ELAS1-mediated inhibition of the cyclin G1-B'γ interaction promotes cancer cell apoptosis via stabilization and activation of p53.

Radiation therapy (RT) is useful for selectively killing cancer cells. However, because high levels of ionizing radiation (IR) are toxic to normal cells, RT cannot be applied repeatedly to cancer patients. Therefore, novel chemicals that enhance the efficacy of chemoradiotherapy (CRT) would be valuable. Here, we report that ELAS1, a peptide corresponding to the protein phosphatase 2A (PP2A) association domain of cyclin G1 (CycG1), can enhance the efficacy of CRT. ELAS1 interacts with the PP2A B'γ-subunit and competitively inhibits association with CycG1, thereby preventing the PP2A holoenzyme from dephosphorylating target proteins, Mdm2 (pT218) and p53 (pS46), following DNA double-strand break (DSB) insults. Doxycycline (Dox)-induced overexpression of Myc-ELAS1 caused γ-irradiation to induce apoptosis in human osteosarcoma (U2OS) cells, at 1/10th the effective dosage of γ-irradiation required for apoptosis in Myc-vector-expressing cells; ELAS1 peptide incorporation into U2OS cells also showed similar apoptotic effects. Moreover, administration of DSB-inducing chemicals, camptothecin (CPT) or irinotecan, to Myc-ELAS1-expressing U2OS cells also induced efficient apoptosis with only 1/100th (CPT) or 1/5th (irinotecan) of the amounts of drugs required for this effect in Myc-vector-expressing cells. Taken together, ELAS1 may be important for the design of ELAS1-mimetic compounds to improve CRT efficacy.

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer.

BACKGROUND: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. METHODS: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. RESULTS: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. CONCLUSIONS: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

Silencing of the Lats2 tumor suppressor overrides a p53-dependent oncogenic stress checkpoint and enables mutant H-Ras-driven cell transformation.

The Lats2 tumor suppressor protein has been implicated earlier in promoting p53 activation in response to mitotic apparatus stress, by preventing Mdm2-driven p53 degradation. We now report that Lats2 also has a role in an ATR-Chk1-mediated stress check point in response to oncogenic H-Ras. Activated mutant H-Ras triggers the translocation of Lats2 from centrosomes into the nucleus, coupled with an increase in Lats2 protein levels. This leads to the induction of p53 activity, upregulation of proapoptotic genes, downregulation of antiapoptotic genes and eventually apoptotic cell death. Many of the cells that survive apoptosis undergo senescence. However, a fraction of the cells escape this checkpoint mechanism, despite maintaining a high mutant H-Ras expression. These escapers display increased genome instability, as evidenced by a substantial fraction of cells with micronuclei and cells with polyploid genomes. Interestingly, such cells show markedly reduced levels of Lats2, in conjunction with enhanced hypermethylation of the Lats2 gene promoter. Our findings suggest that Lats2 might have an important role in quenching H-Ras-induced transformation, whereas silencing of Lats2 expression might serve as a mechanism to enable tumor progression.

Two functional channels from primary visual cortex to dorsal visual cortical areas.

Relationships between the M and P retino-geniculo-cortical visual pathways and "dorsal" visual areas were investigated by measuring the sources of local excitatory input to individual neurons in layer 4B of primary visual cortex. We found that contributions of the M and P pathways to layer 4B neurons are dependent on cell type. Spiny stellate neurons receive strong M input through layer 4Calpha and no significant P input through layer 4Cbeta. In contrast, pyramidal neurons in layer 4B receive strong input from both layers 4Calpha and 4Cbeta. These observations, along with evidence that direct input from layer 4B to area MT arises predominantly from spiny stellates, suggest that these different cell types constitute two functionally specialized subsystems.

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.

Laminar specificity of local circuits in barrel cortex of ephrin-A5 knockout mice.

