Mid-larval Stages Research Articles

Role of BMP signaling during early development of the annelid Capitella teleta

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (≈Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after the birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d ​+ ​12 ​h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue was not greatly affected. Our results compared with those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans.

Expression Changes of MHC and Other Immune Genes in Frog Skin during Ontogeny

Simple SummaryTadpoles undergo many changes in physiology and immunology until metamorphosis into adult frogs. Major histocompatibility complex (MHC) molecules are an important part of vertebrate adaptive immunity, and our study measured the expression of two MHC genes (MHC class I and II) in skin during six tadpole stages of the Montane Brown frog (Rana ornativentris). First, using a qPCR method, we found that both MHC class I and II expression significantly increased between stage 24/25 (‘early’) and stage 28 (‘mid’) tadpole skin. Then, we conducted next-generation sequencing for ‘early’, ‘mid’ and ‘late’ stage tadpole skin mRNA of both R. ornativentris and a model species, Xenopus tropicalis, and confirmed that MHC expression increased from the ‘mid’ stage. We also performed further analyses of transcriptome data and found that several immune-related gene ontology terms were upregulated from the ‘mid’ tadpole stage. Our findings probably support that both MHC class I and II have a functional role during tadpole development.Anuran amphibians undergo major physiological and immunological changes following metamorphosis. Genes of the major histocompatibility complex (MHC) code for receptors important for vertebrate adaptive immunity. We used qPCR to measure skin MHC expression in six different ontological stages of Rana ornativentris (n = 10 per stage); normalized MHC class I and II expression at the mRNA level was significantly higher in stage 28 (mid-larval) compared to stages 24/25 (early-larval) tadpoles. Subsequent transcriptomic analyses of three tadpole (early-, mid-, and late-larval) stages of R. ornativentris and model species Xenopus tropicalis focused on mRNA expression of immune-related genes in the skin. Normalized expression of most MHC class I and II transcripts in both species were significantly higher in mid- and late-larval stages compared to early-larval stage. In addition, gene ontology (GO) analyses of differentially expressed transcripts revealed several immune-related GO terms that were significantly upregulated from the mid-larval stage. Our study provides evidence that both MHC class I and II is expressed during development in both R. ornativentris and X. tropicalis.

The study of larval tail morphology reveals differentiation between two Triturus species and their hybrids

In amphibians, morphological differentiation and disparity at the larval and post-metamorphic ontogenetic stages can diverge, owing to various contrasting environments and different selective pressures. In the monophyletic clade of nine Triturus newt species, five different morphotypes can be recognized, but information on larval morphology is limited. Here we explore divergence of larval morphology in Triturus ivanbureschi, T. macedonicus, and their F1 hybrids. These two genetically and morphologically distinct crested newt species hybridize in nature and form a relatively wide hybrid zone in the central part of the Balkan Peninsula. Using a geometric morphometric approach and multivariate statistics, we evaluated differences of tail size and shape, colouration pattern, and the presence of a tail filament at the mid-larval stage in larvae reared under controlled laboratory conditions. We chose the tail as the main propulsive organ crucial for locomotion, feeding, and escaping predators. We found that Triturus ivanbureschi and T. macedonicus larvae differ in tail shape, but not in tail size. Two groups of F1 hybrid larvae (obtained from reciprocal crossing) were similar to each other, but differed from the parental species in size and shape of the tail, colouration pattern, and the presence of a tail filament. Our results indicate that, like adults, larvae diverge morphologically and hybrid larvae do not exhibit intermediate morphology of the parental species.

It's not all black and white: visual scene parameters influence optokinetic reflex performance in Xenopus laevis tadpoles.

