
Neurogenesis is a complex sequence of coordinated events that involve specification, expansion and differentiation of distinct neuronal subtypes (1, 2). Generation of distinct neuronal subtypes in the developing spinal cord is regulated by signalling gradients originating from dorsal and ventral organizers (3, 4). Shh secreted from notochord and floor plate of the developing ventral neural tube plays a key role in generation of ventral neural subtype, including motor neurons (5-7). Consistently, genetic deletion of Shh leads to severe defects in the neural tube development, including the failure to generate many ventral neuronal subtypes (8). On the other hand, ectopic activation of Shh signalling pathway in dorsal region of neural tube triggers up-regulation of ventral neuronal gene expression accompanied by blocking of dorsal-specific gene expression (7, 9). Importantly, the graded activity of Shh signalling pathway regulates specification of ventral neuronal subtypes through regulation of key transcription factors (2, 10). Thus, tight regulation of Shh signalling pathway is critical for generation of specific neuronal subtypes. Neurons generated in more ventral regions of the neural tube, such as motor neurons require progressively higher concentration of Shh for their induction (2). In response to two- to three-fold changes in Shh concentration, five distinct neuronal subtypes in the spinal cord can be generated
Shh signalling pathway is initiated by Shh binding to the primary receptor Patched-1 (Ptch1) and coreceptors that activate the 7-transmembrane protein, Smo and the downstream transcription factors such as Glioma-associated oncogene (Gli) transcription factors. This signalling activation then induces genes implicated diverse cellular processes including cell specification and differentiation. Cdo and Boc, members of the immunoglobulin (Ig) superfamily, function as Shh coreceptors to induce full activation of Shh signalling pathway together with another coreceptor Gas1 (14-16). Cdo is transiently expressed in the critical organizing regions, such as the prechordal plate and notochord in central nervous system (CNS) development and more persistently in the dorsal region and the roof plate of the developing neural tube (14, 17). Cdo deficiency causes multiple defects in CNS, resulting in holoprosencephaly, hydrocephalus, reduced cortical thickness and reduced ventral neural fate patterning (15, 18). In Cdo-deficient mice, Shh signalling activity is decreased, leading to the defective specification of ventral neural cell fates in the developing spinal cord as well as holoprosencephaly (14, 17, 19). The double mutant mice for two Shh coreceptors Cdo and Gas1 exhibit a complete loss in the specification of progenitors for the floor plate, V3 interneuron and motor neurons that are dependent on Shh. In addition, Cdo and Boc double knockout mice have revealed that the specification of Olig2-positive motor neuron progenitors appears to be normal in developing neural tube at E10.5, while Cdo and Boc are required for the maintenance of Olig2-positive motor neuron progenitors after the initial specification (14). Coreceptors are required to activate the optimal Shh signal strength required for diverse neuronal subtypes. However, the discrete function of Cdo in motor neuron specification is not fully understood.
In this study, we took advantage of an
All mouse work was carried out as previously described (19, 20). The result of mouse genotyping for the current study is presented in Supplementary Fig. S1. The heterozygous Cdo mutant mice were maintained on a C57BL/6 background.
