Ipou/abnormal chemosensory jump 6

EVOLUTIONARY HOMOLOGS (part 2/2)

Mammalian POU domain class IV homologs

Brn-3.0 expression begins at embryonic day 8.5 (E8.5) in a specific set of midbrain tectal neurons. By E9.5, Brn-3.0 is expressed in early CNS neurons in the hindbrain and spinal cord. In the peripheral sensory ganglia, Brn-3.0 expression is first observed at E9.0 in migrating precursors of the trigeminal ganglion, followed by other sensory cranial and dorsal root ganglia, in a rostral to caudal sequence. Brn-3.0 is restricted to the post-mitotic phase of CNS development. In the sensory cranial and dorsal root ganglia, however, Brn-3.0 is expressed in dividing neural precursors, suggesting that the nature or timing of developmental events controlled by Brn-3.0 are distinct in the CNS and peripheral neurons. Restriction of Brn-3.0 expression to post-mitotic CNS neurons demonstrates that Brn-3.0 is not required for neurogenesis or patterning of the neuroepithelium in the CNS, but suggests a role in specification of mature neuronal phenotypes (Fedtsova, 1995).

Brn-3.0, a POU-domain transcription factor, is expressed in specific postmitotic neurons in the dorsal part of the neural tube: these are among the first spinal cord neurons to appear in development. In the mature spinal cord, the Brn-3.0 cells form a numerous population of scattered neurons in the intermediate spinal gray. Ablation of the notochord in chick embryos extends the domain of Brn-3.0 expression into the ventral neural tube, while ectopic grafts of notochord tissue suppress Brn-3.0 expression. The notochord effects on Brn-3.0 expression are reproduced in vivo by the implantation of a local source of recombinant Shh protein. The down-regulation of Brn-3.0 expression in the dorsal spinal cord by the notochord and Shh contrasts with the known inductive effects of these ventral signals on the approximately simultaneous development of the spinal motor neurons. In cultured explants of neural plate from the region of the presumptive spinal cord, Brn-3.0 neurons develop in the absence of surface ectoderm and ventral midline tissue, suggesting that the Brn-3.0 phenotype may represent a "default" developmental pathway for early spinal cord neurons. Together these results advance the understanding of the mechanism of the generation of neuronal diversity in the developing vertebrate CNS (Fedtsova, 1997).

Brn-3.2 exhibits DNA binding properties similar to those of Brn-3.0, but its expression is uniquely regulated by retinoic acid in teratocarcinoma and neuroblastoma cells. In the developing PNS and retina, the expression pattern of Brn-3.2 is similar to that of Brn-3.0. In the caudal CNS (spinal cord, hindbrain, and midbrain) Brn-3.2 and Brn-3.0 are initially coexpressed, but diverge later in development. Rostral to the midbrain, Brn-3.2 and Brn-3.0 exhibit nonoverlapping patterns of expression, suggesting a divergence of gene function in more recently evolved structures. In the CNS Brn-3.2 is selectively expressed in postmitotic neurons, implying a role in specifying terminally differentiated neuronal phenotypes (Turner, 1994).

Brn3c closely resembles Brn3a and Brn3b in its POU-domain; this similarity helps define a family of Unc86-related mammalian POU factors. Both Brn3a and Brn3c are expressed only in the central and peripheral nervous systems. In the neonatal rat, northern blots reveal a 3.6 kb Brn3a transcript in DRG, spinal cord and hindbrain, and a 2.6 kb Brn3c transcript in DRG and spinal cord. In situ hybridization showed that most DRG neurons express Brn3a whereas only a small subset of neurons expresses Brn3c. In the spinal cord, Brn3a is expressed by many dorsal horn neurons. In contrast, Brn3c is expressed by a very small number of cells in laminae 4/5 of the dorsal horn. These data suggest that Brn3-related POU factors may be involved in the development or function of particular subclasses of sensory and spinal cord neurons (Ninkina, 1993).

The Brn-3a POU family transcription factor is expressed only in postmitotic neurons in the central nervous system and identifies the first differentiated neurons to appear in the midbrain, hindbrain, and spinal cord during development. This factor is also induced when undifferentiated proliferating ND7 cells cease dividing and differentiate to a mature neuronal-like phenotype bearing numerous neurite processes. Overexpression of Brn-3a in undifferentiated ND7 cells induces a mature neuronal phenotype characterized by process outgrowth and the induction of genes encoding synaptic proteins, although the cells continue to proliferate. In contrast, the closely related factors Brn-3b and Brn-3c do not have this effect. Although the N-terminal activation domain of Brn-3a is required for maximum induction of neurite outgrowth and gene expression, these effects are primarily dependent on the DNA binding POU domain, which also acts as an activation domain. Overexpression of the isolated POU domain of Brn-3a is sufficient to induce neurite outgrowth, while the ability of full-length Brn-3a to do so is abolished by mutating a single amino acid in the Brn-3a POU homeodomain to its equivalent in Brn-3b. Thus, Brn-3a appears to play a critical role in the specification of the mature neuronal phenotype, acting by stimulating the expression of genes whose products are required for process outgrowth and synapse formation (Smith, 1997a).

