RECK expression was also abundant in the head region of the late embryo as it is preparing to filter feed. During these stages, extensive but specific ECM remodeling is required for the opening of the mouth, formation of the nasal pitts, and branching of the branchial arches (Nieuwkoop and Faber, 1956). All of these processes occur in different regions of the head and require localized ECM remodeling events, thus, there are no global ECM degradation events occurring at this time. Perhaps RECK regulates localized cell migration events rather than global events. Furthermore, MMPs are expressed in a tissue specific manner during development. For example, MMP-11 mRNA is abundantly present in the branchial arches in X. laevis embryos, and less so in the eyes and otic vesicles, whereas MMP-18 mRNA expression is abundant in the ventral head region in X. laevis embryos in an area where the opening to the mouth is forming (Damjanovski et al., 2000). Perhaps RECK expression is linked to the type of MMP that is expressed in a given tissue. For example, in mice, RECK is expressed in lung tissue along with high levels of MMP-2 and MT1-MMP transcripts (Nuttall et al., 2004). Since RECK has previously been shown to inhibit MMP-2 and MT1-MMP activity, perhaps its role in lung tissue is to control ECM remodeling by regulating MMP-2 and MT1-MMP.
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rowhead). At late neurula stage (stage 19), p4ha1 expression is clearly activated in anterior central nervous system (Fig. 3C) and the migrating neural crest cells (Fig. 3C, black arrows). At the same stages, p4ha2 expression appears to be remarkably low, detectable as a diffuse in situ hybridization signal in the anterior neural plate and in the cardiac progenitors’ territory (Fig. 3G, red arrow). At early tailbud stage (stage 22), both genes are highly expressed in the notochord (Fig. 3 D,H) and in the tail bud, which is composed by the chordoneural hinge and the posterior wall (Fig. 3D, H black arrows), with p4ha2 extending in the proctodeum (Fig. 3H, red arrowhead)(Beck and Slack, 1998). Furthermore, p4ha1 is expressed in the central nervous system, including the eyes, in neural crest cells migrated into the branchial arches, which in part express also p4ha2 (Fig. 3 D,H), and, posteriorly to the cement gland, in the presumptive cardiac progenitors’ territory (Fig. 3D, red arrow). As development proceeds, the predominant notochord expression is maintained for both transcripts, accordingly with collagen gene expression and protein synthesis (Su et al., 1991).
Expression of Xenopus CXCR genes during early development XCXCR1/2, XCXCR3, and XCXCRa are expressed zygotically and their expressions increase gradually. Maternal expression of XCXCR5 decreases during the late gastrula and early neurula stages, and increases after the late neurula stage. Expression of XCXCR6 is uniform throughout the early embryonic stages (Fig. 3A). As for CXCL, transcript levels of Xenopus CXC-type chemokine receptors are too low during early embryogenesis to enable their specific localization by whole-mount in situ hybridization. Thus, RT- PCR analysis was performed using region-specific RNA sources. XCXCR1/2 transcripts were distributed uniformly at the gastrula stage, whereas XCXCR3 and XCXCRa are abundantly expressed in the dorsal animal region. Abundant expression of the XCXCR5 transcript is found in the animal and marginal regions, whereas XCXCR6 is abundant in the dorsal vegetal region (Fig. 3B, left panel). During the later stages of embryogenesis, the transcripts of all XCXCR genes are expressed mainly in the head and ventral regions (Fig. 3B, center and right panels).
ABSTRACT Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Mem- brane Spanning) is a conserved scaffold protein that acts as a downstream substrate for protein kinase D and mediates multiple receptor signalling pathways. Despite the dissecting of the function of this protein in mammals, using both in vitro and in vivo studies, a detailed characterization of its gene expression during early phases of embryogenesis has not been described yet. Here, we have used Xenopus laevis as a vertebrate model system to analyze the gene expression and the protein localization of Kidins220/ARMS. We found its expression was dynamically regulated dur- ing development. Kidins220/ARMS mRNA was expressed from neurula to larval stage in different embryonic regions including the nervous system, eye, branchial arches, heart and somites. Similar to the transcript, the protein was present in multiple embryonic domains including the central ner- vous system, cranial nerves, motor nerves, intersomitic junctions, retinal ganglion cells, lens, otic vesicle, heart and branchial arches. In particular, in some regions such as the retina and somites, the protein displayed a differential localization pattern in stage 42 embryos when compared to the earlier examined stages. Taken together our results suggest that this multidomain protein is involved in distinct spatio-temporal differentiative events.
