Forkheadbox N4 (FoxN4) triggers context-dependent differentiation in the developing chick retina and neural tube.

FoxN4, a forkhead box transcription factor, is expressed in the chicken eye field and in retinal progenitor cells (RPCs) throughout development. FoxN4 labelling overlapped with that of Pax6 and Sox2, two crucial transcription factors for RPCs. Later, during neurogenesis in the retina, some cells were intensely and transiently labelled for FoxN4. These cells co-labelled for Lim1, a transcription factor expressed in early-born horizontal cells. The result suggests that high levels of FoxN4 combined with expression of Lim1 define a population of RPCs committed to the horizontal cell fate prior to their last apical mitosis. As these prospective horizontal cells develop, their FoxN4 expression is down-regulated. Previous results suggested that FoxN4 is important for the generation of horizontal and amacrine cells but that it is not sufficient for the generation of horizontal cells (Li et al., 2004). We found that over-expression of FoxN4 in embryonic day 3 chicken retina could activate horizontal cell markers Prox1 and Lim1, and that it generated numerous and ectopically located horizontal cells of both main subtypes. However, genes expressed in photoreceptors, amacrine and ganglion cells were also activated, indicating that FoxN4 triggered the expression of several differentiation factors. This effect was not exclusive for the retina but was also seen when FoxN4 was over-expressed in the mesencephalic neural tube. Combining the results from over-expression and wild-type expression data we suggest a model where a low level of FoxN4 is maintained in RPCs and that increased levels during a restricted period trigger neurogenesis and commitment of RPCs to the horizontal cell fate.

Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina.

During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix-loop-helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.

Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary.

Evolutionary conservation of members of the NGF family in vertebrates was studied by DNA sequence analysis of PCR fragments for NGF, BDNF, and NT-3 from human, rat, chicken, viper, Xenopus, salmon, and ray. The results showed that the three factors are highly conserved from fishes to mammals. Phylogenetic trees reflecting the evolution and speciation of the members of the NGF family were constructed. In addition, the gene for a fourth member of the family, neurotrophin-4 (NT-4), was isolated from Xenopus and viper. The NT-4 gene encodes a precursor protein of 236 amino acids, which is processed into a 123 amino acid mature NT-4 protein with 50%-60% amino acid identity to NGF, BDNF, and NT-3. The NT-4 protein was shown to interact with the low affinity NGF receptor and elicited neurite outgrowth from explanted dorsal root ganglia with no and lower activity in sympathetic and nodose ganglia, respectively. Northern blot analysis of different tissues from Xenopus showed NT-4 mRNA only in ovary, where it was present at levels over 100-fold higher than those of NGF mRNA in heart.

Developmental and regional expression of beta-nerve growth factor receptor mRNA in the chick and rat.

Hybridization probes from the transmembrane region of the chick NGF receptor (NGF-R) that show high homology with the rat NGF-R were used to demonstrate an abundant 4.5 kb NGF-R mRNA in the chick embryo at E3.5. The level remained high until E12 but decreased to adult levels by E18. The highest levels at E8 were in spinal cord, bursa of Fabricius, gizzard, femoralis muscle, and skin. In situ hybridization to E7 embryos showed high expression of the NGF-R gene in spinal cord, particularly the lateral motor column, and in dorsal root, sympathetic, and nodose ganglia. NGF-R mRNA expression was observed throughout brain development and in all regions of the adult brain, with high levels in cerebellum and septum. Lymphoid tissues of chick and rat also expressed the receptor. The complex and widespread expression of NGF-R mRNA in areas not known to be NGF targets suggests broader functions for NGF.

Production and characterization of biologically active recombinant beta nerve growth factor.

DNA fragments encoding either rat or chicken beta nerve growth factor (NGF) were inserted in the expression vector p91023(B) for transient expression in COS cells. The two NGF constructs produced RNA transcripts and proteins of the predicted sizes. Conditioned media from the transfected cells stimulated neurite outgrowth from cultured chicken embryo sympathetic ganglia. The results show that the rat or chicken NGF gene can direct the synthesis of a biologically active NGF protein after transfection of COS cells.

Evolution of the vertebrate neurotrophin and Trk receptor gene families.

Studies of neurotrophins and Trk receptors in jawless fish have shed light on the course of events underlying the formation of these gene families. They evolved early in vertebrate history during major gene duplication events, before the appearance of cartilaginous fish. The existence of multiple genes has permitted the diversification of neurotrophin and Trk receptor expression, and thereby enabling the acquisition of specific functions in selective neuronal populations.

Early identification of retinal subtypes in the developing, pre-laminated chick retina using the transcription factors Prox1, Lim1, Ap2alpha, Pax6, Isl1, Isl2, Lim3 and Chx10.

