Chicken Cranial Motor Axons – Growth, Branching, and Guidance

Cadherin-7 and cadherin-6B differentially regulate the growth, branching and guidance of cranial motor axons


Background

Why study cranial motor axons? Axon growth and guidance has been cited as one of the most spectacular achievements of the developing nervous system (Faissner 1997). The formation and reformation of sufficient networks and the plasticity of synaptic connections are essential for the function and restoration of the nervous system during times of health, as well as disease. Determining the molecular/cellular bases of interneuronal connections could, therefore, lead to new treatments for those suffering from brain damage in the future (Faissner 1997).

Why study chicken cranial motor axons? Not only is the chicken a modern descendant of dinosaurs, it’s also the first non-mammalian amniote to have its genome sequenced (Hillier 2004). The International Chicken Genome Sequencing Consortium, which accomplished this feat of sequencing, discovered that chickens and humans share more than half of their genes! In fact, both genomes contain approximately the same number of genes (20,000-25,000) – which are 75 percent identical on average.

Francis S. Collins, director of the National Human Genome Research Institute, has said that “The chicken genome fills a crucial gap in our scientific knowledge. Located between mammals and fish on the tree of life, the chicken is well positioned to provide us with new insights into genome evolution and human biology. By comparing the genomes of a wide range of animals, we can better understand the structure and function of human genes and, ultimately, develop new strategies to improve human health” (Bethesda 2004).

Differential Regulation of the Cranial Motor Axons via Cadherins

Cadherins are calcium-dependent cell adhesion molecules extensively expressed in the nervous system (Matsunaga et al. 2011).

They are involved in:

  • the growth and differentiation of axons and synapses
  • the guidance of axons
  • the formation of nuclei (Andrews and Mastick, 2003; Boscher and Mege, 2008; Iwai et al., 2002; Marthiens et al., 2005; Salinas and Price, 2005; Togashi et al., 2002).

Type-II cadherins are considered a classical type of cadherin, characterized by an extracellular domain composed of five tandem cadherin repeats (EC1-EC5).

This extracellular domain is how cadherin-dependent adhesion occurs, by interacting with another type II cadherin.

Two cadherins of this type, cadherin-7 (Cad7) and cadherin-6B (Cad6B), have been suggested as modulators of neural activity via the regulation of neuronal morphology (form and structure) (Matsunaga et al. 2011).

Regulation of the actin cytoskeleton and membrane dynamics is dependent upon cadherins; such regulation underscores the role that cadherins possibly play in axon guidance during development. Barnes et al. used fertilized chicken eggs to investigate differential cadherin expression in the growth and guidance of cranial motoneurons.

Specifically, Barnes et al. looked at branchiomotor (BM) motoneurons, a subset of cranial motoneurons. Early BM axon extension has been shown to be dependent upon repellent cues produced by the floor plate, a structure that serves as an organizer to guide neuronal positioning and differentiation along the dorsoventral axis of the neural tube.

Neural Tube

- from the 20th U.S. edition of Gray's Anatomy of the Human Body, originally published in 1918

Hypothesis

Cad7-mediated interactions play a role during the early unbranched outgrowth of cranial motoneurons

-whereas-

Cad6B plays a role during later maturation and branching


Experiments

In vitro

Cad7 was shown to:

  • enhance motor axon outgrowth
  • suppress the formation of multiple axons
  • restrict interstitial branching (branching originating in different centers).

Cad7, therefore, promotes the development of a single unbranched axon – characteristic of differentiating motoneurons.

Cad6B was shown to:

  • promote motor axon branching – typical of mature motoneurons.

In vivo

Gain- and loss-of-function experiments for Cad7 and Cad6B resulted in phenotypes supporting the conclusions of the in vitro experiments.

Specifically, loss of cadherin-mediated interactions led to:

  • dysregulation of cranial motoneuron branching
  • defects in axon navigation.

Materials and Methods

Embryos

Fertilised chicken eggs were incubated to appropriate stages

Plasmids

Control: myristylated-GFP

Extracellular domain deletion: truncated MNcadDnlacZ

Cytoplasmic and transmembrane deletion: truncated Cad7

Full-length Cad7-flag and full-length Cad6B-nlacZ, myristylated-Akt-GFP were used, all utilizing a chick b-actin promoter with a CMV enhancer (pCAGGS).

shRNA plasmids:  Cad7-shRNA-RFP, Cad6B-shRNA-RFP and their control (scrambled sequence) variants, all utilizing a chick U6 promoter

In order to label the electroporated axons: full-length Cad6B-nlacZ, MNcadD-nlacZ and full-length Cad7-flag were co-electroporated with myristylated-GFP

Characterization of shRNA constructs

To verify knockdown of cadherin expression: shRNA constructs for Cad7 or Cad6B (or scrambled controls) were transfected into HEK293 cells with Cad7-flag or Cad6B-nlslacZ using Fugene reagent and immunostained using rabbit anti-flag or chicken anti-b-galactosidase antibodies.

