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Novel lines of Pax6-/- embryonic stem cells exhibit reduced neurogenic capacity without loss of viability.

Quinn JC, Molinek M, Nowakowski TJ, Mason JO, Price DJ - BMC Neurosci (2010)

Bottom Line: The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo.We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons.We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK. jquinn@csu.edu.au

ABSTRACT

Background: Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro.

Results: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- <--> Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- <--> Pax6+/+ chimeras made by embryo aggregation.

Conclusions: We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.

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Pax6-/- cells derived from mutant ES cells integrate in Pax6-/- ↔ Pax6+/+ chimeras and show predictably abnormal phenotypes. (A-D) In these E14.5 chimeras, mutant cells are recognized by a brown nuclear spot, which is the result of DNA-DNA in situ hybridization for a reiterated β-globin transgene. (A,B) Sections of the cerebral cortex showing clusters of Pax6-/- cells in the subventricular zone; these cells are positive for Mash1 (grey staining in B). (C,D) In the eye, chimeras show abnormal retinal morphologies (boxed area) exhibiting extreme segregation between wild type and mutant cells, similar to abnormalities reported previously in aggregation chimeras [20]. The arrowhead in D indicates an example of the boundary between an area of mutant and an area of wild type cells with abnormal retinal folding. Scale bars: A,B,D = 50 μm; C = 100 μm.
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Figure 1: Pax6-/- cells derived from mutant ES cells integrate in Pax6-/- ↔ Pax6+/+ chimeras and show predictably abnormal phenotypes. (A-D) In these E14.5 chimeras, mutant cells are recognized by a brown nuclear spot, which is the result of DNA-DNA in situ hybridization for a reiterated β-globin transgene. (A,B) Sections of the cerebral cortex showing clusters of Pax6-/- cells in the subventricular zone; these cells are positive for Mash1 (grey staining in B). (C,D) In the eye, chimeras show abnormal retinal morphologies (boxed area) exhibiting extreme segregation between wild type and mutant cells, similar to abnormalities reported previously in aggregation chimeras [20]. The arrowhead in D indicates an example of the boundary between an area of mutant and an area of wild type cells with abnormal retinal folding. Scale bars: A,B,D = 50 μm; C = 100 μm.

Mentions: The developmental potential of wild type line wtMM4 and Pax6-/- line SeyD1 was assessed by injection into wild type blastocysts to create chimeras. We produced ten Pax6-/- ↔ Pax6+/+ chimeras aged between embryonic day (E) 10.5 and postnatal day (P) 5 and ten control Pax6+/+ ↔ Pax6+/+ chimeras aged between E10.5 and E14.5. In controls, Tg+ cells derived from wtMM4 were distributed throughout eye and brain tissues, which exhibited no abnormal phenotypes. In Pax6-/- ↔ Pax6+/+ chimeras, Tg+Pax6-/- cells derived from SeyD1 showed abnormal distributions in eye and brain tissues, similar to those reported previously for Pax6-/- cells in Pax6-/- ↔ Pax6+/+ chimeras produced by aggregation of Pax6+/+ and Pax6-/- morulae [9,20,22,25]. For example, Pax6-/- cells derived from SeyD1 formed clusters in the subventricular zone of the embryonic cortex (Figure 1A, B); these clusters expressed inappropriate markers of ventral telencephalic identity, such as Mash1 (Figure 1B), as predicted from previous work [9,22]. Pax6-/- cells derived from SeyD1 failed to contribute to the lens and exhibited extreme segregation from wild type cells in the neural retina, which exhibited areas of characteristic abnormal retinal folding (Figure 1C, D), again as anticipated from previous studies [20]. Clusters of Pax6-/- cells derived from SeyD1 were still present in the brains of postnatal chimeras (Figure 2). These findings indicate that cells derived from Pax6-/- ES cells can contribute in predictably abnormal ways to the eyes and brains of chimeras for periods extending into postnatal life, long after the onset of neurogenesis.