Cortical circuits are characterized by layer-specific axonal arbors. Molecular laminar cues are believed to direct the development of this specificity. We have tested the hypothesis that ephrin-A5 is responsible for preventing layer 2/3 pyramidal cell axons from branching within layer 4 (Castellani et al., 1998) by assessing the laminar specificity of axonal arbors in ephrin-A5 knockout mice. We find that in barrel cortex of knockout mice, layer 2/3 pyramidal neurons form axonal arbors specifically in layers 2/3 and 5, avoiding layer 4. This pattern of arborization is indistinguishable from that of wild-type littermates. Furthermore, we find that in wild-type mice, laminar patterns of ephrin-A5 expression differ between cortical areas despite the similarity of layer-specific local cortical circuits across areas. Most notably, ephrin-A5 is not expressed preferentially in layer 4 of wild-type mouse barrel cortex. We conclude that ephrin-A5 is not responsible for preventing the development of layer 2/3 pyramidal cell axonal arbors in layer 4 of mouse barrel cortex. These observations also suggest that if ephrin-A5 plays a role in the emergence of layer-specific circuits, that role must differ between cortical areas.

Rad24 is essential for proliferation of diploid cells in fission yeast.

The rad24(+) gene of Schizosaccharomyces pombe encodes a ubiquitously expressed 14-3-3 protein. We report here that Deltarad24 cells displayed a defect in diploid colony formation, although they conjugated efficiently. We found that a cumulative deletion of mei2(+) gene almost completely suppressed this defect, and demonstrated using two-hybrid analysis that Rad24 protein directly associates with Mei2 protein by recognizing Ser-438 which is a phosphorylation target of Pat1 kinase. We conclude that constitutive progression to meiosis, caused by lack of Mei2 inhibition due to the absence of Rad24 protein, is the primary cause of the proliferative deficiency observed in Deltarad24 cells.

Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts.

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.

Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F.

Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters.

Cytochrome-oxidase blobs and intrinsic horizontal connections of layer 2/3 pyramidal neurons in primate V1.

Pyramidal neurons in superficial layers of cerebral cortex have extensive horizontal axons that provide a substrate for lateral interactions across cortical columns. These connections are believed to link functionally similar regions, as suggested by the observation that cytochrome-oxidase blobs in the monkey primary visual cortex (V1) are preferentially connected to blobs and interblobs to interblobs. To better understand the precise relationship between horizontal connections and blobs, we intracellularly labeled 20 layer 2/3 pyramidal neurons in tangential living brain slices from V1 of macaque monkeys. The locations of each cell body and the cell's synaptic boutons relative to blobs were quantitatively analyzed. We found evidence for two cell types located at characteristic distances from blob centers: (1) neurons lacking long-distance, clustered axons (somata 130-200 microm from blob centers) and (2) cells with clustered, long-distance axon collaterals (somata < 130 microm or >200 microm from blob centers). For all cells, synaptic boutons close to the cell body were located at similar distances from blob centers as the cell body. The majority of boutons from cells lacking distal axon clusters were close to their cell bodies. Cells located more than 200 microm from blob centers were in interblobs and had long-distance clustered axon collaterals selectively targeting distant interblob regions. Cells located less than 130 microm from blob centers were found within both blobs and interblobs, but many were close to traditionally defined borders. The distant synaptic boutons from these cells were generally located relatively near to blob centers, but the neurons closest to blob centers had synaptic boutons closer to blob centers than those farther away. There was not a sharp transition that would suggest specificity for blobs and interblobs as discrete, binary entities. Instead they appear to be extremes along a continuum. These observations have important implications for the function of lateral interactions within V1.

Functional streams and local connections of layer 4C neurons in primary visual cortex of the macaque monkey.

The primate visual system is composed of multiple, functionally specialized cortical areas. The functional diversity among areas is thought to reflect different contributions from early parallel visual pathways to the area V1 neurons providing input to "higher" cortical areas. The M pathway is believed to provide information about motion and contrast, via layer 4B of V1, to dorsal visual areas. The P pathway is believed to provide information about shape and color, via layer 2/3 of V1, to ventral visual areas, with specialized contributions from cytochrome-oxidase (CO) blob versus interblob neurons. However, the detailed anatomical relationships between the M and P pathways and the neurons in V1 that provide input to higher extrastriate cortical areas are poorly understood. To study these relationships, spiny stellate neurons in the M- and P-recipient layers of V1, 4Calpha and 4Cbeta, respectively, were intracellularly labeled, and their axonal and dendritic arbors were reconstructed. We find that neurons with dendrites in upper layer 4Calpha project axons to layer 4B and CO blobs in layer 2/3, thus relaying M input to these regions. Other neurons in lower layer 4Calpha provide M input to interblobs. These cells have either (1) dendrites restricted to lower layer 4Calpha and axons specifically targeting layer 2/3 interblobs, or (2) dendrites in lower 4Calpha and 4Cbeta and axons targeting blobs and interblobs. P-recipient layer 4Cbeta neurons have dense axonal arbors in both blobs and interblobs but not layer 4B. Quantitative analyses reveal that 4Calpha cells provide approximately five times more synapses than 4Cbeta cells to layer 4B, whereas 4Cbeta cells provide five times more synapses than 4Calpha cells to layer 2/3. These observations imply that M input is dominant in layer 4B. In layer 2/3, both blobs and interblobs receive M and P input, but the P input is dominant, and M input to interblobs derives exclusively from a subpopulation of M afferents that targets lower 4Calpha, not from afferents targeting only upper 4Calpha (cf. Blasdel and Lund, 1983). We speculate that the M and P pathways to interblobs are "X-like" linear systems, whereas blobs also receive nonlinear "Y-like" M input.