Immunostaining of EBs was carried out as previously described (22). Briefly, EBs were fixed and dehydrated with 4% Paraformaldehyde (PFA) and followed by sequential Sucrose incubation. After cryo-embedding, 7
Quantitative real-time PCR (qRT-PCR) analysis was performed as previously described (23). Total RNAs were isolated by using Trizol reagent (Invitrogen) following manufacturer’s instructions. cDNA synthesis was obtained using with PrimeScriptTM RT reagent kit (TaKaRa, Japan) and analyzed by qRT-PCR using SYBR Premix Ex Taq (TaKaRa). The values were normalized to the level of
Coverslips were transferred to a recording chamber mounted to a microscope (Olympus, Tokyo, Japan) for electrophysiological recordings. Experiments were performed on DIV 3 neurons in culture using the whole-cell patch clamp technique. Data were collected using a Multi-Clamp 700B amplifier (Molecular Devices, California, USA), data acquisition system (Digidata 1550, Molecular Devices), and Igor Pro analysis software (Wavemetrics). The cells were superfused at 2∼3 ml/min with solution of the following composition; 143 mM NaCl, 5.4 mM KCl, 5 mM HEPES, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.8 mM CaCl2 and 11.1 mM Glucose (pH 7.4, 300∼305 mOsmol/kg). Whole-cell patch clamp recordings were made at room temperature using micropipettes (3∼5MΩ, Sutter Instrument, Califormia, USA) filled with an internal solution containing 143 mM K-Gluconate, 15 mM HEPES, 7 mM KCl, 0.1 mM EGTA, 4 mM Mg-ATP, 0.3 mM Na-GTP and 4 mM Na-Ascorbate (pH 7.3, 290∼295 mOsmol/kg). The following parameters were measured: (1) the resting membrane potential (RMP), (2) the input resistance (IR, membrane potential changes (V) for given hyperpolarizing current (−30 pA, 500 min) input), (3) after-hyperpolarization (AHP), (4) AP threshold current (current threshold for single action potential generation, 30 min duration), (5) AP amplitude, (6) AP incidence. Voltage-gated sodium and potassium channels were detected in voltage-clamp mode at a holding potential of −70 mV. The holding potential was changed in a stepwise fashion from −60 to +50 mV in 10 mV increments for 1 sec and the voltage-gated peak inward current and sustained outward current (between 800 and 900 min) were measured for each step.
Values are means±SEM or SD as noted in figure legends. Statistical significance was calculated using paired or unpaired two-tailed Student’s
Using a standard protocol as illustrated in Fig. 1a, we induced differentiation of ESCs into motor neurons. To confirm the sucessful motor neuron differentiation, we have analized the differentiation-associated gene expression at subdivided time points for 2 days of NI (NI1, NI2), 5 days of MNS (MNS-1, MNS-3, MNS-5) and Elong. The expression of stemness genes, such as
Before gaining insights into Cdo’s functions in motor neuron specification, we decided to demonstrate clearly that among Shh coreceptors, Cdo is essentially required to motor neuron specification. For that we examined the levels of
Based on the results of RNA-sequencing, we defined the effect of Cdo deficiency on motor neuron specification further. Total RNAs from
To further confirm the result,
In an effort to uncover the role of Cdo as a coreceptor in the regulation of Shh signalling pathway during motor neuron specification, we reactivated Shh signalling pathway in Cdo-deficient ESCs by adding 1
To examine the electrophysiological properties of neurons derived from
The requirement of Shh signalling activity for the efficient generation of ventral neurons has been demonstrated by several previous studies (10, 28, 29). In this study, we attempted to address the defined function of Cdo as a Shh coreceptor in motor neuron generation. Multiple studies have underlined the importance of Cdo in Shh signalling pathwary and neural tube patterning (13, 17, 30). In spite of distinct requirements of three coreceptors, Cdo, Boc, and Gas1 for Shh signalling pathway in a developmental process, the defined function of each coreceptor is still imcompletely understood. Cdo single and Cdo/Boc double mutant mice exhibit defects in neural tube patterning related to impaired Shh signalling pathway (13, 14). However, these coreceptors are not required for other Shh signalling-controlled developmental processes (14). Cdo/Boc/Gas1 triple knockout mice exhibit almost equivalent phenotype of mice lacking Shh, suggesting that these coreceptors are essential for Shh signal transduction. The expression of all three coreceptors in motor neuron generation of ESCs is consistent with their expression pattern in the neural tube development (31, 32). Our data suggest that there is a temporal difference of gene induction which might reflect their action in the multiple stages of motor neuron generation. Cdo expression was induced and stayed high during mortor neuron specification, while Boc expression was progressively increased in motor neuron generation. Gas1 was induced later than Cdo and Boc in this process. Thus, it is likely that Cdo might be the earliest acting Shh coreceptor that might be critical for motor neuron specification.
