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NumbL is essential for Xenopus primary neurogenesis.

Nieber F, Hedderich M, Jahn O, Pieler T, Henningfeld KA - BMC Dev. Biol. (2013)

Bottom Line: Knockdown of NumbL afforded a complete loss of primary neurons and did not lead to an increase in Notch signaling in the open neural plate.Furthermore, we provide evidence that interaction of NumbL with the AP-2 complex is required for NumbL function during primary neurogenesis.We demonstrate an essential role of NumbL during Xenopus primary neurogenesis and provide evidence for a Notch-independent function of NumbL in this context.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Developmental Biochemistry, University of Goettingen, Goettingen, Germany. khennin1@gwdg.de.

ABSTRACT

Background: Members of the vertebrate Numb family of cell fate determinants serve multiple functions throughout early embryogenesis, including an essential role in the development of the nervous system. The Numb proteins interact with various partner proteins and correspondingly participate in multiple cellular activities, including inhibition of the Notch pathway.

Results: Here, we describe the expression characteristics of Numb and Numblike (NumbL) during Xenopus development and characterize the function of NumbL during primary neurogenesis. NumbL, in contrast to Numb, is expressed in the territories of primary neurogenesis and is positively regulated by the Neurogenin family of proneural transcription factors. Knockdown of NumbL afforded a complete loss of primary neurons and did not lead to an increase in Notch signaling in the open neural plate. Furthermore, we provide evidence that interaction of NumbL with the AP-2 complex is required for NumbL function during primary neurogenesis.

Conclusion: We demonstrate an essential role of NumbL during Xenopus primary neurogenesis and provide evidence for a Notch-independent function of NumbL in this context.

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X. laevis NumbL inhibits Notch signaling in reporter assays, but knockdown of NumbL does not influence the expression of Notch target genes in the open neural plate. (A) The influence of NumbL overexpression on a Notch responsive reporter. X. laevis embryos were injected with the Notch-ICD (NICD), Noggin and NumbL mRNA as indicated, together with a Hes1-luciferase reporter. Embryos were cultivated until open neural plate stage and luciferase activity was measured and normalzed to renilla luciferase. Shown is a summary of four independent luciferase experiments. Two batches of embryos containing at least ten embryos per injection mix were collected for each experiment. The error bars represent the standard deviation. (B-E)NumbL MO (12.5 ng) does not lead to an increase in Notch target gene expression at open neural plate stages. (F-I) The NumbL MO induced loss of neuronal differentiation is not rescued by inhibition of Notch signaling. DeltaStu or Su(H)DBM mRNA was injected alone or in combination with 12.5 ng NumbL MO into one blastomere at the two-cell stage and N-tubulin expression analyzed by whole mount in situ hybridization at stage 14. Whole mount in situ probes are indicated in the lower left corner. Embryos are shown in a dorsal view, anterior up. The injected side is marked by X-Gal staining and is always on the right. The midline is indicated with a red arrowhead.
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Figure 3: X. laevis NumbL inhibits Notch signaling in reporter assays, but knockdown of NumbL does not influence the expression of Notch target genes in the open neural plate. (A) The influence of NumbL overexpression on a Notch responsive reporter. X. laevis embryos were injected with the Notch-ICD (NICD), Noggin and NumbL mRNA as indicated, together with a Hes1-luciferase reporter. Embryos were cultivated until open neural plate stage and luciferase activity was measured and normalzed to renilla luciferase. Shown is a summary of four independent luciferase experiments. Two batches of embryos containing at least ten embryos per injection mix were collected for each experiment. The error bars represent the standard deviation. (B-E)NumbL MO (12.5 ng) does not lead to an increase in Notch target gene expression at open neural plate stages. (F-I) The NumbL MO induced loss of neuronal differentiation is not rescued by inhibition of Notch signaling. DeltaStu or Su(H)DBM mRNA was injected alone or in combination with 12.5 ng NumbL MO into one blastomere at the two-cell stage and N-tubulin expression analyzed by whole mount in situ hybridization at stage 14. Whole mount in situ probes are indicated in the lower left corner. Embryos are shown in a dorsal view, anterior up. The injected side is marked by X-Gal staining and is always on the right. The midline is indicated with a red arrowhead.

Mentions: One of the best-described functions for members of the Numb family is their ability to act as an inhibitor of Notch signaling [4,5]. The loss of neuronal differentiation observed upon knock-down of NumbL in X. laevis may therefore be the result of increased Notch signaling, which would then suppress neurogenesis. We first addressed the ability of NumbL to inhibit Notch signaling using a Notch responsive Hes1-luciferase reporter in X. laevis embryos. The reporter was injected into both blastomeres of the two-cell stage embryos together with the indicated mRNAs, and at stage 15 the embryos luciferase activity was measured using the dual luciferase assay. Injection of NICD mRNA gave only a mild activation of the Hes1-luciferase reporter (data not shown). To obtain higher reporter activity, mRNA encoding the neural inducer Noggin that acts through inhibiting BMP signaling, was co-injected with NICD mRNA. As shown in Figure 3A, coinjection of NICD and Noggin mRNA induced the reporter more than 2-fold; indeed, coinjection of NumbL mRNA reduced Notch-mediated induction of the reporter.