Characterization of a maize Ca(2+)-dependent protein kinase phosphorylating phosphoenolpyruvate carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] of plants undergoes regulatory phosphorylation in response to light or nutritional conditions. However, the nature of protein kinase(s) for this phosphorylation has not yet been fully elucidated. We separated a Ca(2+)-requiring protein kinase from Ca(2+)-independent one, both of which can phosphorylate maize leaf PEPC and characterized the former kinase after partial purification. Several lines of evidence indicated that the kinase is one of the characteristic Ca(2+)-dependent but calmodulin-independent protein kinase (CDPK). Although the M(r) of native CDPK was estimated to be about 100 kDa by gel permeation chromatography, in situ phosphorylation assay of CDPK in a SDS-polyacrylamide gel revealed that the subunit has an M(r) of about 50 kDa suggesting dimer formation or association with other protein(s). Several kinetic parameters were also obtained using PEPC as a substrate. Although the CDPK showed an ability of regulatory phosphorylation (Ser-15 in maize PEPC), no significant desensitization to feedback inhibitor, malate, could be observed presumably due to low extent of phosphorylation. The kinase was not specific to PEPC but phosphorylated a variety of synthetic peptides. The possible physiological role of this kinase was discussed.

Structure, expression, and chromosomal localization of human GAK.

We previously cloned a rat cDNA encoding GAK, an association partner of cyclin G and CDK5. Here, we report the cloning of a cDNA encoding human GAK (1311 amino acids) and show that all of the unique motifs that characterize rat GAK, such as the presence of a Ser/Thr kinase domain, a tensin/auxilin homologous domain, and a Tyr phosphorylation target site, are conserved. The expression profiles of GAK and cyclin G during the synchronized HeLa cell cycle showed that GAK expression oscillates slightly, peaking at G1 phase, although the histone H1 kinase activity remains constant throughout the cell cycle. We also found that the kinase activity of immunoprecipitates of anti-cyclin G antibody fluctuates during the cell cycle with a peak at G1 phase, although the expression level of cyclin G remains almost constant. Northern blot analysis showed that GAK is expressed ubiquitously, with the highest level of expression being observed in the testis. By the FISH technique, we assigned the chromosomal localization of GAK to 4p16.

Expression, nuclear localization and interactions of human MCM/P1 proteins.

We report here the comparative analysis of human Mcm/P1 proteins (HsMcm2, -3, -5 and -7), including a characterization of their mutual interactions, cell cycle dependent expression and nuclear localization during the cell cycle and the quiescent state. The mRNA levels of these genes, which undergo cell cycle dependent oscillations with a peak at G1/S phase, may be regulated by E2F motifs, two of which were detected in the 5' upstream region of the HsMCM5 gene. In contrast, the protein levels of these Mcm proteins were found to remain rather constant during the HeLa cell cycle. However, their levels gradually increased in a variable manner as KD cells progressed from GO into the G1/S phase. In the GO stage, the amounts of HsMcm2 and -5 proteins were much lower than those of HsMcm7 and -3 proteins, suggesting that they are not present in stoichiometric amounts, and that only a proportion of these molecules actively participate in cell cycle regulation as part of Mcm/P1 complexes.

Organization of the descending projections from the parabrachial nucleus to the trigeminal sensory nuclear complex and spinal dorsal horn in the rat.