The maintenance of visual acuity during active and passive body motion is ensured by gaze-stabilizing reflexes that aim at minimizing retinal image slip. For the optokinetic reflex (OKR), large-field visual motion of the surround forms the essential stimulus that activates eye movements. Properties of the moving visual world influence cognitive motion perception and the estimation of visual image velocity. Therefore, the performance of brainstem-mediated visuo-motor behaviors might also depend on image scene characteristics. Employing semi-intact preparations of mid-larval stages of Xenopus laevis tadpoles, we studied the influence of contrast polarity, intensity, contour shape and different motion stimulus patterns on the performance of the OKR and multi-unit optic nerve discharge during motion of a large-field visual scene. At high contrast intensities, the OKR amplitude was significantly larger for visual scenes with a positive contrast (bright dots on a dark background) compared with those with a negative contrast. This effect persisted for luminance-matched pairs of stimuli, and was independent of contour shape. The relative biases of OKR performance along with the independence of the responses from contour shape were closely matched by the optic nerve discharge evoked by the same visual stimuli. However, the multi-unit activity of retinal ganglion cells in response to a small single moving vertical edge was strongly influenced by the light intensity in the vertical neighborhood. This suggests that the underlying mechanism of OKR biases related to contrast polarity directly derives from visual motion-processing properties of the retinal circuitry.

Suppression of growth and endopeptidases of red palm weevil,Rhynchophorus ferrugineus (Olivier) infesting coconut using proteinase inhibitors

Investigations on luminal proteinases of grubs of red palm weevil, hynchophorus ferrugineus (Olivier) infesting coconut revealed presence of two endodpeptidases viz., trypsin (BApNA-ase activity) and elastase-like chymotrypsin (SAAPLpNA-ase activity) in all stages of larval development. Highest activity of these proteinases coincided with the active feeding stage (mid-larval stage) of the insect. Aprotinin 50 ìg, Soybean Trypsin Inhibitor (SBTI) 50 ìg and Phenyl Methyl Sulphonyl Fluoride (PMSF) 1700 ìg inhibited trypsin activity of R. ferrugineus by 77.4%, 63.1% and 55.9%, respectively. Serine proteinase inhibitors viz., aprotinin (50 ìg), SBTI (50 ìg) and PMSF (1700 ìg) had a marginal reduction of elastase - like chymotrypsin activity of R. ferrugineus by 32%, 14% and 11%, respectively suggesting the serine nature of the proteinase. In vivo bioassay of 250 ìM aprotinin on coconut petiole method using early stage grubs of R. ferrugineus indicated a significant weight loss of 18.9% due to incorporation of serine proteinase inhibitor, aprotinin in a period of 120 h. Possibility of using serine proteinase inhibitor, aprotinin in the management of R. ferrugineus was suggested.Â

Effect of corticosterone on larval growth, antipredator behaviour and metamorphosis of Hylarana indica

Corticosterone (CORT), a principal glucocorticoid in amphibians, is known to regulate diverse physiological processes including growth and metamorphosis of anuran tadpoles. Environmental stressors activate the neuroendocrine stress axis (hypothalamus–pituitary–interrenal axis, HPI) leading to an acute increase in CORT, which in turn, helps in coping with particular stress. However, chronic increase in CORT can negatively affect other physiological processes such as growth and metamorphosis. Herein, we studied the effect of exogenous CORT on larval growth, antipredator behaviour and metamorphic traits of Hylarana indica. Embryonic exposure to 5 or 20μg/L CORT did not affect their development, hatching duration as well as larval growth and metamorphosis. Exposure of tadpoles to 10 or 20μg/L CORT throughout larval development caused slower growth and development leading to increased body mass at stage 37. However, body and tail morphology of tadpoles was not affected. Interestingly, larval exposure to 5, 10 or 20μg/L CORT enhanced their antipredator response against kairomones in a concentration-dependent manner. Further, larval exposure to increasing concentrations of CORT resulted in the emergence of heavier froglets at 10 and 20μg/L while, delaying metamorphosis at all concentrations. Interestingly, the heavier froglets had shorter hindlimbs and consequently shorter jump distances. Tadpoles exposed to 20μg/L CORT during early, mid or late larval stages grew and developed slowly but tadpole morphology was not altered. Interestingly, exposure during early or mid-larval stages resulted in an enhanced antipredator response. These individuals metamorphosed later but at higher body mass while SVL was unaffected.