The POU domain transcription factor Brn-3a is able to stimulate neurite outgrowth when overexpressed in the neuronal ND7 cell line, whereas the closely related Brn-3b factor does not have this effect. Brn-3a overexpression also enhances the expression of the three neurofilament genes at both the mRNA and protein levels, whereas Brn-3b overexpression has no effect. In addition Brn-3a activates the three neurofilament gene promoters in co-transfection assays in both neuronal and non-neuronal cells. As observed for enhanced neurite outgrowth, the stimulation of neurofilament gene expression and activation of the neurofilament gene promoters is observed with the isolated POU domain of Brn-3a. A single amino acid change in the POU homeodomain of Brn-3a to the equivalent amino acid in Brn-3b abolishes its ability to activate the neurofilament promoters, whereas the reciprocal change converts Brn-3b to an activator of these promoters (Smith, 1997b).

The POU-IV or Brn-3 class of transcription factors exhibit conserved structure, DNA-binding properties, and expression in specific subclasses of neurons across widely diverged species. In the mouse CNS, Brn-3.0 expression characterizes specific neurons from neurogenesis through the life of the cell. This irreversible activation of expression suggests positive autoregulation. To search for cis-acting elements that could mediate autoregulation, a novel method, complex stability screening, was used. It was applied to identify functional Brn-3.0 recognition sites within a large genomic region encompassing the mouse brn-3.0 locus. This method is based on the observation that the kinetic stability of Brn-3.0 complexes with specific DNA sequences, as measured by their dissociation half-lives, is highly correlated with the ability of those sequences to mediate transcriptional activation by Brn-3.0. The principal Brn-3.0 autoregulatory region lies ~5 kb upstream from the Brn-3.0 transcription start site and contains multiple Brn-3.0-binding sites that strongly resemble the optimal binding site for this protein class. This region also mediates transactivation by the closely related protein Brn-3.2, suggesting a regulatory cascade of POU proteins in specific neurons in which Brn-3.2 expression precedes Brn-3.0 (Trieu, 1999).

The estrogen receptor (ER) modulates transcription by forming complexes with other proteins and then binding to the estrogen response element (ERE). An interaction of this receptor with the POU transcription factors Brn-3a and Brn-3b has been identified which is independent of ligand binding. The POU domain of Brn-3a and Brn-3b interacts with the DNA-binding domain of the ER. Brn-3-ER interactions also affect transcriptional activity of an ERE-containing promoter, such that in estradiol-stimulated cells, Brn-3b strongly activates the promoter via the ERE, while Brn-3a has a mild inhibitory effect. The POU domain of Brn-3b, which interacts with the ER is sufficient to confer this activation potential; the change of a single amino acid in the first helix of the POU homeodomain of Brn-3a to its equivalent in Brn-3b can change the mild repressive effect of Brn-3a to a stimulatory Brn-3b-like effect. While Brn-3a and Brn-3b may not form a stable complex with the ERE on their own, interaction with the ER may facilitate binding of the complex to the ERE by helping induce changes in the coformation of a labile ERE structure or by helping stabilize a secondary structure, which the single stranded ERE is thought to form to allow the ER complex to bind more readily (Budhram-Mahadeo, 1998)

Brn3a/Brn-3.0 is a POU-domain transcription factor expressed in primary sensory neurons of the cranial and dorsal root ganglia and in specific neurons in the caudal CNS. Mice lacking Brn3a undergo extensive sensory neural death late in gestation and die at birth. To further examine Brn3a expression and the abnormalities that accompany its absence, a transgene was constructed containing 11 kb of Brn3a upstream regulatory sequence linked to a LacZ reporter. These regulatory sequences direct transgene expression specifically to Brn3a peripheral sensory neurons of the cranial and dorsal root ganglia. Furthermore, expression of the 11 kb/LacZ reporter in the sensory neurons of the mesencephalic trigeminal, but not other Brn3a midbrain neurons, demonstrates that cell-specific transgene expression is targeted to a functional class of neurons rather than to an anatomical region. The 11 kb/LacZ reporter strain was interbred with mice carrying a null mutant allele of Brn3a to generate 11 kb/LacZ, Brn3a knock-out mice. ß-Galactosidase expression in these mice reveals significant axonal growth defects, including excessive and premature branching of the major divisions of the trigeminal nerve and a failure to correctly innervate whisker follicles, all of which precede sensory neural death in these mice. These defects in Brn3a-/- mice resemble strongly those seen in mice lacking the mediators of sensory pathfinding semaphorin 3A and neuropilin-1. It is shown, however, that sensory neurons are able to express neuropilin-1 in the absence of Brn3a (Eng, 2001).