NumbL is essential for Xenopus primary neurogenesis.

Nieber F, Hedderich M, Jahn O, Pieler T, Henningfeld KA - BMC Dev. Biol. (2013)

X. laevis NumbL inhibits Notch signaling in reporter assays, but knockdown of NumbL does not influence the expression of Notch target genes in the open neural plate. (A) The influence of NumbL overexpression on a Notch responsive reporter. X. laevis embryos were injected with the Notch-ICD (NICD), Noggin and NumbL mRNA as indicated, together with a Hes1-luciferase reporter. Embryos were cultivated until open neural plate stage and luciferase activity was measured and normalzed to renilla luciferase. Shown is a summary of four independent luciferase experiments. Two batches of embryos containing at least ten embryos per injection mix were collected for each experiment. The error bars represent the standard deviation. (B-E)NumbL MO (12.5 ng) does not lead to an increase in Notch target gene expression at open neural plate stages. (F-I) The NumbL MO induced loss of neuronal differentiation is not rescued by inhibition of Notch signaling. DeltaStu or Su(H)DBM mRNA was injected alone or in combination with 12.5 ng NumbL MO into one blastomere at the two-cell stage and N-tubulin expression analyzed by whole mount in situ hybridization at stage 14. Whole mount in situ probes are indicated in the lower left corner. Embryos are shown in a dorsal view, anterior up. The injected side is marked by X-Gal staining and is always on the right. The midline is indicated with a red arrowhead.
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Related In: Results  -  Collection

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Figure 3: X. laevis NumbL inhibits Notch signaling in reporter assays, but knockdown of NumbL does not influence the expression of Notch target genes in the open neural plate. (A) The influence of NumbL overexpression on a Notch responsive reporter. X. laevis embryos were injected with the Notch-ICD (NICD), Noggin and NumbL mRNA as indicated, together with a Hes1-luciferase reporter. Embryos were cultivated until open neural plate stage and luciferase activity was measured and normalzed to renilla luciferase. Shown is a summary of four independent luciferase experiments. Two batches of embryos containing at least ten embryos per injection mix were collected for each experiment. The error bars represent the standard deviation. (B-E)NumbL MO (12.5 ng) does not lead to an increase in Notch target gene expression at open neural plate stages. (F-I) The NumbL MO induced loss of neuronal differentiation is not rescued by inhibition of Notch signaling. DeltaStu or Su(H)DBM mRNA was injected alone or in combination with 12.5 ng NumbL MO into one blastomere at the two-cell stage and N-tubulin expression analyzed by whole mount in situ hybridization at stage 14. Whole mount in situ probes are indicated in the lower left corner. Embryos are shown in a dorsal view, anterior up. The injected side is marked by X-Gal staining and is always on the right. The midline is indicated with a red arrowhead.
Mentions: One of the best-described functions for members of the Numb family is their ability to act as an inhibitor of Notch signaling [4,5]. The loss of neuronal differentiation observed upon knock-down of NumbL in X. laevis may therefore be the result of increased Notch signaling, which would then suppress neurogenesis. We first addressed the ability of NumbL to inhibit Notch signaling using a Notch responsive Hes1-luciferase reporter in X. laevis embryos. The reporter was injected into both blastomeres of the two-cell stage embryos together with the indicated mRNAs, and at stage 15 the embryos luciferase activity was measured using the dual luciferase assay. Injection of NICD mRNA gave only a mild activation of the Hes1-luciferase reporter (data not shown). To obtain higher reporter activity, mRNA encoding the neural inducer Noggin that acts through inhibiting BMP signaling, was co-injected with NICD mRNA. As shown in Figure 3A, coinjection of NICD and Noggin mRNA induced the reporter more than 2-fold; indeed, coinjection of NumbL mRNA reduced Notch-mediated induction of the reporter.

Bottom Line: Knockdown of NumbL afforded a complete loss of primary neurons and did not lead to an increase in Notch signaling in the open neural plate.Furthermore, we provide evidence that interaction of NumbL with the AP-2 complex is required for NumbL function during primary neurogenesis.We demonstrate an essential role of NumbL during Xenopus primary neurogenesis and provide evidence for a Notch-independent function of NumbL in this context.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Developmental Biochemistry, University of Goettingen, Goettingen, Germany. khennin1@gwdg.de.

ABSTRACT

Background: Members of the vertebrate Numb family of cell fate determinants serve multiple functions throughout early embryogenesis, including an essential role in the development of the nervous system. The Numb proteins interact with various partner proteins and correspondingly participate in multiple cellular activities, including inhibition of the Notch pathway.

Results: Here, we describe the expression characteristics of Numb and Numblike (NumbL) during Xenopus development and characterize the function of NumbL during primary neurogenesis. NumbL, in contrast to Numb, is expressed in the territories of primary neurogenesis and is positively regulated by the Neurogenin family of proneural transcription factors. Knockdown of NumbL afforded a complete loss of primary neurons and did not lead to an increase in Notch signaling in the open neural plate. Furthermore, we provide evidence that interaction of NumbL with the AP-2 complex is required for NumbL function during primary neurogenesis.

Conclusion: We demonstrate an essential role of NumbL during Xenopus primary neurogenesis and provide evidence for a Notch-independent function of NumbL in this context.

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