A survey of the effectiveness of non-cell autonomous RNAi throughout development in the sawfly, Athalia rosae (Hymenoptera)

RNA interference (RNAi) is a powerful and convenient tool not only for functional analysis of specific genes, but also for large-scale screening of gene function in insects; however, reports on its efficiency throughout development in a single species are limited. We demonstrate here that non-cell autonomous RNAi by injection of double-stranded RNA (dsRNA) knocks down targeting genes in most developmental stages in the sawfly, Athalia rosae. Injection of dsRNA targeting the green fluorescence protein (gfp) gene into eggs of a transgenic strain carrying the constitutively expressing gfp gene resulted in the absence of GFP fluorescence during embryogenesis, while a portion of the gfp dsRNA-injected embryos began exhibiting GFP fluorescence at late embryogenesis. When gfp dsRNA was injected into parental female pupae, the RNAi effect was carried over to all embryos of the next generation and the effect lasted until mid-larval stages. Parental injection of dsRNA was more efficient than embryonic injection in terms of penetrance of the effect and the survival rate. After injection of gfp dsRNA into last instar larvae, the RNAi effect was sustained during prepupal and pupal stages and in adults. The gfp gene transcript markedly decreased in these knockdown phenotypes. It was revealed by employing fluorescence-labeled dsRNA that injected dsRNA was taken up in internal organs. Knockdown of an endogenous gene, Distal-less (Dll), resulted in typical phenotypes represented by the lack and malformation of Dll-expressing organs, such as distal parts of the appendages and wing edges without showing off-target effects. In contrast, RNAi by dsRNA injection seems to be hardly effective in mid- to late-larval stages.

Determinants of the Drosophila Odorant Receptor Pattern

In most olfactory systems studied to date, neurons that express the same odorant receptor (Or) gene are scattered across sensory epithelia, intermingled with neurons that express different Or genes. In Drosophila, olfactory sensilla that express the same Or gene are dispersed on the antenna and the maxillary palp. Here we show that Or identity is specified in a spatially stereotyped pattern by the cell-autonomous activity of the transcriptional regulators Engrailed and Dachshund. Olfactory sensilla then become highly motile and disperse beneath the epidermis. Thus, positional information and cell motility underlie the dispersed patterns of Drosophila Or gene expression.

Change in prey selectivity during the larval life of Atlantic cod in the southern Gulf of St Lawrence

Atlantic cod larvae showed strong selectivity for the medium-size calanoid copepod Pseudocalanus spp. Naupliar stages comprised 62.4% of total prey number in the gut of first-feeding larvae. Selectivity shifted to Pseudocalanus spp. females at the mid-larval stage, representing 43% of all prey ingested by > 12 mm larvae.

Effects of bacterial treatment at early stages of Atlantic cod (Gadus morhuaL.) on larval survival and development

To assess the effects of bacterial treatment at the earliest stages of cod rearing on the microbial load, larval development and performance, testing three bacterial strains (Carnobacterium divergens V41, Arthrobacter sp. and Enterococcus sp.) in vivo that were previously shown to have inhibitory potential towards fish pathogens in vitro. A bacterial mixture was added eight times to the rearing water from the prehatch to the mid-larval stage (a 38-day period). Microbiological analysis of ova, larvae and rearing water was performed regularly. Larval performance and development were evaluated by survival rate, hypersalinity tolerance and physiological measurements. Different larval survival rates were observed within and between treatments, and possibly explained by variations in larval microflora and established probionts. Larvae from one silo, which had been bathed in the bacterial suspension, showed the highest survival rate (42.1%), lowest Vibrio levels, and were significantly heavier (19.3%) and more stress tolerant than control larvae (P < 0.01). This coincided with the intestinal establishment of two of the tested bacteria. Arthrobacter and Enterococcus strains added regularly to the rearing water from the postfertilized egg stage can become established in larval gastrointestinal tract. The Enterococcus strain was associated with increased larval growth, performance and microflora control, indicating its probiotic nature. Regular application of autochthonous probionts may promote larval welfare, development and stress tolerance at early stages, hence increasing production yield in intensive cod larviculture.

