Takako Shimada

Expression pattern of LRR and Ig domain-containing protein (LRRIG protein) in the early mouse embryo.

The combination of leucine-rich repeat (LRR) and immunoglobulin-like (Ig) domains is found in the domain architecture of the Trk neurotrophin receptor protein. Recently dozens of such proteins simultaneously carrying LRR and Ig domains as the Trk receptors have been identified. Given the significant biological roles of Trk and such newly identified proteins, we have searched the public database for human proteins with LRR and Ig domains (collectively termed the leucine-rich repeat and Ig domain-containing protein, LRRIG protein, in this study), and have analyzed the mRNA expression pattern of mouse orthologs of obtained human LRRIG proteins at embryonic day 10. The list of the LRRIG proteins includes 36 human proteins: four LINGO, three NGL, five SALM, three NLRR, three Pal, two ISLR, three LRIG, two GPR, two Adlican, two Peroxidasin-like proteins, three Trk neurotrophin receptors, a yet unnamed protein AAI11068, and three AMIGO. Some molecules (LINGO2, LINGO4, NGL1, SALM1, SALM5, and TrkB) were expressed exclusively in neuronal tissues, whereas others (ISLR1, GPR124, and Adlican2) exhibited non-neuronal expression profiles. However, the majority of LRRIG protein family exhibited broad mRNA tissue-expression profiles.

Caspase activity is involved in, but is dispensable for, early motoneuron death in the chick embryo cervical spinal cord

We examined the role of caspases in the early programmed cell death (PCD) of motoneurons (MNs) in the chick embryo cervical cord between embryonic day (E) 4 and E5. An increase in caspase-3-like activity in MNs was observed at E4.5. Treatment with an inhibitor of caspase-3-like activity, Ac-DEVD-CHO, for 12 h blocked this increase and revealed that caspase-3-like activity is mainly responsible for DNA fragmentation and the nuclear changes during PCD but not for degenerative changes in the cytoplasm. When a more broad-spectrum caspase inhibitor was used (bocaspartyl (OMe)-fluoromethyl ketone, BAF), the appearance of degenerative changes in the cytoplasm was delayed by at least 12 h. However, following treatment with either Ac-DEVD-CHO or BAF for 24 h, the number of surviving healthy MNs did not differ from controls, indicating a normal occurrence of PCD despite the inhibition of caspases. These results suggest that caspase cascades that occur upstream of and are independent of the activation of caspase-3-like activity are responsible for the degenerative changes in the cytoplasm of dying cervical MNs. These data also suggest that, although one function of caspases may be to facilitate the kinetics of PCD, caspases are nonetheless dispensable for at least some forms of normal neuronal PCD in vivo.

Differential expression of the GDNF family receptors RET and GFRα1, 2, and 4 in subsets of motoneurons: A relationship between motoneuron birthdate and receptor expression

Previous studies have demonstrated the expression of specific members of the glial cell line‐derived neurotrophic factor (GDNF) receptor family α (GFRα) in subsets of motoneurons (MNs) in the developing mouse spinal cord. We examined the expression pattern of GFRα and RET in the avian lumbar spinal cord during the period of programmed cell death (PCD) of MNs by using double labeling in situ hybridization and immunohistochemistry. In the lateral motor column (LMC) of the lumbar spinal cord, a laminar organization of GFRα expression was observed: GFRα1‐positive MNs were located in the medial LMC; GFRα1‐, 2‐, and 4‐positive MNs were situated in the lateral LMC; and GFRα4‐positive MNs were located in the intermediate LMC. The species of GFRα receptor that was expressed in MNs was found to be related to their birthdates. The expression of subpopulation‐specific transcriptional factors was also used to define MNs that express a specific pattern of GFRα. This analysis suggests that motor pools as defined by these transcriptional factors have unique expression patterns of GFRα receptor. Early limb bud ablation did not affect the expression of GFRα in the spinal cord, indicating that regulation of receptor expression is independent of target‐derived signals. Finally, GDNF mRNA expression was found in the limb during the PCD period of MNs. In conclusion, these results indicate that time of withdrawal from the mitotic cycle may specify the expression pattern of GFRα in subsets of MNs and that GDNF may function as a target‐derived neurotrophic factor for specific subpopulations of MNs. J. Comp. Neurol. 456:245–259, 2003.

Elucidation of target muscle and detailed development of dorsal motor neurons in chick embryo spinal cord

The avian cervical spinal cord includes motoneurons (MNs) that send their axons through the dorsal roots. They have been called dorsal motoneurons (dMNs) and assumed to correspond to MNs of the accessory nerve that innervate the cucullaris muscle (SAN‐MNs). However, their target muscles have not been elucidated to date. The present study sought to determine the targets and the specific combination of transcription factors expressed by dMNs and SAN‐MNs and to describe the detailed development of dMNs. Experiments with tracing techniques confirmed that axons of dMNs innervated the cucullaris muscle. Retrogradely labeled dMNs were distributed in the ventral horn of C3 and more caudal segments. In most cases, some dMNs were also observed in the C2 segment. It was also demonstrated that SAN‐MNs existed in the ventral horn of the C1–2 segments and the adjacent caudal hindbrain. Both SAN‐MNs and dMNs expressed Isl1 but did not express Isl2, MNR2, or Lhx3. Rather, these MNs expressed Phox2b, a marker for branchial motoneurons (brMNs), although the intensity of expression was weaker. Dorsal MNs and SAN‐MNs were derived from the Nkx2.2‐positive precursor domain and migrated dorsally. Dorsal MNs remain in the ventral domain of the neural tube, unlike brMNs in the brainstem. These results indicate that dMNs and SAN‐MNs belong to a common MN population innervating the cucullaris muscle and also suggest that they are similar to brMNs of the brainstem, although there are differences in Phox2b expression and in the final location of each population. J. Comp. Neurol. 521: 2987–3002, 2013.

