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Bhlhb5::flpo allele uncovers a requirement for Bhlhb5 for the development of the dorsal cochlear nucleus.

Cai X, Kardon AP, Snyder LM, Kuzirian MS, Minestro S, de Souza L, Rubio ME, Maricich SM, Ross SE - Dev. Biol. (2016)

Bottom Line: Using an intersectional genetic strategy, we labeled cartwheel cells, thereby providing proof of concept that subpopulations of Bhlhb5-expressing neurons can be genetically targeted.Moreover, fate-mapping experiments using this allele revealed that Bhlhb5 is required for the proper development of the DCN, since mice lacking Bhlhb5 showed a dramatically diminished number of neurons, including unipolar brush and cartwheel cells.Intriguingly, the Bhlhb5::flpo allele also genetically labels numerous other regions of the nervous system that process sensory input, including the dorsal horn, the retina, and the nucleus of the lateral olfactory tract, hinting at a more general role for Bhlhb5 in the development of neurons that mediate sensory integration.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Pittsburgh, 200 Lothrop St., Pittsburgh, PA 15213, USA; The Pittsburgh Center for Pain Research, University of Pittsburgh, 200 Lothrop St., Pittsburgh, PA 15213, USA.

No MeSH data available.


Dual intersectional strategy distinguishes subpopulations of Bhlhb5-expressing cells: cartwheel and unipolar brush neurons. (A) Schematic illustrating the use of the Bhlhb5::flpo with RCE::FRT to label Bhlhb5-expressing neurons. (B–I) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (J) Schematic illustrating the use of Bhlhb5::flpo, Ptf1a::cre and RCE::dual to label a population of neurons at the genetic intersection, namely the inhibitory subset of Bhlhb5-expressing neurons. (L–R) Coronal sections of the DCN from an adult Ptf1a::cre; Bhlhb5::flpo; RCE::dual mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (S–U) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, CamKIIα, as indicated. Images are single confocal optical plane of coronal sections from six- to eight-week old mice. Arrowheads and arrows indicate examples of double-labeled inhibitory and excitatory neurons, respectively. Scale bars: B, K = 200 µm; all others = 50 µm.
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Figure 4: Dual intersectional strategy distinguishes subpopulations of Bhlhb5-expressing cells: cartwheel and unipolar brush neurons. (A) Schematic illustrating the use of the Bhlhb5::flpo with RCE::FRT to label Bhlhb5-expressing neurons. (B–I) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (J) Schematic illustrating the use of Bhlhb5::flpo, Ptf1a::cre and RCE::dual to label a population of neurons at the genetic intersection, namely the inhibitory subset of Bhlhb5-expressing neurons. (L–R) Coronal sections of the DCN from an adult Ptf1a::cre; Bhlhb5::flpo; RCE::dual mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (S–U) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, CamKIIα, as indicated. Images are single confocal optical plane of coronal sections from six- to eight-week old mice. Arrowheads and arrows indicate examples of double-labeled inhibitory and excitatory neurons, respectively. Scale bars: B, K = 200 µm; all others = 50 µm.

Mentions: Intersectional genetic approaches give us a way to label subsets of neurons that express two different genes of interest. With the Bhlhb5::flpo tool in hand, we were now positioned to test the feasibility of using this strategy to label subsets of Bhlhb5-expressing neurons. Here, we coupled the Ptf1a::cre allele, which is selective to inhibitory neurons (Fujiyama et al., 2009), to the Bhlhb5::flpo allele in order to selectively label the inhibitory subset of Bhlhb5-expressing cells in the DCN. This allowed us to compare the recombination pattern using RCE reporters that were either singly responsive (i.e., to Bhlhb5::flpo; Fig. 4A) or dually responsive (i.e., to Bhlhb5::flpo and Ptf1a::cre; Fig. 4J). Using this strategy, we found that the Bhlhb5::flpo allele caused eGFP expression in at least two cell types. The first are medium-sized, Pax2-positive neurons whose cell bodies are found predominantly between the fusiform cell and the molecular layers and that co-express parvalbumin (Fig. 4B–F); arrowheads). In addition, we found a population of small neurons that are found predominantly in the fusiform and deep layers that lack both Pax2 and parvalbumin expression (Fig. 4B–F); arrows). However, these small neurons co-express Tbr2 (Fig. 4G–I; arrows), a marker of unipolar brush cells (Dino and Mugnaini, 2008).


Bhlhb5::flpo allele uncovers a requirement for Bhlhb5 for the development of the dorsal cochlear nucleus.