To clarify direct descending projections from the parabrachial nucleus (PB) to the trigeminal sensory nuclear complex (TSNC) and spinal dorsal horn (SpDH), the origin and termination of descending tract cells were examined by the anterograde and retrograde transport methods. Phaseolus vulgaris leucoagglutinin (PHA-L) and Fluorogold (FG) or dextran-tetramethylrhodamine (Rho) were used as neuronal tracers for the anterograde and retrograde transport, respectively. The ventrolateral PB, including Kölliker-Fuse nucleus (KF), sent axons terminating mainly in the ventrolateral parts of rostral trigeminal nuclei of the principalis (Vp), oralis (Vo), and interpolaris (Vi) as well as in the inner lamina II of the medullary (nucleus caudalis, Vc) and SpDH. Although the descending projections were bilateral with an ipsilateral dominance, TSNC received a more dominant ipsilateral projection than SpDH. The cells of origin of the descending tracts were located mainly in KF, but TSNC received fewer projections from the KF than SpDH. Namely, TSNC received a considerable projection from the medial subnucleus of PB and the ventral parts of lateral subnuclei of PB, such as the central lateral subnucleus and lateral crescent area. The other difference noted between TSNC and SpDH was that the former received projections mainly from the caudal two thirds of KF and the latter from the rostral two thirds of KF. These results demonstrate the existence of direct parabrachial projections to TSNC and SpDH that are organized in a distinct manner and suggest that both pathways are involved in the control of nociception.

HsMCM6: a new member of the human MCM/P1 family encodes a protein homologous to fission yeast Mis5.

BACKGROUND: The tight regulatory mechanism that prevents more than one round of chromosomal DNA replication per cell cycle is thought to require the function of Mcm/P1 proteins. We report here the structural and functional analyses of HsMcm6, a human homologue of the Mis5 of Schizosaccharomyces pombe. RESULTS: We demonstrate here that the transcription of the HsMCM6 gene was repressed in quiescent cells but was rapidly induced at the G1/S phase by growth factor stimulation. The 5' regulatory region of the HsMCM6 gene was found to harbour four putative E2F binding motifs, and these were responsible for the promoter activity. The HsMcm6 protein level oscillated during the cell cycle, with a peak at the G1/S phase. We also showed that the cell-cycle dependent change of subcellular localization of HsMcm6 resembles those of other Mcm/P1 proteins. HsMcm6 consists of two forms, a form extractable by Nonidet P-40 and the nucleus-bound form. A demonstration of the association of HsMcm6 with HsMcm2 and HsMcm7 in vivo supports the idea that they behave as a heteromeric complex. We mapped the HsMCM6 gene at 2q12-14. CONCLUSION: The results indicate that the behaviour of HsMcm6 is reminiscent of replication licensing factor like other Mcm/P1 family members.

Electron microscopic observation of synaptic connections of jaw-muscle spindle and periodontal afferent terminals in the trigeminal motor and supratrigeminal nuclei in the cat.

Previous studies indicate that the trigeminal motor nucleus (Vmo) and supratrigeminal nucleus (Vsup) receive direct projections from muscle spindle (MS) and periodontal ligament (PL) afferents. The aim of the present study is to examine the ultrastructural characteristics of the two kinds of afferent in both nuclei using the intracellular horseradish peroxidase (HRP) injection technique in the cat. Our observations are based on complete or near-complete reconstructions of 288 MS (six fibers) and 69 PL (eight fibers) afferent boutons in Vmo, and of 93 MS (four fibers) and 188 PL (four fibers) afferent boutons in Vsup. All the labeled boutons contained spherical synaptic vesicles and were presynaptic to neuronal elements, and some were postsynaptic to axon terminals containing pleomorphic, synaptic vesicles (P-endings). In Vmo neuropil, MS afferent boutons were distributed widely from soma to distal dendrites, but PL afferent boutons predominated on distal dendrites. Most MS afferent boutons (87%) formed synaptic specialization(s) with one postsynaptic target while some (13%) contacting two or three dendritic profiles; PL afferents had a higher number of boutons (43%) contacting two or more dendritic profiles. A small but significant number of MS afferent boutons (12%) received contacts from P-endings, but PL afferent boutons (36%) received three times as many contacts from P-endings as MS afferents. In Vsup neuropil, most MS (72%) and PL (87%) afferent boutons formed two contacts presynaptic to one dendrite and postsynaptic to one P-ending, and their participation in synaptic triads was much more frequent than in Vmo neuropil. The present study indicates that MS and PL afferent terminals have a distinct characteristic in synaptic arrangements in Vmo and Vsup and provides evidence that the synaptic organization of primary afferents differs between the neuropils containing motoneurons and their interneurons.