Neurogenin2 expression together with NeuroM regulates GDNF family neurotrophic factor receptor α1 (GFRα1) expression in the embryonic spinal cord

In many regions of the nervous system, the combinatorial action of transcriptional factors specifies the individual fate of neuronal subtypes. Contrary to this, we report that a single transcriptional factor controls a phenotype shared by different subtypes of neurons, namely the expression of a neurotrophic factor receptor in the spinal cord. Along the dorsoventral axis of the chick embryo spinal cord, the expression pattern of a specific receptor for glial cell line derived-neurotrophic factor (GDNF family of receptors α1: GFRα1) was related to that of two basic helix-loop-helix (bHLH) transcriptional factors (NeuroM and Neurogenin2: Ngn2). In ovo electroporation in the chick embryo revealed that the overexpression of NeuroM alone was sufficient to induce ectopic GFRα1 expression without overt neuronal differentiation, whereas the suppression of NeuroM activity resulted in the specific loss of GFRα1 expression, indicating that NeuroM may act as a differentiation factor for GFRα1 expression. Ngn2 overexpression was also sufficient to induce precocious GFRα1 expression. However, the forced expression of both obligate suppressor and activator forms of Ngn2 also induced aberrant GFRα1 expression. Thus, any deviation from an optimum level of Ngn2 expression resulted in aberrant GFRα1 expression. Consistent with this, manipulation of Ngn2 expression levels by other bHLH factors also resulted in ectopic GFRα1 expression. For example, the downregulation by Ascl1 and the upregulation by Ptf1a induced ectopic GFRα1 expression, irrespective of endogenous expression patterns of Ascl1 and Ptf1a (Ascl1/Ptf1) in the spinal cord. The suppression of Ascl1/Ptf1a activities abolished Ngn2 and GFRα1 expression, even in Ascl1/Ptf1a-negative regions. These data indicate the presence of a distinct regulatory sequence for a determinant of GFRα1 expression, in which Ascl1/Ptf1a may competitively intervene to stochastically modulate default Ngn2 expression levels. Thus, Ngn2 together with NeuroM serves as readout to regulate GFRα1 expression, which occurs in multiple subtypes of spinal neurons.

Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1+ MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1+ MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1+ MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1+ MNs committed to LMC neurons, depending on the Hox expression pattern. Summary: Hox-dependent apoptosis in the limbless neck region of the chick embryo eliminates a transient population of spinal motor neurons that express Foxp1 and have characteristics of limb-innervating LMC neurons.

Regulation of GFRα1 expression by Ptf1a in the dorsal spinal cord

Xanthosine, a component of roasted rice bran extracts, is not polyphenol but has a potent antioxidant activity. We analyzed whether this chemical alleviates neuronal damage induced by a high dosage of ethanol. Rats were treated with 20% ethanol plus 0.1% xantosine for 42 weeks from the age of 6 weeks, and their CNS neurons were compared with those treated with or without 20% ethanol alone by immunoblotting, immunohistochemistry and electron microscopy. Addition of xanthosine to ethanol reduced distribution density of hepatic lipid droplets augmented by ethanol. In the CNS, xanthosine reduced appearance of lipofuscin granules and neurofilaments, markers for the neuronal damage, enhanced expressions of synaptophysin, superoxide dismutase and Bcl-xL related with activation of neuronal function, and decreased neurofilament proteins, GFAP and Bax associated with neurodeterioration. We first demonstrate that xanthosine is beneficial to the neurons.

BCL-2 introduced with retrograde transport of adenovirus vectors rescues motoneurons from cell death in vivo

To examine whether overexpression of Bcl-2 rescues motoneurons from programmed cell death in the developing chick spinal cord, we have generated adenovirus vectors capable of overexpressing Bcl-2. Following injection of Bcl-2 vectors into right leg buds of the chick embryos on embryonic day (E) 4.5-5 (mst 29, many motoneurons in the right lateral motor column (LMC) in lumbosacral segments strongly expressed Bcl-2 by E7. On E7 colchicine was injected into the same leg buds to induce motoneuron cell death due to depletion of target-derived neurotrophic factors. Virtually all motoneurons in the LMC died by El0 in the control embryos that received injection of colchicine without preceding injection of Bcl-2 vectors. By contrast, remaining motoneurons were observed in the LMC of the embryos that received injection of Bcl-2 vectors followed by injection of colchicine. Most of the remaining motoneurons expressed Bcl-2. These results suggest that Bcl-2 adenovirus vectors can be retrogradely transported from the periphery to cell bodies, and that overexpression of Bcl-2 can rescue motoneurons from cell death in viva.