region has been defined containing six transcription factor binding sites; three CCAAT (sites for CCAAT box family transcription factors), two GC (GGGCGG binding sites for transcription factor Sp1), and a TATA box (Hatch and Bonner, 1996). In addition to this proximal promoter two more distal transcription factor binding sites exist between -234 to -361bp from the transcription start site (Hatch and Bonner, 1990). Some sites are conserved between species (Bernstein and Hake, 2006). In X. laevis the major H2A.Z transcript is ~869bp not including the poly-A tail (Iouzalen et al., 1996) with a ~381bp translated region (Iouzalen et al., 1996). H2A.Z transcription is not limited to S phase as with canonical core histones; a conclusion supported by the transcript’s lack of the 3’ stem loop structure found in S-phase transcribed histones (Hatch and Bonner, 1990). The transcribed region contains introns; four in human and chicken (Hatch and Bonner, 1990). Intron and exon lengths vary between species (Hatch and Bonner, 1990). In the human gene these introns are 276 to 438bp in size (Hatch and Bonner, 1990). The gene also contains a polyadenylation signal (Ernst et al., 1987; Hatch and Bonner, 1990; Iouzalen et al., 1996) and the transcript is polyadenylated in most species (Hatch and Bonner, 1990). In X. laevis, H2A.Z mRNA has polyadenylated and non- polyadenylated forms (Iouzalen et al., 1996), which is likely to affect mRNA stability and translation in vivo (Allende et al., 1974; Beilharz and Preiss, 2007).
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Oct-1. The high degree of relatedness of the two homologues indicates that these are likely to be copies of the same gene, which arose during the theoretical genome duplication event in X. laevis evolution. X. laevis and human Oct-1 display strong evolutionary conservation (85% Identity over a stretch of 750 amino acids), which presumably means that X. laevis has a similar, if not identical function to human Oct-1.
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The variation observed in gap junction expression in the Xenopus embryo during development supports the concept that these junctions transmit the inducing signals that control regional specification of the embryo (see Section 1.3d). Warner et a l (1984) used a channel blocking antibody against the 27,000 Mr protein to inject into one cell of the eight cell Xenopus embryo. The antibody blocked gap junction communication in the progeny cells and caused clear developmental defects that were consistent with the notion that the block of gap junctional communication interferes with neural induction (Warner fife fil, 1984). However, a direct role for gap junctions as channels for the neural inducing signal has been ruled out in view of the fact that the early stages of neural induction can occur in the absence of any cell contact (Toivonen fife fil, 1975). Experiments have also indicated that inhibition of gap junction activity did not prevent the induction of muscle gene activation in the Xenopus embryo (Warner and Gurdon, 1987). The precise role of gap junctions in embryonic induction remains unclear. It is thought that gap junctions may be involved in mediating the inducing signal in homeogenetic induction ( i . e the spread of neural inducing signal from nervous system to induce further nervous system. Mangold, 1933). However the presence of homeogenetic induction in Xenopus embryos and the associated role of gap junctions remains unproven.