Ontogenic change of gill chloride cells in leptocephalus and glass eel stages of the Japanese eel, Anguilla japonica

The development of gill chloride cells was examined in premetamorphic larvae (leptocephali) and juveniles (glass eels) of the Japanese eel, Anguilla japonica. Branchial chloride cells were detected by immunocytochemistry using an antiserum specific for Na+,K+-ATPase. The specificity and availability of the antiserum for the detection of Japanese eel chloride cells were confirmed by Western blot analysis. The chloride cells first appeared on the developing gill filaments in a mid larval stage of leptocephalus (32.2 mm). Both immunoreactivity and the number of chloride cells gradually increased as the fish grew to a late stage of leptocephalus over 54 mm. In glass eels just after metamorphosis, gill lamellae developed from the gill filaments, and a rich population of chloride cells was observed in the gill filaments. In glass eels collected at a coastal area, chloride cells were extensively distributed in the gill filaments. The chloride cell size decreased progressively in glass eels transferred from seawater (SW) to freshwater (FW), whereas there was no difference in cell number. In contrast, some Na+,K+-ATPase immunoreaction distinct from typical chloride cells was observed in the gill lamellae throughout FW-transferred fish, but disappeared in control fish maintained in SW for 14 days. These findings indicate that the gill and gill chloride cells developed slowly during the extremely long larval stage, followed by rapid differentiation during a short period of metamorphosis. The excellent euryhalinity of glass eels may be due to the presence of the filament chloride cells and lamellar Na+,K+-ATPase-immunoreaction, presumably being responsible for SW and FW adaptation, respectively.

Alternatively spliced Robo2 isoforms in zebrafish and rat

Robo2, a member of the robo gene family, functions as a repulsive axon guidance receptor as well as a regulator of cell migration and tissue morphogenesis in different taxa. In this study, a novel isoform of the zebrafish robo2 (robo2_tv2), which included an otherwise alternatively spliced exon (CAE), has been characterized. Robo2_tv2 is expressed differentially in most non-neuronal tissues of adult zebrafish whereas robo2_tv1 expression to a great extent is restricted to the brain and eye. In zebrafish, robo2_tv2 exhibits a very-low-level basal expression starting from 1 day post fertilization until the mid-larval stages, at which time its expression increases dramatically and could be detected throughout adulthood. Our findings demonstrate that the amino acid sequence coded by CAE of the robo2 gene is highly conserved between zebrafish and mammals, and also contains conserved motifs shared with robo1 and robo4 but not with robo3. Furthermore, we provide an account of differential transcription of the CAE homolog in various tissues of the adult rat. These results suggest that the alternatively spliced robo2 isoforms may exhibit tissue specificity.