Shh signalling pathway has been proposed to play a stage-specific function in the specification of ventral neuronal subtypes (5, 12, 31, 33). In the early stage, Shh signalling pathway is critical for the specification of the most ventral cell type Nkx2.2-positive V3 interneuron and later, it is required for the maintenance of Olig2-positive motor neuron progenitors (34). This is consistent with the current data showing that Olig2 levels were mildly decreased in Cdo-deficient cells at MNS-5 while subsequently at Elong stage, the levels were significantly affected. Unlike Olig2, the expression of Nkx6.1 together with Isl1 and Chx10 was greatly reduced in Cdo-deficient cells at MNS-5. Our data is consistent with the reported role of Nkx6.1 in motor neuron specification. Nkx6.1 is expressed in motor neuron, V2 and V3 interneuron progenitors. Also, mice lacking Nkx6.1 display an impairment in motor neuron and V2 interneuron specification with diminished expression of Hb9 and Isl1 (35). Previous studies have reported that weakened Shh signalling activity causes a shift of ventral to dorsal neuronal patterning (8, 31, 32). Consistently, Cdo-deficient cells express significantly elevated dorsal interneuron markers, such as Brn3a, Pax2, Lbx1 and Pax3 in response to Shh addition. The ventral to doral shift in Cdo-deficient cells was alleviated by the reactivation of Shh signalling pathway by SAG treatment. Among ventral markers, Olig2, Isl1 and Chx10 were expressed higher in Cdo-deficient cells in response to SAG, relative to the wildtype cells. Especially the level of Olig2 was still slightly reduced in Cdo-deficient cells at MNS-5 which was greatly elevated at the late stage of differentiation, likely reflecting a delayed motor neuron specfication. In addition, the dorsal neuron marker, Brn3a expression was greatly decreased while the level of Pax2 and Lbx1 was not fully suppressed in Cdo-deficient cells at MNS-5 in response to SAG treatment. These data suggest that Cdo might regulate a Shh-independent mechanism to suppress dorsal cell fate. RA and Shh signallings are essential for motor neuron specification (28, 36) and the inhibition of Notch signalling has been shown to provide a permissive signal for the motor neuron specification. Especially the forced expression of a Notch effector, Hes5 inhibits motor neuron differentiation (37). The residual ventral to dorsal shift observed in Cdo-deficient cells likely reflects deregulated Notch signalling pathway. Consistently, the close examination of RNA sequencing result reveals that the level of Hes5 was elevated in Cdo-deficient cells relative to the wildtype cells (date not shown). Currently it is unkown whether Cdo regulates Notch signalling pathway. Other possibility is that Cdo might suppress Wnt signalling pathway critical for dorsal cell fate specification (4, 38). We have previously shown that Cdo inhibits Wnt signalling pathwat by interaction with a Wnt signal coreceptor, Lrp6 in the control of forebrain development (39). Cdo-deficiency causes hyperactive Wnt signalling resulting in expansion of dorsal cell fate. Considering the graded activity of Wnt signalling pathway is critical for dorsal ventral patterning, Cdo deficiency causes dysregulation of Wnt signalling contributing to ventral to dorsal shift. Our data further refine the function of Cdo in motor neuron specification, providing the mechanisms regulating differentiation of pluripotent stem cells into motor neurons. Discoveries along these lines would facilitate pluripotent stem cell-based regenerative medicine applications.
Supplementary data including one table and four figures can be found with this article online at http://pdf.medrang.co.kr/paper/pdf/IJSC/IJSC-13-s20037.pdf.
This research was supported by the National Research Foundation of Korea Grant funded by the Korean Govern-ment (MSIP) (NRF-2019R1A2C2006233, NRF-2017M3A9 D8048710 and NRF-2016R1A5A2945889 to JSK, NRF-201 6H1A2A1908679 to HK, and NRF-2018R1D1A1B0704166 1 to YEL).
The authors have no conflicting financial interest.
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