Neuronal differentiation in the developing human spinal ganglia

Abstract

The spatiotemporal developmental pattern of the neural crest cells differentiation toward the first appearance of the neuronal subtypes was investigated in developing human spinal ganglia (SG) between the fifth and tenth developmental week using immunohistochemistry and immunofluorescence methods. First neurofilament-200-(NF200, likely myelinated mechanoreceptors) and isolectin-B4-positive neurons (likely unmyelinated nociceptors) appeared already in the 5/6th developmental week and their number subsequently increased during the progression of development. Proportion of NF200-positive cells was higher in the ventral parts of the SG than in the dorsal parts, particularly during the 5/6th and 9/10th developmental weeks (Mann-Whitney, P 5 0.040 and P 5 0.003). NF200 and IB4 colocalized during the whole investigated period. calcitonin gene-related peptide (CGRP; nociceptive responses), vanilloid receptor-1 (VR1; polymodal nociceptors), and calretinin (calcium signaling) cell immunoreactivity first appeared in the sixth week and eighth week, respectively, especially in the dorsal parts of the SG. VR1 and CGRP colocalized with NF00 during the whole investigated period. Our results indicate the high potential of early differentiated neuronal cells, which slightly decreased with the progression of SG differentiation. On the contrary, the number of neuronal subtypes displayed increasing differentiation at later developmental stage. The great diversity of phenotypic expression found in the SG neurons is the result of a wide variety of influences, occurring at different stages of development in a large potential repertory of these neurons. Understanding the pathway of neural differentiation in the human, SG could be important for the studies dealing with the process of regeneration of damaged spinal nerves or during the repair of pathological changes within the affected ganglia.

Time course and expression pattern of the neuronal markers in the developing human spinal cord

“…Since the myelinization still does not occur in the presented investigated periods, NF200 positive fibers could be described as nonnociceptive ones. The same was found in developing human spinal ganglia (Vukojevic et al, 2016) and in the SC (Foster et al, 1987). Moreover, NF200 was expressed in mechanoreceptors of dural afferents in mouse, pointing that all trigeminal ganglion neurons expressing NF200 innervate mouse dura (Ren et al, 2018).…”

“…In the present study CGRP was expressed in all three SC zones, especially during the 5th–6th DW indicating cessation of the neural differentiation into sensory neurons. Our previous study on SG has also shown that first signs of the CGRP appeared in the sixth week of development (Vukojevic et al, 2016), confirming that the fibers which terminate in the superficial laminae of the dorsal SC are from neurons located in the DGR (Ribeiro da Silva, 1995; Ribeiro da Silva and De Koninck, 2008). These small peptidergic afferents are associated to the transmission of the pain related informations.…”

“…Following the procedure for paraffin embedded tissues (Vukojevic et al, 2016), primary antibodies were applied: rabbit Anti‐200 kD Neurofilament Heavy antibody (Ab8135, 1:300, Abcam, Cambridge, UK), goat anti‐CGRP antibody (ab36001, 1: 1,000, Abcam, Cambridge, UK), sheep polyclonal to VR1 (ab63103, 1:100, Abcam, Cambridge, UK) and rabbit polyclonal anti‐Calretinin antibody (ab702, 1:300, Abcam, Cambridge, UK). After overnight incubation and washing in PBS, primary antibodies were visualized with appropriate secondary antibodies: donkey anti goat‐rhodamine conjugated (Jackson ImmunoResearch Laboratories, Inc., Baltimore, PA, USA; 705‐295‐003); donkey anti‐rabbit IgG H&L (Alexa Fluor ® 488) (ab150073, Abcam, Cambridge, UK) and donkey anti‐sheep IgG‐ FITC (713‐095‐147 Jackson ImmunoResearch Laboratories, Inc., Baltimore, PA, USA), at dilution of 1:300.…”

“…It is expressed by primary afferent nociceptors (Andoh et al, 1997; Caterina et al, 1997) and it is probably necessary for transduction of nociceptive stimuli (Karai et al, 2004) and neuropathic pain (Hwang and Valtschanoff, 2003). VR1 receptors were found in the SC sensory neurons, in dorsal root ganglion (DRG) (Vukojevic et al, 2016) in the SC visceral afferents of the rat (Huang et al, 2002; Hwang and Valtschanoff, 2003), and human SC ventral horn as well, but with less intensity (Anand et al, 2008). Calretinin is a member of calcium‐binding protein family (CaBPs) present in the neurons and nerve fibers of birds and mammals (Résibois and Rogers, 1992; Castro et al, 2006) involved in somatosensory and proprioceptive pathways (Ren et al, 1993; Li et al, 2000; Ichikawa et al, 2005).…”

“…Various neuronal subpopulations in zebrafish (Castro et al, 2006), lamprey's SC (Viloria et al, 2008), Purkinje cells and migrating cerebellar interneurons in human (Yew et al, 1997; Pibiri et al, 2017) expressed calretinin. Since we have long time experience in the investigation of the neurulation mechanisms in human development, especially those of spinal cord (Saraga‐Babić et al, 1995; Saraga Babic et al, 1996; Saraga‐Babic et al, 2002; Vukojevic et al, 2016) in the present study we tried to elucidate the spatio‐temporal distribution of several neuronal markers in the different zones of the human SC at early embryonic stages. These markers are usually expressed by certain subtype of neurons so they could provide more information about the order of appereance of different neuron subtypes in developing SC.…”