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Embryonic motor activity and implications for regulating motoneuron axonal pathfinding in zebrafish.

Menelaou E, Husbands EE, Pollet RG, Coutts CA, Ali DW, Svoboda KR - Eur. J. Neurosci. (2008)

Bottom Line: Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development.When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos.These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

ABSTRACT
Zebrafish embryos exhibit spontaneous contractions of the musculature as early as 18-19 h post fertilization (hpf) when removed from their protective chorion. These movements are likely initiated by early embryonic central nervous system activity. We have made the observation that narrowminded mutant embryos (hereafter, nrd(-/-)) lack normal embryonic motor output upon dechorionation. However, these mutants can swim and respond to tactile stimulation by larval stages of development. nrd(-/-) embryos exhibit defects in neural crest development, slow muscle development and also lack spinal mechanosensory neurons known as Rohon-Beard (RB) neurons. At early developmental stages (i.e. 21-22 hpf) and while still in their chorions, nrd siblings (nrd(+/?)) exhibited contractions of the musculature at a rate similar to wild-type embryos. Anatomical analysis indicated that RB neurons were present in the motile embryos, but absent in the non-motile embryos, indicating that the non-motile embryos were nrd(-/-) embryos. Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development. These errors were reversed when nrd(-/-) embryos were raised in high [K(+)] beginning at 21 hpf, indicating that the abnormal axonal phenotypes may be related to a lack of depolarizing activity early in development. When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos. These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

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Narrowminded mutants do not exhibit an increase in musculature twitch rates upon dechorionation. (A) Two 22-hpf embryos that were dechorionated are shown in the field of view. In these experiments, embryos were observed and videotaped for 30 min. We noticed in our initial experiments that some embryos lacked movements when dechorionated (embryo denoted by black arrow). (B) Behavioral examination quantifying the number of bends that occurred for each embryo for each minute. The embryos that lacked movement were separated from the rest of the group and along with the motile embryos, processed for immunohistochemistry. (C and D) The motile embryos possessed aat- or zn12-positive RB neurons arrows at 48 hpf. Those that did not exhibit any movements lacked RB neurons. Scale bars, 20 μm.
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fig01: Narrowminded mutants do not exhibit an increase in musculature twitch rates upon dechorionation. (A) Two 22-hpf embryos that were dechorionated are shown in the field of view. In these experiments, embryos were observed and videotaped for 30 min. We noticed in our initial experiments that some embryos lacked movements when dechorionated (embryo denoted by black arrow). (B) Behavioral examination quantifying the number of bends that occurred for each embryo for each minute. The embryos that lacked movement were separated from the rest of the group and along with the motile embryos, processed for immunohistochemistry. (C and D) The motile embryos possessed aat- or zn12-positive RB neurons arrows at 48 hpf. Those that did not exhibit any movements lacked RB neurons. Scale bars, 20 μm.

Mentions: Zebrafish embryos display bends of the musculature when removed from their chorions as early as 18 hpf. The frequency of these contractions peaks at about 19–20 hpf and then declines gradually (Saint-Amant & Drapeau, 1998). When we first began studying narrowminded, we dechorionated embryos between 23 and 26 hpf and noticed that a fraction of the embryos did not move upon dechorionation. We wondered if the non-motile embryos were in fact nrd−/− embryos. To determine this, we combined behavioral and anatomical analyses of embryos spawned from heterozygote carriers of the narrowminded mutation. Embryos that did not exhibit spontaneous contractions of the musculature upon dechorionation were videotaped (Fig. 1A arrow, Fig. 1B), segregated from the motile embryos and raised until 48 hpf. Non-motile embryos did not respond to tactile stimulation applied to their tails at 30 hpf. This behavioral response is mediated by RB neurons (Ribera & Nusslein-Volhard, 1998). Immunohistochemistry was performed on these embryos using aat and zn12 to confirm the presence or absence of RB neurons, as nrd−/− embryos lack RB neurons (Artinger et al., 1999). Anatomical analysis confirmed that the embryos identified as non-motile at 21–22 hpf (Fig. 1B), lacking tail evoked touch responses at 30 hpf, did not have RB neurons, indicating that they were nrd−/− embryos (n= 37; Fig. 1C and D). The nrd mutants also have alterations in pigmentation (Artinger et al., 1999) and this phenotype was seen in non-motile embryos. However, we felt it was initially important to directly confirm the presence or absence of RB neurons when determining the identity of non-motile embryos, and thus focused on the RB cells. The embryos that were motile at 21–22 hpf (n= 146) exhibited tail evoked touch responses and had RB neurons.