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The importance of facilitative glucose transporters for fish remains undefined. To study its functional properties, we expressed the putative glucose transporter OnmyGLUT1 in Xenopus laevis oocytes. This heterologous expression system has been very useful for characterizing mammalian and protozoan hexose transporters. The relative abundance of OnmyGLUT1 transcripts in rainbow trout embryos suggested that this protein might play an important role in development. In mammals, GLUT1 is referred to as an early isoform because it is expressed at high levels in embryos and foetuses, being gradually substituted for other GLUT types during the course of development (Santalucia et al., 1992; Postic et al., 1994). We have analysed the expression of OnmyGLUT1 at a number of developmental stages (blastula, early and late gastrula, and during somitogenesis and the formation of the vitelline plexus) using whole-mount in situ hybridization. We also measured
The present study aims at characterizing the class 3 POU tran- scription factor complement of vertebrates, with a special focus on the pou3f genes from the frog X. laevis. Amino acid alignments revealed the general conservation of vertebrate Pou3f class pro- teins. Based on the fact that non-vertebrates generally encode only a single pou3f ortholog, the results of our phylogenetic and synteny analyses further indicate that the four vertebrate pou3f genes result from two successive duplications of a single ancestral pou3f gene in the last common ancestor of all vertebrates. The evolutionary history of the vertebrate pou3f class is thus consistent with the hypothesis that two whole-genome duplication (WGD) events occurred during early vertebrate evolution (Van de Peer et al., 2017). The existence of two pou3f2 and pou3f3 paralogs in teleost fish further supports the scenario of an additional, third, WGD marking the evolutionary diversification of the teleost fish lineage (Van de Peer et al., 2017). Intriguingly, we also found two examples of lineage-specific pou3f gene losses in teleost fish: pou3f4 in the zebrafish and pou3f2a in the medaka. Future work will have to address, how widespread these lineage-specific pou3f gene losses are in teleost fish, whether they are directly linked to the third WGD and thus to the increased teleost fish pou3f complement, and whether they compensate, for example, gene dosage effects. Our results show that all four X. laevis pou3f genes are ex- pressed during neurulation in the forming neural tube and that their expression is maintained in the brain, mostly in the dorsal part, at tailbud stages. Neural expression of Xenopus pou3f3 has already been described and our results largely confirm the pub- lished data (Square et al., 2015). Our results further highlight an evolutionary conserved expression of pou3f members in neural tissues. In mice, for example, pou3f1 is expressed in the anterior neuro-ectoderm
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and bony fish (Hanington et al., 2007); and CSF1Rs have been identified, in birds (Garceau et al., 2010) and teleosts (Barreda et al., 2005; Honda et al., 2005; How et al., 1996; Parichy et al., 2000; Pettersen et al., 2008). However, it appears that the bony fish CSF1 ligand-receptor axis is distinct to that of mammals (Hanington et al., 2007; Wang et al., 2008; Wang et al., 2014). Indeed, fish spe- cies possess multiple distinct gene copies of both the CSF1 ligand and receptor, which contrasts to single copies seen across higher vertebrates. Owing to the key position occupied by amphibians in vertebrate evolution, it would be interesting to determine whether the Xenopus CSF1 ligand-receptor system is more reminiscent of those seen in fish, mammals or alternatively a hybrid intermediate. Moreover, amphibian macrophage development remains to be fully characterized. Notably, monopoiesis of most vertebrate species occurs within designated hematopoietic tissues/organs such as the bone marrow of birds and mammals (Garceau et al., 2010; Tushinski et al., 1982) and the teleost head kidney (Belosevic et al., 2006; Neumann et al., 2000). By contrast, although the Xenopus liver periphery clearly functions as primary site of hematopoiesis (Hadji-Azimi et al., 1987; Hadji-Azimi et al., 1990; Lane and Sheets, 2002), our recent findings indicate that rather than the subcapsular liver, the bone marrow functions as the primary site of X. laevis macrophage development and contains CSF1 responsive mac- rophage precursors (Grayfer and Robert, 2013).