Cai X, Kardon AP, Snyder LM, Kuzirian MS, Minestro S, de Souza L, Rubio ME, Maricich SM, Ross SE - Dev. Biol. (2016)

Dual intersectional strategy distinguishes subpopulations of Bhlhb5-expressing cells: cartwheel and unipolar brush neurons. (A) Schematic illustrating the use of the Bhlhb5::flpo with RCE::FRT to label Bhlhb5-expressing neurons. (B–I) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (J) Schematic illustrating the use of Bhlhb5::flpo, Ptf1a::cre and RCE::dual to label a population of neurons at the genetic intersection, namely the inhibitory subset of Bhlhb5-expressing neurons. (L–R) Coronal sections of the DCN from an adult Ptf1a::cre; Bhlhb5::flpo; RCE::dual mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (S–U) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, CamKIIα, as indicated. Images are single confocal optical plane of coronal sections from six- to eight-week old mice. Arrowheads and arrows indicate examples of double-labeled inhibitory and excitatory neurons, respectively. Scale bars: B, K = 200 µm; all others = 50 µm.
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Figure 4: Dual intersectional strategy distinguishes subpopulations of Bhlhb5-expressing cells: cartwheel and unipolar brush neurons. (A) Schematic illustrating the use of the Bhlhb5::flpo with RCE::FRT to label Bhlhb5-expressing neurons. (B–I) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (J) Schematic illustrating the use of Bhlhb5::flpo, Ptf1a::cre and RCE::dual to label a population of neurons at the genetic intersection, namely the inhibitory subset of Bhlhb5-expressing neurons. (L–R) Coronal sections of the DCN from an adult Ptf1a::cre; Bhlhb5::flpo; RCE::dual mouse immunostained with antibodies against GFP, Pax2, parvalbumin (PV), or Tbr2, as indicated. (S–U) Coronal sections of the DCN from an adult Bhlhb5::flpo; RCE::FRT mouse immunostained with antibodies against GFP, CamKIIα, as indicated. Images are single confocal optical plane of coronal sections from six- to eight-week old mice. Arrowheads and arrows indicate examples of double-labeled inhibitory and excitatory neurons, respectively. Scale bars: B, K = 200 µm; all others = 50 µm.
Mentions: Intersectional genetic approaches give us a way to label subsets of neurons that express two different genes of interest. With the Bhlhb5::flpo tool in hand, we were now positioned to test the feasibility of using this strategy to label subsets of Bhlhb5-expressing neurons. Here, we coupled the Ptf1a::cre allele, which is selective to inhibitory neurons (Fujiyama et al., 2009), to the Bhlhb5::flpo allele in order to selectively label the inhibitory subset of Bhlhb5-expressing cells in the DCN. This allowed us to compare the recombination pattern using RCE reporters that were either singly responsive (i.e., to Bhlhb5::flpo; Fig. 4A) or dually responsive (i.e., to Bhlhb5::flpo and Ptf1a::cre; Fig. 4J). Using this strategy, we found that the Bhlhb5::flpo allele caused eGFP expression in at least two cell types. The first are medium-sized, Pax2-positive neurons whose cell bodies are found predominantly between the fusiform cell and the molecular layers and that co-express parvalbumin (Fig. 4B–F); arrowheads). In addition, we found a population of small neurons that are found predominantly in the fusiform and deep layers that lack both Pax2 and parvalbumin expression (Fig. 4B–F); arrows). However, these small neurons co-express Tbr2 (Fig. 4G–I; arrows), a marker of unipolar brush cells (Dino and Mugnaini, 2008).

Bottom Line: Using an intersectional genetic strategy, we labeled cartwheel cells, thereby providing proof of concept that subpopulations of Bhlhb5-expressing neurons can be genetically targeted.Moreover, fate-mapping experiments using this allele revealed that Bhlhb5 is required for the proper development of the DCN, since mice lacking Bhlhb5 showed a dramatically diminished number of neurons, including unipolar brush and cartwheel cells.Intriguingly, the Bhlhb5::flpo allele also genetically labels numerous other regions of the nervous system that process sensory input, including the dorsal horn, the retina, and the nucleus of the lateral olfactory tract, hinting at a more general role for Bhlhb5 in the development of neurons that mediate sensory integration.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Pittsburgh, 200 Lothrop St., Pittsburgh, PA 15213, USA; The Pittsburgh Center for Pain Research, University of Pittsburgh, 200 Lothrop St., Pittsburgh, PA 15213, USA.

No MeSH data available.