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Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development.

Bai T, Seebald JL, Kim KE, Ding HM, Szeto DP, Chang HC - BMC Dev. Biol. (2010)

Bottom Line: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns.Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene.In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 915 W, State St,, West Lafayette, Indiana 47907-2054, USA.

ABSTRACT

Background: The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.

Results: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.

Conclusion: In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

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The expression patterns of GAK and auxilin during zebrafish embryonic development. (A, D) Lateral, (B, E) dorsal, and (C, F) anterior views of wild-type embryos at the 15-somite stage. (A-C) zGAK is expressed broadly in the hindbrain (B), forebrain and eyes (C). (D-F) zAux is expressed mostly in neural tissues as described in the main text. (G, H) Dorsal views of 19-somite stage embryos. (G) zGAK is still ubiquitously expressed and (H) zAux remains specific to bilateral stripes of neural cells. (I, K, L, N) Lateral and (J, M) dorsal views of 24 hpf embryos. (I-K) zGAK is seen in the brain, vasculature and otic vesicles. (L-N) zAux remains concentrated in bilateral stripes of neural cells. Panels K and N are close-up views of the posterior regions of the embryos shown in (I) and (L), respectively. In all the images, anterior is to the left, and in all the lateral views, dorsal is up. ey, eye; is, intersomitic vessel; lg, lateral line ganglion; nt, neural tube; ot, otocyte; ov, otic vesicle; sag, statoacoustic ganglion; Scn, Spinal cord neuron; tg, trigeminal ganglion; v, vasculature; vd, ventral diencephalon. Scale Bar, 100 μm.
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Figure 5: The expression patterns of GAK and auxilin during zebrafish embryonic development. (A, D) Lateral, (B, E) dorsal, and (C, F) anterior views of wild-type embryos at the 15-somite stage. (A-C) zGAK is expressed broadly in the hindbrain (B), forebrain and eyes (C). (D-F) zAux is expressed mostly in neural tissues as described in the main text. (G, H) Dorsal views of 19-somite stage embryos. (G) zGAK is still ubiquitously expressed and (H) zAux remains specific to bilateral stripes of neural cells. (I, K, L, N) Lateral and (J, M) dorsal views of 24 hpf embryos. (I-K) zGAK is seen in the brain, vasculature and otic vesicles. (L-N) zAux remains concentrated in bilateral stripes of neural cells. Panels K and N are close-up views of the posterior regions of the embryos shown in (I) and (L), respectively. In all the images, anterior is to the left, and in all the lateral views, dorsal is up. ey, eye; is, intersomitic vessel; lg, lateral line ganglion; nt, neural tube; ot, otocyte; ov, otic vesicle; sag, statoacoustic ganglion; Scn, Spinal cord neuron; tg, trigeminal ganglion; v, vasculature; vd, ventral diencephalon. Scale Bar, 100 μm.

Mentions: Although it is known that mammalian GAK and auxilin are expressed in different tissues [24], their temporal and spatial expressions during development have not been fully investigated. To better understand the requirement of zGAK and zAux, we examined their expression at various stages of zebrafish development using whole-mount in situ hybridization. Zygotic expressions of zGAK and zAux, which were also maternally expressed (data not shown), were detected in diverse tissues, but were most prominently associated with neural tissues. Between 8- to 15-somite stages, zGAK was broadly expressed in the dorsal region of the embryo, and the expression of zAux appeared to be restricted to the bilateral cell clusters on the dorsal side of the embryo (Figure 5A-F). As a control, hybridization using sense probes did not yield detectable signals (data not shown). At the 19-somite stage, zGAK still expressed broadly in the entire embryo, whereas zAux was predominantly found in hindbrain neurons, spinal chord neurons, and otic vesicles (Figure 5G and 5H). At 24-hour post fertilization (hpf), zGAK was enriched in brains, eyes, otic vesicles, and vasculature, and zAux expression was detected in hindbrain neurons, spinal chord neurons, otic vesicles and posterior otic capsules (Figure 5I-N). The fact that the expression patterns of zGAK and zAux did not coincide completely suggests that they have overlapping and distinct functions during development.


Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development.