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In order to avoid excessive hydration, FW amphibians excrete large volumes of dilute urine (Henderson et al., 1972; Shpun and Katz, 1995). Under such conditions, the epithelium of the urinary bladder is kept relatively ‘tight’ in order to prevent the passive flow of salts from hyperosmotic body fluids into the dilute contents of the bladder (Claude and Goodenough, 1973; Reuss and Finn, 1975). In saline conditions, the composition of ureteral urine changes substantially, with osmolality and solute concentrations increasing in accordance with environmental conditions (Shpun and Katz, 1995). Furthermore, a comparison of ureteral urine with urinary bladder urine collected from saline-adapted Bufo demonstrated that urine generated by the kidney is additionally subject to modification by the bladder, such that bladder urine can have a higher concentration of salts than ureteral urine (Shpun and Katz, 1995). While this could be the result of an increase in water reabsorption from the bladder, for example by increased expression of water channels (e.g. FA-CHIP) in saltwater-acclimated frogs (Verbavatz et al., 1992; Abrami et al., 1995), in our studies, decreased occludin expression in the Xenopus urinary bladder suggests that this epithelium also becomes ‘leakier’ under saline conditions (Fig. 6B). Since amphibian urine can be, at most, iso-osmotic with plasma, these data support the idea that salts may also be able to move into the bladder through the paracellular pathway (i.e. from serosa to mucosa). Indeed, isolated urinary bladders from Bufo bathed on the mucosal surface with increasing salt concentrations exhibit a reduction in TER and increased paracellular Na + flux into the bladder
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Female albino Xenopus frogs were primed one week ahead with 30-40 units human chorionic gonadotropin (hCG) and induced for ovulation with 400 units hCG. Two percent cysteine pH7.8 was used to take off the jelly coat. Embryos were staged according to the Nieuwkoop normal table (Nieuwkoop and Faber J, 1994). Different stage embryos were fixed at room temperature (R/T) with MEMFA (0.1M MOPS, pH 7.4, 1 mM EGTA, 2 mM MgSO4, and 3.7% formaldehyde) for 45 minutes to 90 minutes. The embryos were dehydrated at R/T by sequential washings in 25% methanol/PBSW, 50% methanol/PBSW, 75% methanol/PBSW for 5 minutes, and finally 100% methanol two times, 5 minutes each time. Then the embryos were stored at –20 o C for up to 6 months ready to be used for in situ hybridization.
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rnmt cDNA sequence contains an ORF of 1209 bases, encoding a putative protein of 402 amino acids. The alignment of the predicted amino acid sequences of known homologues reveals high similar- ity in the region essential for RNA (guanine-7-) methyltransferase activity (100 %). The similarity over the whole length of the protein is >65% among vertebrates (Fig. 1A). It is important to note that the length and sequence of different N-terminal regions differs substantially (Fig. 1A). In human RNMT, the N-terminal residues 1 to 120 are dispensable for enzymatic activity but they are nec- essary for the recruitment of RNMT to the transcription initiation site (Aregger and Cowling, 2013) and for nuclear localization of the methyltransferase (Fig. 1A) (Shafer et al., 2005). However, Xenopus Rnmt, has a shorter N-terminus, lacking the two nuclear localization signal (NLS) motifs present in the human protein. It has been shown that a third NLS motif at K126 of the human protein sequence is sufficient for alternative nuclear localization (Shafer et al., 2005). This NLS motif is also present in the Xenopus laevis protein at position K53. Similarly, the SAM (S-adenosylmethionine) binding site as well as the sites necessary for methyltransferase activity are well conserved between mammals and amphibians (Fig. 1A) (Bujnicki et al., 2001; Saha et al., 1999). Further, we identified the sequence of a paralogue (rnmt-b) within the Xenopus laevis genome. Sequence comparison of the paralogues revealed a large un-sequenced region, which lies between the first and the third exon including exon 2 (Fig. 2C). A search for Xenopus laevis EST clones lead to the identification of a cDNA sequence, encoding a truncated protein (Supplementary Fig. S1) thus making it difficult for any further analysis.
sexes the gonads were mature, with sperm present in testes, and eggs in ovaries. Oocytes in giant tadpoles have yolk, which is normally produced in the liver (Duellman and Trueb, 1986), thus proving that the giants’ reproductive systems are mature (if not functional). This is in contrast to previous findings (Wangh and Schneider, 1982) that without TH X. laevis is unable to synthesize vitellogenin and produce eggs. It is thought that estrogens from the ovary induce vitellogenin synthesis and secretion by the liver only after the liver has been exposed to TH (Hayes, 1997b). There is a slim possibility that our giant tadpoles had ectopic thyroid tissue that we failed to find, and thus may have produced some hormone that influenced liver function. However, if this were true, we would expect to see more metamorphic changes in giant tadpoles, and their development to proceed beyond NF stage 56.