In this study, antibodies toward the transcription factors Prox1, Lim1, Ap2alpha, Pax6, Isl1, Isl2, Lim3 and Chx10 were used to identify and distinguish between developing cell types in the pre-laminated chick retina. The spatio-temporal expression patterns were analysed from embryonic day 3 (E3) to E9, thus covering a time-span from the onset of retinal cell-fate determination to when retinal laminas can be distinguished. Most transcription factors were found at early stages of development, enabling us to trace various precursor cell populations throughout the lamination process. With time, each transcription factor expression became restricted to distinct laminas or sub-laminas of the maturing retina. These early emerging patterns were compared and found to be consistent with those of the hatched chick retina, where the outer nuclear layer label for Lim3, Isl1 and Isl2. In the inner nuclear layer, horizontal cells labeled for Prox1, Lim1, Isl1, Ap2alpha and Pax6, bipolar cell labeled for Lim3, Isl1 and Chx10 and amacrine cells labeled for Ap2alpha, Isl1 and Pax6. The ganglion cell layer labeled for Isl1, Pax6 and Isl2. The immunolabeling patterns of Lim3 and Isl2 have not previously been described in detail.

Distribution of BDNF and trkB mRNA in the otic region of 3.5 and 4.5 day chick embryos as revealed with a combination of in situ hybridization and tract tracing.

We have used a recently developed technique which combines fluorescent tract tracing and in situ hybridization to study co-localization of neurotrophin mRNA and neurotrophin receptor mRNA expression simultaneously with the pattern of innervation in the developing chick ear. Efferent and afferent fibersfrom the VII/VIIIth cranial nerves were retrogradely and anterogradely filled with Dextran amines conjugated to Texas red and the brain stem was incubated for 2 hours in tissue culture medium. The tissue was subsequently fixed, sectioned frozen, mounted and subjected to in situ hybridization analysis using probes for brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, trkB. The results show that afferent and efferent fibers to the ear innervate areas of the developing otocyst which express BDNF mRNA. We also found that neurons in the stato-acoustic ganglion express high levels of trkB mRNA whereas the subset of facial motor neurons that is efferent to the ear only had no or very low levels of trkB mRNA. From our results we conclude that chicken otic efferent fibers preferentially project to areas with BDNF mRNA expression. The very low levels of trkB mRNA in these motor neurons compared to afferent neurons innervating the same region suggest that other factors, perhaps co-expressed with BDNF, may support efferents. A possible involvement of afferents in guiding efferents to specific areas of the ear is suggested.

Expression of neurotrophin-4 mRNA during oogenesis in Xenopus laevis.

Neurotrophin-4 (NT-4), a recently discovered novel member of the family of neurotrophic factors structurally related to nerve growth factor (NGF), is abundantly expressed in the Xenopus laevis ovary. In this study we have localized NT-4 mRNA expressing cells in the Xenopus ovary by in situ hybridization and have used this technique together with Northern blot analyses to quantify NT-4 mRNA expression during oogenesis in Xenopus. In situ hybridization of sections through the Xenopus ovary using an alpha-[35S]-dATP labeled Xenopus NT-4 mRNA specific probe showed an intense labeling over the cytoplasm of oocytes with a diameter of 50-200 microns corresponding to stage I according to Dumont (1972). Labeling was also seen over the cytoplasm of stages II to IV although with a lower intensity than over stage I oocytes. No labeling was seen over more mature oocytes of stages V and VI. NT-4 mRNA could not be detected in the early embryo from the onset of cleavage division to the neurula stage suggesting that the NT-4 gene is not expressed during Xenopus early embryogenesis. The confinement of NT-4 mRNA in the Xenopus ovary to immature oocytes suggests that NT-4 mRNA expression is strictly regulated during oogenesis and that the NT-4 protein could play a role as a maturation factor for immature oocytes.

Neurotrophins and their receptors in chicken neuronal development.

A review on current studies of chicken neurotrophins and their receptors is given. Chicken NGF, BDNF and NT-3 have been cloned and sequences have been used to synthesize oligonucleotides for specific localization of expression during development. Also, chicken TrkA, TrkB and TrkC have been cloned, sequenced and studied by in situ hybridization. Recombinant NT-3 was applied to chicken ganglia at different developmental stages to examine acquirement of responsiveness to NT-3 compared to NGF. Phylogenetic analyses of the chicken neurotrophins and Trk receptors were carried out based on parsimony. Finally, some data on apoptosis in chicken embryo sympathetic ganglia are presented.

The p53 co-activator Zac1 neither induces cell cycle arrest nor apoptosis in chicken Lim1 horizontal progenitor cells.