Embryonic motor activity and implications for regulating motoneuron axonal pathfinding in zebrafish.

Menelaou E, Husbands EE, Pollet RG, Coutts CA, Ali DW, Svoboda KR - Eur. J. Neurosci. (2008)

Narrowminded mutants do not exhibit an increase in musculature twitch rates upon dechorionation. (A) Two 22-hpf embryos that were dechorionated are shown in the field of view. In these experiments, embryos were observed and videotaped for 30 min. We noticed in our initial experiments that some embryos lacked movements when dechorionated (embryo denoted by black arrow). (B) Behavioral examination quantifying the number of bends that occurred for each embryo for each minute. The embryos that lacked movement were separated from the rest of the group and along with the motile embryos, processed for immunohistochemistry. (C and D) The motile embryos possessed aat- or zn12-positive RB neurons arrows at 48 hpf. Those that did not exhibit any movements lacked RB neurons. Scale bars, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2741004&req=5

fig01: Narrowminded mutants do not exhibit an increase in musculature twitch rates upon dechorionation. (A) Two 22-hpf embryos that were dechorionated are shown in the field of view. In these experiments, embryos were observed and videotaped for 30 min. We noticed in our initial experiments that some embryos lacked movements when dechorionated (embryo denoted by black arrow). (B) Behavioral examination quantifying the number of bends that occurred for each embryo for each minute. The embryos that lacked movement were separated from the rest of the group and along with the motile embryos, processed for immunohistochemistry. (C and D) The motile embryos possessed aat- or zn12-positive RB neurons arrows at 48 hpf. Those that did not exhibit any movements lacked RB neurons. Scale bars, 20 μm.
Mentions: Zebrafish embryos display bends of the musculature when removed from their chorions as early as 18 hpf. The frequency of these contractions peaks at about 19–20 hpf and then declines gradually (Saint-Amant & Drapeau, 1998). When we first began studying narrowminded, we dechorionated embryos between 23 and 26 hpf and noticed that a fraction of the embryos did not move upon dechorionation. We wondered if the non-motile embryos were in fact nrd−/− embryos. To determine this, we combined behavioral and anatomical analyses of embryos spawned from heterozygote carriers of the narrowminded mutation. Embryos that did not exhibit spontaneous contractions of the musculature upon dechorionation were videotaped (Fig. 1A arrow, Fig. 1B), segregated from the motile embryos and raised until 48 hpf. Non-motile embryos did not respond to tactile stimulation applied to their tails at 30 hpf. This behavioral response is mediated by RB neurons (Ribera & Nusslein-Volhard, 1998). Immunohistochemistry was performed on these embryos using aat and zn12 to confirm the presence or absence of RB neurons, as nrd−/− embryos lack RB neurons (Artinger et al., 1999). Anatomical analysis confirmed that the embryos identified as non-motile at 21–22 hpf (Fig. 1B), lacking tail evoked touch responses at 30 hpf, did not have RB neurons, indicating that they were nrd−/− embryos (n= 37; Fig. 1C and D). The nrd mutants also have alterations in pigmentation (Artinger et al., 1999) and this phenotype was seen in non-motile embryos. However, we felt it was initially important to directly confirm the presence or absence of RB neurons when determining the identity of non-motile embryos, and thus focused on the RB cells. The embryos that were motile at 21–22 hpf (n= 146) exhibited tail evoked touch responses and had RB neurons.

Bottom Line: Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development.When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos.These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

ABSTRACT
Zebrafish embryos exhibit spontaneous contractions of the musculature as early as 18-19 h post fertilization (hpf) when removed from their protective chorion. These movements are likely initiated by early embryonic central nervous system activity. We have made the observation that narrowminded mutant embryos (hereafter, nrd(-/-)) lack normal embryonic motor output upon dechorionation. However, these mutants can swim and respond to tactile stimulation by larval stages of development. nrd(-/-) embryos exhibit defects in neural crest development, slow muscle development and also lack spinal mechanosensory neurons known as Rohon-Beard (RB) neurons. At early developmental stages (i.e. 21-22 hpf) and while still in their chorions, nrd siblings (nrd(+/?)) exhibited contractions of the musculature at a rate similar to wild-type embryos. Anatomical analysis indicated that RB neurons were present in the motile embryos, but absent in the non-motile embryos, indicating that the non-motile embryos were nrd(-/-) embryos. Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development. These errors were reversed when nrd(-/-) embryos were raised in high [K(+)] beginning at 21 hpf, indicating that the abnormal axonal phenotypes may be related to a lack of depolarizing activity early in development. When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos. These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

Show MeSH
Related in: MedlinePlus