Novel lines of Pax6-/- embryonic stem cells exhibit reduced neurogenic capacity without loss of viability.

Quinn JC, Molinek M, Nowakowski TJ, Mason JO, Price DJ - BMC Neurosci (2010)

Pax6-/- cells derived from mutant ES cells integrate in Pax6-/- ↔ Pax6+/+ chimeras and show predictably abnormal phenotypes. (A-D) In these E14.5 chimeras, mutant cells are recognized by a brown nuclear spot, which is the result of DNA-DNA in situ hybridization for a reiterated β-globin transgene. (A,B) Sections of the cerebral cortex showing clusters of Pax6-/- cells in the subventricular zone; these cells are positive for Mash1 (grey staining in B). (C,D) In the eye, chimeras show abnormal retinal morphologies (boxed area) exhibiting extreme segregation between wild type and mutant cells, similar to abnormalities reported previously in aggregation chimeras [20]. The arrowhead in D indicates an example of the boundary between an area of mutant and an area of wild type cells with abnormal retinal folding. Scale bars: A,B,D = 50 μm; C = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Pax6-/- cells derived from mutant ES cells integrate in Pax6-/- ↔ Pax6+/+ chimeras and show predictably abnormal phenotypes. (A-D) In these E14.5 chimeras, mutant cells are recognized by a brown nuclear spot, which is the result of DNA-DNA in situ hybridization for a reiterated β-globin transgene. (A,B) Sections of the cerebral cortex showing clusters of Pax6-/- cells in the subventricular zone; these cells are positive for Mash1 (grey staining in B). (C,D) In the eye, chimeras show abnormal retinal morphologies (boxed area) exhibiting extreme segregation between wild type and mutant cells, similar to abnormalities reported previously in aggregation chimeras [20]. The arrowhead in D indicates an example of the boundary between an area of mutant and an area of wild type cells with abnormal retinal folding. Scale bars: A,B,D = 50 μm; C = 100 μm.
Mentions: The developmental potential of wild type line wtMM4 and Pax6-/- line SeyD1 was assessed by injection into wild type blastocysts to create chimeras. We produced ten Pax6-/- ↔ Pax6+/+ chimeras aged between embryonic day (E) 10.5 and postnatal day (P) 5 and ten control Pax6+/+ ↔ Pax6+/+ chimeras aged between E10.5 and E14.5. In controls, Tg+ cells derived from wtMM4 were distributed throughout eye and brain tissues, which exhibited no abnormal phenotypes. In Pax6-/- ↔ Pax6+/+ chimeras, Tg+Pax6-/- cells derived from SeyD1 showed abnormal distributions in eye and brain tissues, similar to those reported previously for Pax6-/- cells in Pax6-/- ↔ Pax6+/+ chimeras produced by aggregation of Pax6+/+ and Pax6-/- morulae [9,20,22,25]. For example, Pax6-/- cells derived from SeyD1 formed clusters in the subventricular zone of the embryonic cortex (Figure 1A, B); these clusters expressed inappropriate markers of ventral telencephalic identity, such as Mash1 (Figure 1B), as predicted from previous work [9,22]. Pax6-/- cells derived from SeyD1 failed to contribute to the lens and exhibited extreme segregation from wild type cells in the neural retina, which exhibited areas of characteristic abnormal retinal folding (Figure 1C, D), again as anticipated from previous studies [20]. Clusters of Pax6-/- cells derived from SeyD1 were still present in the brains of postnatal chimeras (Figure 2). These findings indicate that cells derived from Pax6-/- ES cells can contribute in predictably abnormal ways to the eyes and brains of chimeras for periods extending into postnatal life, long after the onset of neurogenesis.

Bottom Line: The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo.We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons.We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK. jquinn@csu.edu.au

ABSTRACT

Background: Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro.

Results: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- <--> Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- <--> Pax6+/+ chimeras made by embryo aggregation.

Conclusions: We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.

Show MeSH
Related in: MedlinePlus