Chicken horizontal progenitor cells are able to enter their final mitosis even in the presence of DNA damage despite having a functional p53-p21 system. This suggests that they are resistant to DNA damage and that the regulation of the final cell cycle of horizontal progenitor cells is independent of the p53-p21 system. The activity of p53 is regulated by positive and negative modulators, including the zinc finger containing transcription factor Zac1 (zinc finger protein that regulates apoptosis and cell cycle arrest). Zac1 interacts with and enhances the activity of p53, thereby inducing cell cycle arrest and apoptosis. In this work, we use a gain-of-function assay in which mouse Zac1 (mZac1) is overexpressed in chicken retinal progenitor cells to study the effect on the final cell cycle of horizontal progenitor cells. The results showed that overexpression of mZac1 induced expression of p21 in a p53-dependent way and arrested the cell cycle as well as triggered apoptosis in chicken non-horizontal retinal progenitor cells. The negative regulation of the cell cycle by mZac1 is consistent with its proposed role as a tumour-suppressor gene. However, the horizontal cells were not affected by mZac1 overexpression. They progressed into S- and late G2/M-phase despite overexpression of mZac1. The inability of mZac1 to arrest the cell cycle in horizontal progenitor cells support the notion that the horizontal cells are less sensitive to events that triggers the p53 system during their terminal and neurogenic cell cycle, compared with other retinal cells. These properties are associated with a cell that has a propensity to become neoplastic and thus with a cell that may develop retinoblastoma.

Expression of neurotrophins and trk receptors in the avian retina.

Using the RNase protection assay, we have found that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are expressed in the avian retina during development. The expression peaks around embryonic days 12-15, with decreasing levels at later stages of development. Abundant levels of NGF and BDNF but low levels of NT-3 mRNA were found in the adult retina. We also found that light/darkness regulated the levels of NGF and BDNF mRNAs but not the levels of NT-3 mRNA in the 5-day-old chicken retina. It was demonstrated that NGF and BDNF mRNA levels were up-regulated by light exposure. The cellular localization of mRNA expression for the neurotrophins and neurotrophin receptors TrkA, TrkB, and TrkC in the retina was studied using in situ hybridization. The patterns of NGF and trkA mRNA expression were very similar and were localized to the external part of the inner nuclear layer on the border with the outer plexiform layer and corresponded to the localization of horizontal cells. NT-3 labeling was also found over the external part of the inner nuclear layer, whereas trkC mRNA was found over all layers in the retina. BDNF labeling was found over all layers in the retina, whereas TrkB labeling was intense over cells in the ganglion cell layer, which is in agreement with the response of ganglion cells to BDNF stimulation. Functional neurotrophin receptors were suggested by the response of retinal explants to neurotrophin stimulation. These data indicate that the neurotrophins play local roles in the retina that involve interactions between specific neuronal populations, which were identified by the localization of the Trk receptor expression. The data also suggest that NGF and BDNF expression is regulated by normal neuron usage in the retina.

Nerve growth factor receptor TrkA is expressed by horizontal and amacrine cells during chicken retinal development.

Nerve growth factor is known to stimulate neurite outgrowth and support neuronal survival during embryonic development. We have studied the expression of the nerve growth factor receptor, TrkA, at both mRNA and protein levels during the course of chicken retinal development. Furthermore, we have compared the expression of trkA mRNA with that of the 75-kD low-affinity neurotrophin receptor (p75NTR). RNase protection assay identified peak-levels of trkA mRNA in the late embryonic retina. Using in situ hybridization and immunohistochemistry, we found cells expressing TrkA in both the internal and the external part of the inner nuclear layer, corresponding to amacrine and horizontal cells, respectively. The TrkA-expressing amacrine cell has a unistratified dendritic arborization in the second sublamina of the inner plexiform layer, and may represent the stellate amacrine cell described by Cajal. The horizontal cells, possessing arciform dendrite processes in the outer plexiform layer, showed strong TrkA immunoreactivity in both dendrites and cell bodies. During the course of retinal development, the TrkA-expressing amacrine cells decreased in number, whereas the TrkA-expressing horizontal cells persisted. Because nerve growth factor was expressed where the horizontal cells, but not where the amacrine cells were located, these findings raise the question of whether nerve growth factor could locally support the survival of TrkA-expressing interneurons during retinal development.

Kainic acid, tetrodotoxin and light modulate expression of brain-derived neurotrophic factor in developing avian retinal ganglion cells and their tectal target.