Bai T, Seebald JL, Kim KE, Ding HM, Szeto DP, Chang HC - BMC Dev. Biol. (2010)

The expression patterns of GAK and auxilin during zebrafish embryonic development. (A, D) Lateral, (B, E) dorsal, and (C, F) anterior views of wild-type embryos at the 15-somite stage. (A-C) zGAK is expressed broadly in the hindbrain (B), forebrain and eyes (C). (D-F) zAux is expressed mostly in neural tissues as described in the main text. (G, H) Dorsal views of 19-somite stage embryos. (G) zGAK is still ubiquitously expressed and (H) zAux remains specific to bilateral stripes of neural cells. (I, K, L, N) Lateral and (J, M) dorsal views of 24 hpf embryos. (I-K) zGAK is seen in the brain, vasculature and otic vesicles. (L-N) zAux remains concentrated in bilateral stripes of neural cells. Panels K and N are close-up views of the posterior regions of the embryos shown in (I) and (L), respectively. In all the images, anterior is to the left, and in all the lateral views, dorsal is up. ey, eye; is, intersomitic vessel; lg, lateral line ganglion; nt, neural tube; ot, otocyte; ov, otic vesicle; sag, statoacoustic ganglion; Scn, Spinal cord neuron; tg, trigeminal ganglion; v, vasculature; vd, ventral diencephalon. Scale Bar, 100 μm.
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Related In: Results  -  Collection

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Figure 5: The expression patterns of GAK and auxilin during zebrafish embryonic development. (A, D) Lateral, (B, E) dorsal, and (C, F) anterior views of wild-type embryos at the 15-somite stage. (A-C) zGAK is expressed broadly in the hindbrain (B), forebrain and eyes (C). (D-F) zAux is expressed mostly in neural tissues as described in the main text. (G, H) Dorsal views of 19-somite stage embryos. (G) zGAK is still ubiquitously expressed and (H) zAux remains specific to bilateral stripes of neural cells. (I, K, L, N) Lateral and (J, M) dorsal views of 24 hpf embryos. (I-K) zGAK is seen in the brain, vasculature and otic vesicles. (L-N) zAux remains concentrated in bilateral stripes of neural cells. Panels K and N are close-up views of the posterior regions of the embryos shown in (I) and (L), respectively. In all the images, anterior is to the left, and in all the lateral views, dorsal is up. ey, eye; is, intersomitic vessel; lg, lateral line ganglion; nt, neural tube; ot, otocyte; ov, otic vesicle; sag, statoacoustic ganglion; Scn, Spinal cord neuron; tg, trigeminal ganglion; v, vasculature; vd, ventral diencephalon. Scale Bar, 100 μm.
Mentions: Although it is known that mammalian GAK and auxilin are expressed in different tissues [24], their temporal and spatial expressions during development have not been fully investigated. To better understand the requirement of zGAK and zAux, we examined their expression at various stages of zebrafish development using whole-mount in situ hybridization. Zygotic expressions of zGAK and zAux, which were also maternally expressed (data not shown), were detected in diverse tissues, but were most prominently associated with neural tissues. Between 8- to 15-somite stages, zGAK was broadly expressed in the dorsal region of the embryo, and the expression of zAux appeared to be restricted to the bilateral cell clusters on the dorsal side of the embryo (Figure 5A-F). As a control, hybridization using sense probes did not yield detectable signals (data not shown). At the 19-somite stage, zGAK still expressed broadly in the entire embryo, whereas zAux was predominantly found in hindbrain neurons, spinal chord neurons, and otic vesicles (Figure 5G and 5H). At 24-hour post fertilization (hpf), zGAK was enriched in brains, eyes, otic vesicles, and vasculature, and zAux expression was detected in hindbrain neurons, spinal chord neurons, otic vesicles and posterior otic capsules (Figure 5I-N). The fact that the expression patterns of zGAK and zAux did not coincide completely suggests that they have overlapping and distinct functions during development.

Bottom Line: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns.Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene.In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 915 W, State St,, West Lafayette, Indiana 47907-2054, USA.

ABSTRACT

Background: The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.

Results: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.

Conclusion: In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

Show MeSH
Related in: MedlinePlus