Controls: cells were transfected with tagged cadherin constructs only.

Dissociated neuron cultures and NIH3T3 cell co-cultures

Cranial motoneurons were grown on laminin-coated coverslips or on monolayers of transfected NIH3T3 cells.

Glass coverslips were coated with poly-D-ornithine and laminin.

NIH3T3 cells were transfected with Cad7-flag or Cad6B-nlacZ and plated in chamber slides.

For preparation of cranial motoneurons: the ventral portion of the hindbrain was used, which is enriched in cranial motoneurons at E3 (stage 16-17) or E5 (stage 25-26).

Dissociated neurons were plated on laminin-coated coverslips or on NIH3T3 cell monolayers in chamber slides.

In some experiments, neurons were transfected with full-length Cad7 or full-length Cad6B constructs and myr-GFP,

Control: neurons transfected with the myr-GFP construct alone.

In some experiments, dissociated BM neurons grown on laminin were treated with soluble Cad7 protein, which was generated from medium conditioned by HEK293 cells transfected by a construct encoding Cad7 lacking its cytoplasmic and transmembrane domains.

Controls: treated with medium collected from mock-transfected HEK293 cells.

To investigate PI3K signaling: E5 neurons cultured on a Cad6B-expressing monolayer,  treated with the PI3K inhibitor

The inhibitor was diluted in pre-warmed culture medium.

Controls: treated with dimethyl sulphoxide (DMSO) vehicle.

Immunohistochemistry on cultures

Coverslips/chamber slides were fixed, rinsed in PBS and blocked using PBS containing 1% sheep serum and 0.5% Triton X-100.

For quantitation: cultures were immunostained using mouse anti-Islet1/2, and rabbit anti-neurofilament H antibodies diluted in blocking solution.

Secondary antibodies were Alexa Fluor 568 anti-rabbit and Alexa Fluor 488 anti-mouse

At least 30 Islet1/2-positive neurons per condition were imaged in three separate experiments.

Total axon length was quantitated using SimplePCI software; the number of branch points per neuron and the number of axons extending from the cell body (‘polarity’) were counted manually.

Statistical analysis utilized: Student’s t-test (two-tailed) or the Mann-Whitney U-test.

Some neurons were immunostained using mouse anti-Cad7 or anti-Cad6B antibodies together with rabbit anti-Islet1/2 antibody

Electroporation of chick embryos in ovo

Electroporation was performed at stage 10-11 (E2) and embryos were incubated to E4, E5 or E6.

Immunohistochemistry was performed on whole-mount or cryosections using rabbit or chicken anti-GFP, mouse anti-Islet1/2, mouse anti-SC1, chicken anti-b-galactosidase, antisarcomeric myosin antibody MF20 (1:250; DSHB) and rabbit anti-phospho-Akt.

Visualization of motor axon trajectories in hindbrains electroporated with cadherin gain- and loss-of-function constructs: achieved by co-electroporation of a myristylated-GFP (myr-GFP) construct.

To verify co-expression of the constructs in the cells and to confirm expression by motoneuron: hindbrains were immunostained for the cadherin tag, GFP and Islet1/2, and/or SC1.

Results

The molecular cues that mediate stages of branchiomotor neuron development remain largely unknown, but Barnes et al. demonstrated that Cad7 and Cad6B play distinct roles.

  • Cad7 is expressed during early phases of chick BM neuron development.
  • Cad7 promotes the growth of a single unbranched axon and regulates axon guidance.
  • Cad6B is expressed during late phases of chick BM neuron development.
  • Cad6B promotes axon branching.

Cad7 & Cad6B Expression Patterns

Fig. 1. Motoneuron development and Cad7 and Cad6B expression in the chick hindbrain. (A) Diagrams of flat-mounted brainstem showing the positions of cranial nerves and motor nuclei. Dorsally projecting branchiomotor (BM) neurons are shown in red, ventrally projecting somatic motoneurons in blue. Roman numerals indicate cranial nerves (III, oculomotor; IV, trochlear; V, trigeminal; VI, abducens; aVI, accessory abducens; VII/VII, facial/vestibuloacoustic; IX, glossopharyngeal; X, vagus; XI, cranial accessory; XII, hypoglossal; gV-X indicate sensory ganglia and cranial nerves as above). Rhombomeres are numbered. (B) Transverse section through the branchial region, showing hindbrain neuroepithelium and branchial arches (BA), with dorsally projecting BM (red) and ventrally projecting somatic motor (blue) motor axons. Adapted with permission from Guthrie (Guthrie, 2007). (C,D) Diagram of flat-mounted even- and odd-numbered rhombomere pairs at early (C) and late (D) developmental stages, with respect to BM axon outgrowth. (C) Cad7 (red) is expressed from E2-5 by the hindbrain neuroepithelium, extending cranial motoneurons and the boundary cap cells at the nerve exit point. (D) Cad6B (green) is expressed by mature motoneurons and their axons between E5 and E9. Arrows indicate the direction of migration of BM neuron somata. Expression data derived from Ju et al. (Ju et al., 2004). MB, midbrain; HB, hindbrain; FP, floor plate; OV, otic vesicle; BA, branchial arch; VE, ventricle; G, ganglion; PX, pharynx; EP, exit point; RL, rhombic lip.