Increasing evidence underlies the importance of neurotrophins as neuron-derived trophic signals in the developing visual system, although their precise roles are still undefined. Here we show that brain-derived neurotrophic factor messenger RNA is simultaneously expressed in a subpopulation of retinal ganglion cells and in their target during late embryogenesis. Moreover, light as well as the excitotoxin; kainic acid, induced an increase of the brain-derived neurotrophic factor messenger RNA, which could be blocked by the sodium-channel blocker; tetrodotoxin. Messenger RNA for trkB, a receptor for brain-derived neurotrophic factor, was found in the retinal ganglion cells expressing brain-derived neurotrophic factor showing that certain retinal ganglion cells express messenger RNA both for brain-derived neurotrophic factor and trkB. Furthermore, trkB messenger RNA was found in tectum, in the same layers as the brain-derived neurotrophic factor messenger RNA. These findings suggest that brain-derived neurotrophic factor expression is regulated in an activity-dependent manner during the phase of development when neuronal activity plays an important role.

MAP kinase phosphatase-1 mRNA is expressed in embryonic sympathetic neurons and is upregulated after NGF stimulation.

The family of Tyr/Thr protein phosphatases, called dual-specificity phosphatases, have been implicated in the feedback regulation of the MAP kinase cascade by dephosphorylating the MAP kinases. Using low stringent cDNA screening we have isolated a chicken homologue of the CL100 phosphatase also called MAP kinase phosphatase 1 (MKP-1). The chicken MKP-1 has 84% and 85.5% identity to the rat and human amino acid sequence, respectively. Using RNase protection assay and in situ hybridization we have found that MKP-1 mRNA is expressed at low levels in most tissues during development. In embryonic dorsal root and sympathetic ganglia MKP-1 mRNA expression increases with age. The expression in large cells in dorsal root ganglia suggests that it is neurons which express MKP-1 mRNA. We also show that MKP-1 mRNA is induced in dissociated embryonic sympathetic neurons after nerve growth factor stimulation. In addition, our results show that MKP-1 mRNA is induced after NGF stimulation of fibroblasts expressing the NGF receptor TrkA, suggesting that MKP-1 is upregulated after activation of the TrkA receptor. These data show that the MKP-1 gene is regulated in a tissue and temporal specific fashion with strong expression in the developing peripheral ganglia, and suggest that the activation of MKP-1 mRNA expression by NGF is a ubiquitously induced response to TrkA activation, independent of the cellular origin or type on which the TrkA receptor is active.

Molecular cloning and cellular localization of trkC in the chicken embryo.

Degenerate primers directed against conserved regions of the trk and trkB amino acid sequences were used in the polymerase chain reaction to isolate a 455 bp fragment from embryonic day 3 chicken cDNA encoding the trkC. This fragment was subsequently used to synthesize an anti-sense trkC cRNA probe which was used in a RNase protection assay of total RNA from chicken embryos. trkC mRNA was found in the E2 embryo with increasing levels later in development. In the E9 embryo highest levels were found in brain and spinal cord with intermediate levels in eye, heart, gut and muscle. Low levels were found in kidney, liver, skin and yolk sac. Using the 455 bp trkC fragment as a probe in RNA blot analyses of poly A+ RNA, a major transcript of 6.3 kb and two minor transcripts of 3 kb and 10 kb were found. In situ hybridization was performed on embryos taken at three stages of development (embryonic day 3, 9 and 19), using a 48-mer antisense oligonucleotide probe for chicken trkC. Within the sensory nervous system trkC mRNA expression at all ages was confined to the ventrolateral neurons of the spinal sensory and trigeminal ganglia as well as distal ganglia associated with the VIIth, IXth and Xth cranial nerves. Labelling for trkC mRNA was also observed within the developing CNS at E3 and the ganglion of Remak at E19. A barely detectable level of expression was observed in the sympathetic chain and no labelling was evident in the proximal ganglia of the cranial nerves. These results suggest that neurons have a very early capacity to respond to neurotrophin-3 which continues throughout embryonic development. The early expression of trkC mRNA also support the growing evidence suggesting a role for neurotrophins in neuronal differentiation.

A simple and reliable technique to combine oligonucleotide probe in situ hybridization with neuronal tract tracing in vertebrate embryos.

We describe a simple and reliable combination of in situ hybridization with neuronal tracing. The technique uses recent advances in the field of neuronal tract tracing including fast diffusing, low molecular weight dextran amines and fade resistant fluorescent dyes, and combines them with in situ hybridization using a sensitive oligonucleotide probe. Using this technique we have investigated the mRNA encoding the trkB receptor for brain-derived neurotrophic factor in identified facial and vestibular afferent and efferent neurons. We found very low levels of trkB mRNA in facial efferent neurons, whereas in the vestibular afferent neurons, clear labeling for the trkB mRNA could be seen. This technique can be applied to the developing embryo to study topology of a variety of cellular markers with reference to neuronal population or fibers identified by their origin or target.