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microinjection of EXP1 or GlyR (± IVM treatment) ion channel mRNA in the dorsal or ventral two cells (red arrows) of the four-cell Xenopus embryo. A high incidence of malformed eyes is observed in dorsal injections in comparison to controls or ventral injections. (B) (i) Four-cell Xenopus embryos were injected (red arrow indicates injected cell). (ii,iii) Stage 42 control (uninjected) tadpoles. (iv,v) Stage 42 tadpoles injected with GlyR mRNA plus IVM treatment. lacZ lineage tracer mRNA was co-injected with the ion channel mRNA. Yellow arrowhead indicates normal eye in the absence of - gal (blue-green), whereas the red arrowhead indicates an absent eye on the contralateral side of the same tadpole in the presence of -gal. (C) Quantification of tadpoles with malformed eyes at stage 42 after microinjecting GlyR plus IVM treatment in two dorsal cells at the four-cell stage and incubation in different concentrations of choline chloride (0, 20 and 60 mM). 60 mM is the isomolar concentration. A depolarization dose- dependent decrease in malformed eye incidence is observed. Values are mean ± s.e.m. (n3). *, P<0.05; **, P<0.01; one-way ANOVA with Tukey’s post test. (D) Stage 22 control (uninjected) embryos (i,iv,vii) and embryos microinjected with GlyR mRNA plus IVM treatment (ii,v,viii) or EXP1 mRNA (iii,vi,ix) in the two dorsal cells at the four-cell stage. In situ hybridization for Otx2 (i-iii), Rx1 (iv-vi) and Pax6 (vii-ix) shows a significantly disrupted expression (yellow arrowheads) of Rx1 [GlyR+IVM, 53% disrupted (n15); EXP1, 50% disrupted (n18)] and Pax6 [GlyR+IVM, 56% disrupted (n9); EXP1, 57% disrupted (n7)], whereas Otx2 expression remains unchanged (green arrowheads) [GlyR+IVM, 92% normal (n26); EXP1, 94% normal (n17)]. The black arrowheads indicate Pax6 expression in the forebrain and the spinal cord, which remain largely unchanged. (E) CC2-DMPE staining showing the relative V mem of cells of the indicated region (i) in the stage 19 Xenopus embryo. Red arrowheads mark the specific bilateral
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Like other processes during metamorphosis, T3 sign- aling is required for intestinal metamorphosis, includ- ing the formation of adult intestinal stem cells, and this effect of T3 is mediated by TR. Many studies using X. laevis have demonstrated that the proliferating intesti- nal stem cells are formed de novo via the dedifferentia- tion of a very small fraction of the larval epithelial cells in a process that requires T3 signaling in both intestinal epithelial and non-epithelial tissues [34, 48]. Further- more, TR is both necessary and sufficient for mediating the effect of T3 for the formation of such adult stem cells [39, 49]. Thus, to determine the molecular mechanisms underlying adult stem cell formation in the epithelium, it is critical to identify genes that are regulated by T3 in the epithelium as well as in the non-epithelial tissues. A genome-wide microarray analysis of the epithelial and non-epithelial tissues during intestinal remodeling  revealed many T3-inducible genes that are likely involved in the formation of the adult stem cells, such as PRMT1 , AMDHD1 , HAL2 [53, 54], Sox3 , and Evi1 [56, 57]. Of particular interest among such candidate stem cell genes is Mad1, which has previously been asso- ciated with cell differentiation and anti-apoptotic but not with stem cells [11, 20–22]. Our recent studies reveal an interesting role for Mad1 during intestinal remodeling .
PDGF activates the expression of mesodermal marker genes To determine whether PDGF is mesoderm inducer, we exam- ined the expression of mesoderm specific markers, MyoD family of genes including XMyoD, XMyf5, and XMRF4 by RT-PCR. In normal Xenopus embryos, the expression of XMyoD and XMyf5 begins weakly at the early gastrula stage (Hopwood et al., 1992), whereas XMRF4 expression is first detected only in somites at stage 18 (neural fold stage) and becomes maximum at stage 22- 23 (Jennings, 1992). The XMyoD family of genes was expressed in the animal caps treated with PDGF. Similar results were ob- tained in the cases of activin and FGF, which are well known as mesoderm inducers (Fig. 3). The expression of α-cardiac actin was also assayed by RT-PCR. This is supporting evidence for the in situ study (Fig. 1). Somite mesodermal markers, such as related serum responsive factor (RSRF) family, SL1 (MEF2D), and a cardiac mesodermal marker, Xenopus myosin light chain (XMLC2) were studied by RT-PCR. SL1 and XMLC2 were both expressed in the animal cap explants treated with PDGF, activin and FGF (Fig. 3). With the mesoderm induction from the PDGF treated animal caps, neural induction also occurred. Microinjection of the XPDGF receptor antisense RNA inhibits the expression of mesoderm-specific genes in animal cap explants. To confirm the above results, the signal of XPDGF receptor was blocked by microinjection of XPDGF receptor antisense RNA. After microinjection at 1 cell stage into Xenopus embryos, the animal caps were dissected at the blastula stage (st. 8-9) and RT-PCR assay was carried out. When the control animal caps microinjected with neo pa RNA (microinjection control) were treated with PDGF, XMyoD, XMyf5, XMRF4 and α-cardiac actin were expressed. On the contrary, the animal caps microinjected with antisense RNA of XPDGF receptor and cultured with PDGF did not express any of these genes (Fig. 4).
