trkB encodes a receptor tyrosine kinase activated by three neurotrophins—brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4/5. In vivo , three isoforms of the receptor are generated by differential splicing—gp145 trkB or the full-length trkB receptor, and trkB.T1 and trkB.T2, two cytoplasmically truncated receptors that lack kinases, but contain unique C termini. Although the truncated receptors appear to be precisely regulated during nervous system development and regeneration, their role in neurotrophin signaling has not been directly tested. In this paper, we studied the signaling properties and interactions of gp145 trkB , trkB.T1, and trkB.T2 by expressing the receptors in a Xenopus oocyte microinjection assay. We found that oocytes expressing gp145 trkB , but not trkB.T1 or trkB.T2, were capable of eliciting 45 Ca efflux responses (a phospholipase C-γ-mediated mechanism) after stimulation by BDNF. When trkB.T1 and trkB.T2 were coexpressed with gp145 trkB , they acted as dominant negative receptors, inhibiting the BDNF signal by forming nonfunctional heterodimers with the full-length receptors. An ATP-binding mutant of gp145 trkB had similar dominant inhibitory effects. Our data suggest that naturally occurring truncated trkB receptors function as inhibitory modulators of neurotrophin responsiveness. Furthermore, the homodimerization of gp145 trkB appears to be an essential step in activation of the BDNF signaling cascade.
ABSTRACT Mice lacking the POU domain-containing transcription factor Brn-3a have several neuronal deficits. In the present paper, we show that Brn-3a plays two distinct roles during development of the trigeminal ganglion. In this ganglion, neurons expressing the neurotrophin receptors, TrkB and TrkC, are born between E9.5 and E11.5. In the absence of Brn-3a, very few neurons ever express TrkC, but TrkB- expressing neurons are present at E12.5 in elevated numbers, suggesting that Brn-3a may be a constituent of a regulatory circuit determining which Trk receptor is expressed by these early-born neurons. Most neurons expressing the neurotrophin receptor TrkA are generated between E11.5 and E13.5 in this ganglion and their initial generation is not prevented by absence of Brn-3a. However, after E12.5, absence of Brn-3a results in a progressive loss in neuronal TrkA and TrkB expression, which leads to a massive wave of apoptosis that peaks at E15.5. Despite complete absence of the Trk receptors at E17.5 and P0, approximately 30% of the normal complement of neurons survive to birth in Brn-3a mutants. Approximately 70% of these express the GDNF receptor subunit, c-ret; many can be sustained by GDNF, but not by NGF in culture. Thus, the vast majority of surviving neurons are probably sustained in vivo by trophic factor(s) whose receptors are not regulated by Brn-3a. In conclusion, our data indicate the specific functions of Brn-3a in controlling the survival and differentiation of trigeminal neurons by regulating expression of each of the three Trk receptors.
Neural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Focal adhesion kinase (FAK) mediates signal transduction by integrin and growth factor receptors, each of which is important for normal cardiovascular development. To investigate the role of FAK in NCC morphogenesis, we deleted it in murine NCCs using Wnt1cre, yielding craniofacial and cardiovascular malformations resembling those observed in individuals with DiGeorge syndrome. In these mice, we observed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and impaired cardiac outflow tract rotation, which resulted in a dextroposed aortic root. Moreover, within the conotruncal cushions, Fak-deficient NCCs formed a less organized mesenchyme, with reduced expression of perlecan and semaphorin 3C, and exhibited disorganized F-actin stress fibers within the aorticopulmonary septum. Additionally, absence of Fak resulted in reduced in vivo phosphorylation of Crkl and Erk1/2, components of a signaling pathway essential for NCC development. Consistent with this, both TGF-β and FGF induced FAK and Crkl phosphorylation in control but not Fak-deficient NCCs in vitro. Our results indicate that FAK plays an essential role in cardiac outflow tract development by promoting the activation of molecules such as Crkl and Erk1/2.
The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.
In developing tissues, cell polarity and tissue architecture play essential roles in the regulation of proliferation and differentiation. During cerebral cortical development, adherens junctions link highly polarized radial glial cells in a neurogenic niche that controls their behavior. How adherens junctions regulate radial glial cell polarity and/or differentiation in mammalian cortical development is poorly understood. Conditional deletion of Afadin, a protein required for formation and maintenance of epithelial tissues, leads to abnormalities in radial glial cell polarity and subsequent loss of adherens junctions. We observed increased numbers of obliquely-oriented progenitor cell divisions, increased exit from the ventricular zone neuroepithelium, and increased production of intermediate progenitors. Together, these findings indicate that Afadin plays an essential role in regulating apical-basal polarity and adherens junction integrity of radial glial cells, and suggest that epithelial architecture plays an important role in radial glial identity by regulating mitotic orientation and preventing premature exit from the neurogenic niche.
ABSTRACT Animals lacking neurotrophin-3 (NT-3) are born with deficits in almost all sensory ganglia. Among these, the trigeminal ganglion is missing 70% of the normal number of neurons, a deficit which develops during the major period of neurogenesis between embryonic stages (E) 10.5 and E13.5. In order to identify the mechanisms for this deficit, we used antisera specific for TrkA, TrkB, and TrkC to characterize and compare the expression patterns of each Trk receptor in trigeminal ganglia of wild type and NT-3 mutants between E10.5 and E15.5. Strikingly, TrkA, TrkB, and TrkC proteins appear to be exclusively associated with neurons, not precursors. While some neurons show limited co-expression of Trk receptors at E11.5, by E13.5 each neuron expresses only one Trk receptor. Neuronal birth dating and cell counts show that in wild-type animals all TrkB- and TrkC-expressing neurons are generated before E11.5, while the majority of TrkA-expressing neurons are generated between E11.5 and E13.5. In mice lacking NT-3, the initial formation of the ganglion, as assessed at E10.5, is similar to that in wild-type animals. At E11.5, however, the number of TrkC-expressing neurons is dramatically reduced and the number of TrkC-immunopositive apoptotic profiles is markedly elevated. By E13.5, TrkC-expressing neurons are virtually eliminated. At E11.5, compared to wild type, the number of TrkB-expressing neurons is also reduced and the number of TrkB immunoreactive apoptotic profiles is increased. TrkA neurons are also reduced in the NT-3 mutants, but the major deficit develops between E12.5 and E13.5 when elevated numbers of TrkA-immunoreactive apoptotic profiles are detected. Normal numbers of TrkA- and TrkB-expressing neurons are seen in a TrkC-deficient mutant. Therefore, our data provide evidence that NT-3 supports the survival of TrkA-, TrkB- and TrkC-expressing neurons in the trigeminal ganglion by activating directly each of these receptors in vivo.
Many molecules regulate synaptogenesis, but intracellular signaling pathways required for their functions are poorly understood. Afadin is a Rap-regulated, actin-binding protein that promotes cadherin complex assembly as well as binding many other cell adhesion molecules and receptors. To examine its role in mediating synaptogenesis, we deleted afadin ( mllt1 ), using a conditional allele, in postmitotic hippocampal neurons. Consistent with its role in promoting cadherin recruitment, afadin deletion resulted in 70% fewer and less intense N-cadherin puncta with similar reductions of β-catenin and αN-catenin puncta densities and 35% reduction in EphB2 puncta density. Its absence also resulted in 40% decreases in spine and excitatory synapse densities in the stratum radiatum of CA1, as determined by morphology, apposition of presynaptic and postsynaptic markers, and synaptic transmission. The remaining synapses appeared to function normally. Thus, afadin is a key intracellular signaling molecule for cadherin recruitment and is necessary for spine and synapse formation in vivo .