BMP Receptor Activates LIMK to Stabilize Synapses

The neuromuscular junction (NMJ) in Drosophila was used as a model system to uncover novel signaling processes that control synaptic stability. Mutations that disrupt bone morphogenic protein (BMP) signaling inhibit synaptic growth, leading to a reduced number of boutons and an increase in the number of "synaptic footprints," sites where the nerve has retracted from a synapse. Eaton and Davis report that these two functions of the BMP system can be separated and that synaptic stability relies on signaling through the type II BMP receptor [Wishful Thinking (Wit) to LIM kinase 1 (DLIMK1 in Drosophila )], whereas synaptic growth requires the canonical BMP pathway acting through Smads. The authors used genetic analysis to show that the canonical BMP components--Wit, Tkv (thick veins, a type I BMP receptor), Mad, and Medea (Smads)--increase synaptic footprints and decrease synaptic boutons at the fly NMJ. Expression of a truncated form of the receptor Wit that lacked a portion of the C-terminal region that is dispensable for Smad signaling restored synaptic bouton number, but these NMJ continued to exhibit elevated numbers of footprints, suggesting that the synaptic stability defect was not rescued. This C-terminal region interacted with the LIM and LIM-PDZ domains of DLIMK1 in yeast two-hybrid experiments, and the two proteins colocalized when expressed in S2 cells. In the motoneurons, DLIMK1 was localized to a cytosolic filamentous compartment that was near, but not identical to or dependent on, microtubules. Deficiency of DLIMK caused an increase in synaptic footprints without affecting the number of synaptic boutons. Expression of a dominant-negative DLIMK1 in neurons promoted synapse retraction, whereas expression in muscle did not. Overexpression of wild-type DLIMK1 in the wit background completely rescued not only synaptic stability but also synaptic growth and animal viability, with large numbers of mobile adult flies emerging; the wit flies typically die in a midlarval stage. This viability and synaptic growth and function rescue are perplexing but may be the result of hyperstabilization of synapses in the presence of excess DLIMK1, which then allows a second growth factor pathway to assume the role of synaptic growth stimulation. These results provide evidence for a Smad-independent signaling pathway from BMP receptors and provide in vivo support and physiological relevance for the in vitro data from mammals, suggesting that BMP receptors stimulate LIMK1. B. A. Eaton, G. W. Davis, LIM kinase1 controls synaptic stability downstream of the type II BMP receptor. Neuron 47 , 695-708 (2005). [PubMed]

Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia.

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes.

The neural receptor protein tyrosine phosphatase DPTP69D is required during periods of axon outgrowth in Drosophila.

We have isolated and characterized a series of 18 chemically induced alleles of Ptp69D ranging in strength from viable to worse than null, which represent unique tools for probing the structure, function, and signaling pathway of DPTP69D. Three alleles are strongly temperature sensitive and were used to define the developmental periods requiring DPTP69D function; adult health requires DPTP69D during the mid- to late-pupal stage, eclosion requires DPTP69D during the early to mid-larval stage, and larval survival requires DPTP69D during embryogenesis. Mutations predicted to abolish the phosphatase activity of the membrane proximal D1 domain severely reduce but do not abolish DPTP69D function. Six alleles appear null; only 20% of null homozygotes pupate and <5% eclose, only to fall into the food and drown. One allele, Ptp69D(7), confers axon and viability defects more severe than those of the null phenotype. Sequence analysis predicts that Ptp69D(7) encodes a mutant protein that may bind but not release substrate. Like mutations in the protein tyrosine phosphatase gene Dlar, strong Ptp69D alleles cause the ISNb nerve to bypass its muscle targets. Genetic analysis reveals that the bypass defect in Dlar and Ptp69D mutants is dependent upon DPTP99A function, consistent with the hypothesis that DPTP69D and DLAR both counteract DPTP99A, allowing ISNb axons to enter their target muscle field.

Development of the Anterior Vertebrae of Chanos chanos (Ostariophysi: Gonorynchiformes)

The development of the anterior vertebrae were examined in late larval and early juvenile Chanos chanos, with a focus on character states that have been used in ostariophysan systematics such as the expansion of dorsal cartilages of the anterior neural arches. In Chanos, these changes take place in juvenile fishes beginning at about 12.0 mm SL. This contrasts sharply with the ontogeny of the otophysan Weberian apparatus, which occurs in early to midlarval stages. A supraneural space is present in Chanos, between neural arch 1 and the occiput. It is into this space that the cartilages of neural arch 1 and the supraoccipital expand. We also found paired nodular cartilages are present in the supraneural space but are incorporated into the supraoccipital cartilage by about 15.5 mm SL. These cartilages may represent an accessory neural arch (ANA).