Light microscopic observations of the contacts made between two spindle afferent types and alpha-motoneurons in the cat trigeminal motor nucleus.

Previous studies indicate that cat jaw-muscle spindle afferents can be divided into two types (type I and II) on the basis of their axonal trajectories. The present study examined the relationship between spindle afferent fibers and their target masseter alpha-motoneurons in the cat by using the intracellular horseradish peroxidase (HRP) injection technique, and provided several new findings on the synaptic organization generated between the two. Five type I afferent fiber-motoneuron pairs and nine type II afferent-motoneuron pairs were well stained with HRP. The following conclusions were drawn: 1) A motoneuron received contacts from only one collateral of any given spindle afferent. 2) The number of contacts made between an afferent and a motoneuron ranged from one to three. 3) The contacts made by a spindle afferent were on the same dendrite or dendrites branching from the same primary dendrite. 4) The vast majority of the contacts made by an afferent on a motoneuron were distributed in the dendritic tree within 600 microns from the soma, i.e., in the proximal three fourths of the dendritic tree. The differences observed between the two afferent types were as follows. First, type II afferent terminals made contacts on more distal dendrites of the motoneurons than did type I afferent terminals. Second, the contacts made between a type I afferent and a motoneuron were clustered together, but those made between a type II afferent and a motoneuron were widely dispersed. The present results provided the general rules of synaptic contacts between the spindle afferents and masseter alpha-motoneurons, and demonstrated that the spatial distribution of synaptic contacts on the dendritic tree was different between type I and type II afferents.

NADPH-diaphorase in the developing rat: lower brainstem and cervical spinal cord, with special reference to the trigemino-solitary complex.

A previous study indicated that in adult rat, a distinctive neuronal group in the dorsomedial division of the subnucleus oralis of the spinal trigeminal nucleus (SpVo) and the rostrolateral part of the nucleus of the solitary tract (Sn) is stained for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), and suggested that the labeled structures are involved with sensorimotor reflexive functions. This study aimed to characterize the developmental expression of NADPH-d in SpVo and Sn, including other areas of the lower brainstem and cervical spinal cord, by means of the enzyme histochemical staining technique, from the prenatal through the postnatal period. On embryonic day 12 (E12), no neurons in the brain were stained for NADPH-d, whereas blood vessels were stained. Labeling in the vessels was consistently present throughout pre- and postnatal periods but decreased with development. On E15, labeled neurons appeared in the dorsomedial part of SpVo and the rostrolateral part of Sn, but not in the other nuclei. The labeled neurons in both nuclei increased in numbers drastically to E17. Postnatally, they tended to increase gradually in Sn, but to decrease slightly in SpVo. The cell size of labeled neurons reached a plateau at E17 in SpVo, but at postnatal day 4 (P4) in Sn. In other nuclei on E17, labeling appeared in the lateral paragigantocellular reticular, intermediate reticular, medullary reticular, pedunculopontine tegmental, and spinal vestibular nuclei, and laminae V, VI, and X of the cervical spinal cord. On E20 and P0, labeling appeared in the dorsal column, laterodorsal tegmental, raphe obscurus, parvocellular reticular, ventral gigantocellular reticular, and parahypoglossal nuclei, and laminae IX of the cervical spinal cord. On P4 labeling appeared in the parabrachial and median raphe nuclei, medial and caudolateral Sn, the magnocellular zone of subnucleus caudalis of the spinal trigeminal nucleus (SpVc), and laminae III/IV of the cervical spinal cord. On P10, labeling appeared in the paratrigeminal and dorsal raphe nuclei, the superficial zone of SpVc, and laminae I/II of the cervical spinal cord. No newly labeled neurons appeared in any nuclei after P14. The very early appearance of NADPH-d staining in SpVo and Sn, which precedes the appearance of NADPH-d elsewhere in the brainstem, suggests that the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system has an important role for primitive orofacial sensorimotor reflexive functions. Furthermore, the pattern of developmental expression of NADPH-d in SpVo and Sn suggests that the NO/cGMP system is organized in a distinct manner in different nuclei.