leads to reduced expression of Olig2 in the spinal cord at both embryonic [32] and early postnatal stages ( Figure S2), it is plausible that the defective astrocyte specification and differentiation in Nkx6.1 mutants may be caused by reduction in Olig2 expression. Consistently, inhibition of Olig2 expression was also observed in primary astrocytes when Nkx6.1 expression is knocked down ( Figure 6B). Moreover, the expression of STAT3 was also reduced in the astrocytes treated with siRNA-Nkx6.1 ( Figure 6B). It raised the possibility that Nkx6.1 regulated astrocyte differentiation through the JaK/STAT signaling pathway, given that STAT3 is required for astrocyte differentiation [31]. Figure S1 Normal Reelin expression in spinal cords at embryo stages of Nkx6.1 mutant. Spinal cord sections from E12.5, E14.5, E16.5 and E18.5 were subjected to ISH with Reelin riboprobe in WT and Nkx6.12/2 mice. Reelin was similarly expressed in WT and Nkx6.12/2 mice at all stages (A-H). I,J: The insets are the higher magnification of ventral astrocyte subtypes 2 (VA2) of WT and Nkx6.12/2 spinal cord at E18.5.Scale bars: 100 mm. Error bar, standard deviation (n = 3, **P,0.01, n.s. = no significant). (TIF)