The Vascular Anatomy of the Developing Zebrafish: An Atlas of Embryonic and Early Larval Development

We have used confocal microangiography to examine and describe the vascular anatomy of the developing zebrafish, Danio rerio. This method and the profound optical clarity of zebrafish embryos make it possible to view the entire developing vasculature with unprecedented resolution. A staged series of three-dimensional images of the vascular system were collected beginning shortly after the onset of circulation at 1 day postfertilization through early- to midlarval stages at approximately 7 days postfertilization. Blood vessels in every region of the animal were imaged at each stage, and detailed “wiring patterns” were derived describing the interconnections between every major vessel. We present an overview of these data here in this paper and in an accompanying Web site “The interactive atlas of zebrafish vascular anatomy” online at (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We find a highly dynamic but also highly stereotypic pattern of vascular connections, with different sets of primitive embryonic vessels severing connections and rewiring in new configurations according to a reproducible plan. We also find that despite variation in the details of the vascular anatomy, the basic vascular plan of the developing zebrafish shows strong similarity to that of other vertebrates. This atlas will provide an invaluable foundation for future genetic and experimental studies of vascular development in the zebrafish.

Daf-12encodes a nuclear receptor that regulates the dauer diapause and developmental age inC. elegans

The daf-12 gene acts at the convergence of pathways regulating larval diapause, developmental age, and adult longevity in Caenorhabditis elegans. It encodes a nuclear receptor most closely related to two C. elegans receptors, NHR-8 and NHR-48, Drosophila DHR96, and vertebrate vitamin D and pregnane-X receptors. daf-12 has three predicted protein isoforms, two of which contain DNA- and ligand-binding domains, and one of which contains the ligand-binding domain only. Mutations cluster in DNA- and ligand-binding domains, but correspond to distinct phenotypic classes. DAF-12 is expressed widely in target tissues from embryo to adult, but is upregulated during midlarval stages. In the adult, expression persists in nervous system and somatic gonad, two tissues that regulate adult longevity. We propose that DAF-12 integrates hormonal signals in cellular targets to coordinate major life history traits.

Maturation of Lobster Stomatogastric Ganglion Rhythmic Activity

The stomatogastric ganglion of the adult lobster, Homarus americanus generates extremely regular pyloric rhythms with a characteristic period of 0.5-1.5 Hz. To study the changes in the pyloric rhythm during embryonic and larval development, we recorded excitatory junctional potentials evoked by lateral pyloric (LP) neuron activity. Early in development the motor discharge of the LP neuron was often irregular, preventing use of conventional analysis methods that rely on extracting burst times to calculate cycle frequency and its variability. Instead, cycle frequency was determined for the LP neuron from the peak of the power spectrum obtained from the occurrence times of excitatory junctional potentials in the p1 muscle. The ratio of the power in the peak to the power from 0 to 3 Hz was used as a relative measure of the regularity of the rhythm. Throughout embryonic and the first larval stage, LP neuron activity is slow, irregular, and only weakly periodic. The regularity of the rhythm increased during midlarval stages, and both the frequency and regularity increased considerably by the postlarval stage LIV.

Late-generated cells in the lateral motor columns of developing frog spinal cord

Spinal motor neurons of larval frogs continue to be generated for the lateral motor columns (LMCs) even after their peak numbers have been reached and during the major period of normal neuronal loss. This study elucidates the extent to which relatively late generated motor neurons survive to the end of larval development. Bromodeoxyuridine was injected into Rana pipiens tadpoles at mid-larval stages twice a day for 5 days in order to label LMC neurons arising during the period of greatest neuron loss. Counts of labeled neurons visualized by immunocytochemical methods revealed that substantial numbers of late generated motor neurons survive to the end of larval development and may be retained in greater proportion than early generated motor neurons (prior to the onset of normally occurring neuron loss). Tadpoles that had been exposed to exogenous thyroxine so as to increase proliferative activity and thereby label more motor neurons during the injection period, exhibited an increase in labeled LMC cells although the total number of neurons remained comparable to controls. Protracted neurogenesis concurrent with cell loss during the later phases of development is a major determinant of the final population of the LMC.