IgG, Thermo Fisher Scientific) diluted 1:200. Slides were mounted as described in Section 2.2.4. Antibody validation for rabbit anti-RECK, rabbit anti-MT1-MMP, and mouse anti-TIMP-2 was performed using Western blotting with either X. laevis whole embryo lysate or X. laevis A6 cell lysate (Supplementary Fig. S3.2). Of note, the epitope region of mouse anti-TIMP-2 shares 82% sequence identity with the X. laevis TIMP-2 amino acid sequence. Serial sections were probed with pairs of antibodies: RECK with MT1-MMP, RECK with TIMP-2, and TIMP-2 with MT1-MMP. Thus, the fidelity of an antibody’s localization could be confirmed across 2 colocalization studies. Negative controls, following the above protocol, but without primary antibody, were performed to analyze nonspecific binding of the secondary antibody (Supplementary Fig. S3.3). Slides were visualized using a Leica DM16000 B microscope with Hamamatsu camera controller (C10600) using Leica automated inverted microscope and analyzed using Image J 2.0.0 software (NIH, Bethesda). 4 sections per embryo, from 3 embryos per stage were analyzed for each pair of antibodies. Images are representative of 1 section from 1 embryo; however, consistent staining patterns were seen between the sections from each embryo.
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ment in animal cap cells from Xenopus laevis and Xenopus tropicalis. The qualitative analysis using RT-PCR presented here revealed no difference in the dose of activin required to induce gene expression between Xenopus tropicalis and Xenopus laevis, suggesting that the rate and speed of signal transduction may be regulated by a common system in Xenopus laevis and Xenopus tropicalis. Interestingly, the quantitative analysis using real-time RT-PCR revealed several differences in the levels of individual marker gene expression activated by activin treatment between the two species, indicating that transcriptional regulation might differ between these two species. The expression levels of the organizer marker, cer was the same in the two species, indicating that the induction of these genes might be conserved during development. Differences in the amounts and peaks of expres- sion of other marker genes (chd, cer and Xbra) between the two species suggested that there are different mechanisms to define expression levels in a germ layer- (tissue or position) or gene- specific manner between Xenopus laevis and Xenopus laevis.
Figure 1. The Xenopus tadpole and its spinal neurons. A Xenopus tadpole at the time of hatching (stage 37/38), with its head to the left, the border between midbrain and hindbrain (m/h), and the spinal cord lying under segmented swimming muscles. Below, diagram of a length of spinal cord viewed from the side to show the neurons. Sensory pathway: dlc (orange) and dla (brown) are excited by skin mechanosensory RB neurons that have no dendrites. Motor circuit: glycinergic aINs (dark blue) have ascending and descending axons that can inhibit other swimming CPG components: mn (grey), excitatory dINs (red), glycinergic reciprocal inhibitory (cINs; blue). aINs also can form inhibitory synapses with the sensory dla and dlc INs. For each neuron class, the circle/oval is the soma; oblique lines indicate the dorso-ventral extent of the dendrites; and the thin line shows the axon projection (asterisk indicates that the axon crosses ventrally to the opposite side). Each class of neuron actually forms a longitudinal column of 50- 150 neurons on each side of the cord. Note Kolmer-Agduhr cells are not included in diagram. (Adapted from Li et al. 2004)
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