Fig. 1A and 1B

  • diagrams essentially showing that BM motoneurons/axons project dorsally in the chick hindbrain, shown as small red circles.

Fig. 1C and 1D

Fig. 1C

  • shows the early developmental stage of BM axon outgrowth, from embryonic days (E) 2 to 5 – also known as stages 11-25.
  • During this time of axon extension, BM neurons express Cad7 (shown in red).
  • Around stage 25 (embryonic day 5), Cad7 begins to be downregulated
  • Cad7 continues to be expressed until stage 30 (embryonic day 8), when the majority of BM neurons have formed nuclei adjacent to the exit points (EP).

Fig. 1D

  • shows the expression of Cad6B (shown in green) from embryonic days 5 to 9 by mature motoneurons/axons.
  • Cad6B expression is upregulated during the downregulation of Cad7

Cad7 and Cad6B are expressed by dissociated cranial motoneurons and have contrasting effects on cranial motoneuron growth/branching (in vitro)

Fig. 2. Expression of cadherins and responses of cranial motoneurons to culture on cadherin-expressing substrata. (A-D) Immunohistochemistry using anti-Cad7 and anti-Cad6B antibodies in chick E5 cultured cranial motoneurons (red). Insets in A and B show Islet1/2 expression (green). (E-G) E5 cranial motoneurons cultured on control, Cad7- or Cad6B-expressing NIH3T3 cell monolayers. (H-K) The effects of cadherin-mediated interactions on cranial motoneuron morphology (length, polarity and branching). Significant differences from controls are indicated by asterisks (*, P<0.05; ***, P<0.001; Student’s t-test in all cases, Xxxxxxx? test); n=90 neurons per condition. bps, branch points. Scale bars: in D, 20 mm for A,B and 10 mm for C,D; in G, 20 mm for E-G.

To mimic in vitro the cadherin environments that cranial motoneurons are exposed to in vivo:

Stage 25 (embryonic day 5) neurons were

  • dissociated and cultured for 48 hours
  • labeled with (for visualization)
  1. anti-Cad7 antibodies (red) and anti-Islet 1/2 antibodies (green)
  2. anti-Cad6B antibodies (red) and anti-Islet 1/2 antibodies (green)

Both Cad7 and Cad6B were expressed at this stage.

Fig. 2A & 2C

  • Cad7 expression appeared in a proximal-to-distal gradient from the cell body

Fig. 2B & 2D

  • Cad6B was expressed uniformly (expressed on both cell bodies and axons)
  • Cad6B was strongly expressed within the growth cone (a dynamic, actin-supported extension of a developing axon seeking its synaptic target) – shown by the small white arrow in Fig. 2D

Fig. 2H-J

Three aspects of axonogenesis were quantitated per condition:

    • Total neuron length
    • Total number of branch points per neuron
    • Number of axons extending from the cell body
      1. A measure of neuronal polarity
  • When compared to the control
    • Cad7- and Cad6B-expressing cells showed no significant difference in axon length
    • Cad7-expressing cells showed a reduction in the number of branch points and the number of processes extending from the cell body (polarity)
    • Cad6B-expressing cells showed increased branching
    • Cad6B-expressing cells showed no difference in the number of axons extending from the cell body
      • –> Cad6B promotes interstitial branching instead of the development of multiple axons

Fig. 2K

  • The modal number of branch points:
  1. For control neurons = 1
  2. For neurons grown on Cad7-expressing cells = 0
  3. For neurons grown on Cad6B-expressing cells = 2

–> Cad7-expressing cells have less than control number of branch points, while Cad6B-expressing cells have more

Evaluation

Minor weaknesses in the paper include not putting the study into a modern context; at no point did the authors mention greater implications of this study, nor did they outline possible future research that could be stem from this study. Additionally, the authors mention many experimental techniques, as well as results, but did not include the data in the article. This data may have made some of the other experiments they elaborate upon more clear.

Take Home Message

Cad7 and Cad6B play crucial roles in distinct aspects of cranial motoneuron development.

Cad7 interactions promote unbranched outgrowth and guidance, whereas Cad6B promotes branching.


References

Andrews, G. L. and Mastick, G. S. (2003). R-cadherin is a Pax6-regulated, growth-promoting cue for pioneer axons. J. Neurosci. 23, 9873-9880. http://www.jneurosci.org/content/23/30/9873.full

Barnes, S. H., Price S. R., Wentzel, C., and Guthrie, S. C. (2010). Cadherin-7 and cadherin-6b differentially regulate the growth, branching and guidance of cranial motor axons. Development 137, 805-814. http://dev.biologists.org/content/137/5/805.full

Bethesda, Md. 2004. Researchers Compare Chicken, Human Genomes: Analysis of First Avian Genome Uncovers Differences Between Birds and Mammals. National Human Genome Research Institute. Retrieved April 27, 2012 from http://www.genome.gov/12514316

Boscher, C. and Mege, R. M. (2008). Cadherin-11 interacts with the FGF receptor and induces neurite outgrowth through associated downstream signalling. Cell Signal. 20, 1061-1072.

Faissner, A. 1997. Glial Derived Extracellular Matrix Components: Important Roles in Axon Growth and Guidance. Neuroscientist 3(6): 371-380. http://nro.sagepub.com/content/3/6/371.abstract

Hamburger, V. and Hamilton, H. L. (1992). A series of normal stages in the development of the chick embryo. Dev. Dyn. 195, 231-272. http://homepage.univie.ac.at/brian.metscher/Hamburger51_ChickStages.pdf

Hillier, L.W., et al. 2004. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432: 695-716. http://www.nature.com/nature/journal/v432/n7018/full/nature03154.html

Iwai, Y., Hirota, Y., Ozaki, K., Okano, H., Takeichi, M. and Uemura, T. (2002). DN-cadherin is required for spatial arrangement of nerve terminals and ultrastructural organization of synapses. Mol. Cell. Neurosci. 19, 375-388.

Matsunaga, E., Kurotani, T., Suzuki, K., and Okanoya, K. (2011). Type-II cadherins modulate neural activity in cultured rat hippocampal neurons. Neuroreport 22(13), 629-632.

Marthiens, V., Gavard, J., Padilla, F., Monnet, C., Castellani, V., Lambert, M. and Mege, R. M. (2005). A novel function for cadherin-11 in the regulation of motor axon elongation and fasciculation. Mol. Cell. Neurosci. 28, 715-726.

Salinas, P. C. and Price, S. R. (2005). Cadherins and catenins in synapse development. Curr. Opin. Neurobiol. 15, 73-80.

Togashi, H., Abe, K., Mizoguchi, A., Takaoka, K., Chisaka, O. and Takeichi, M. (2002). Cadherin regulates dendritic spine morphogenesis. Neuron 35, 77-89.

Andrews, G. L. and Mastick, G. S. (2003). R-cadherin is a Pax6-regulated,

growth-promoting cue for pioneer axons. J. Neurosci. 23, 9873-9880.

Barnes, S. H., Price S. R., Wentzel, C., and Guthrie, S. C. (2010). Cadherin-7 and cadherin-6b differentially regulate the growth, branching and guidance of cranial motor axons. Development 137, 805-814.

Boscher, C. and Mege, R. M. (2008). Cadherin-11 interacts with the FGF receptor

and induces neurite outgrowth through associated downstream signalling. Cell

Signal. 20, 1061-1072.

Hamburger, V. and Hamilton, H. L. (1992). A series of normal stages in the

development of the chick embryo. Dev. Dyn. 195, 231-272.

Iwai, Y., Hirota, Y., Ozaki, K., Okano, H., Takeichi, M. and Uemura, T. (2002).

DN-cadherin is required for spatial arrangement of nerve terminals and

ultrastructural organization of synapses. Mol. Cell. Neurosci. 19, 375-388.

Matsunaga, E., Kurotani, T., Suzuki, K., and Okanoya, K. (2011). Type-II cadherins modulate neural activity in cultured rat hippocampal neurons. Neuroreport 22(13), 629-632.

http://journals.lww.com/neuroreport/Abstract/2011/09140/Type_II_cadherins_modulate_neural_activity_in.1.aspx

Marthiens, V., Gavard, J., Padilla, F., Monnet, C., Castellani, V., Lambert, M.

and Mege, R. M. (2005). A novel function for cadherin-11 in the regulation of

motor axon elongation and fasciculation. Mol. Cell. Neurosci. 28, 715-726.

Salinas, P. C. and Price, S. R. (2005). Cadherins and catenins in synapse

development. Curr. Opin. Neurobiol. 15, 73-80.

Togashi, H., Abe, K., Mizoguchi, A., Takaoka, K., Chisaka, O. and Takeichi,

M. (2002). Cadherin regulates dendritic spine morphogenesis. Neuron 35, 77-

89.

1 Response to Chicken Cranial Motor Axons – Growth, Branching, and Guidance

  1. akbar wiguna says:

    thanks for your article this it a good and nice article

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