Mammalian POU domain class IV homologs and midbrain development

The vertebrate midbrain consists of dorsal and ventral domains, the tectum and tegmentum, which execute remarkably different developmental programs. Tectal development is characterized by radial migration of differentiating neurons to form a laminar structure, while the tegmentum generates functionally diverse nuclei at characteristic positions along the neural axis. Neurons appearing early in the development of the tectum are characterized either by the expression of the POU-domain transcription factor Brn3.0, or by members of the Pax and LIM families. Early neurons of the rostral tegmentum co-express Brn3.0 and Lim1/2, and caudal tegmental neurons express Islet1/2. Notochord tissue or Shh-transfected epithelial cells, transplanted to the developing tectum, suppress the development of tectal neurons, and induce the differentiation of multiple tegmental cell types. The distance from the midbrain-hindbrain boundary (MHB) determines the specific markers expressed by the tegmental neurons induced in the tectum, and the transplantation of MHB tissue adjacent to the rostral tegmentum also induces caudal markers, demonstrating the role of MHB signals in determining the phenotype of these early midbrain neurons. Co-culture of isolated midbrain neuroepithelium with Shh-expressing cells demonstrates that Shh is sufficient to convert tectal neurons to a tegmental fate. In mice lacking Shh, Brn3.0- and Pax7-expressing neurons typical of the tectum develop throughout the ventral midbrain, and gene expression patterns characteristic of early tegmental development do not appear (Fedtsova, 2001).

The paired domain transcription factor Pax2 is required for the formation of the isthmic organizer (IsO) at the midbrain-hindbrain boundary, where it initiates expression of the IsO signal Fgf8. To gain further insight into the role of Pax2 in mid-hindbrain patterning, novel Pax2-regulated genes were sought by cDNA microarray analysis of FACS-sorted GFP⁺ mid-hindbrain cells from wild-type and Pax2^–/– embryos carrying a Pax2^GFP BAC transgene. Five genes have been identified that depend on Pax2 function for their expression in the mid-hindbrain boundary region. These genes code for the transcription factors En2 and Brn1 (Pou3f3), the intracellular signaling modifiers Sef and Tapp1, and the non-coding RNA Ncrms. The Brn1 gene was further identified as a direct target of Pax2; two functional Pax2-binding sites in the promoter and in an upstream regulatory element of Brn1 are essential for lacZ transgene expression at the mid-hindbrain boundary. Moreover, ectopic expression of a dominant-negative Brn1 protein in chick embryos implicates Brn1 in Fgf8 gene regulation. Together, these data defined novel functions of Pax2 in the establishment of distinct transcriptional programs and in the control of intracellular signaling during mid-hindbrain development (Bouchard, 2005).

The rostral part of the dorsal midbrain, known as the superior colliculus in mammals or the optic tectum in birds, receives a substantial retinal input and plays a diverse and important role in sensorimotor integration. However, little is known about the development of specific subtypes of neurons in the tectum, particularly those that contribute tectofugal projections to the thalamus, isthmic region, and hindbrain. This study shows that two homeodomain transcription factors, Brn3a and Pax7, are expressed in mutually exclusive patterns in the developing and mature avian midbrain. Neurons expressing these factors are generated at characteristic developmental times, and have specific laminar fates within the tectum. In mice expressing βgalactosidase targeted to the Pou4f1 (Brn3a) locus, Brn3a-expressing neurons contribute to the ipsilateral but not the contralateral tectofugal projections to the hindbrain. Using misexpression of Brn3a and Pax7 by electroporation in the chick tectum, combined with GFP reporters, it was shown that Brn3a determines the laminar fate of subsets of tectal neurons. Furthermore, Brn3a regulates the development of neurons contributing to specific ascending and descending tectofugal pathways, while Pax7 globally represses the development of tectofugal projections to nearly all brain structures (Fedtsova, 2008).

Brn3 POU-domain transcription factors and the inner ear

The Brn-3 subfamily of POU-domain transcription factor genes consists of three highly homologous members (Brn-3a, Brn-3b, and Brn-3c) that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells; the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes play distinctive roles in the somatosensory, visual, and auditory/vestibular systems (Xiang, 1997).

The inner ear is a complex sensory organ responsible for balance and sound detection in vertebrates. It originates from a transient embryonic structure, the otic vesicle, which contains all of the information to develop autonomously into the mature inner ear. The development of the otic vesicle is reviewed here, bringing together classical embryological experiments and recent genetic and molecular data. The specification of the prospective ectoderm and its commitment to the otic fate are very early events and can be related to the expression of genes with restricted expression domains. A combinatorial gene expression model for placode specification and diversification, based on classical embryological evidence and gene expression patterns, is discussed. The formation of the otic vesicle is dependent on inducing signals from endoderm, mesoderm and neuroectoderm. Ear induction consists of a sequence of discrete instructions from those tissues that confer the final identity on the otic field, rather than a single all-or-none process. The head ectoderm develops three pairs of sensory placodes from anterior to posterior -- nose, lens and ear -- along with those placodes that generate the neurons of some cranial sensory ganglia. Homeobox genes of the Sine oculis (six) and Distal-less related Dlx families are expressed but these factors are expressed in more than one sensory placode. Specific combinations of genes rather than single gene expression thus appears to be characteristic for each placode. The important role of the neural tube in otic development is highlighted by the abnormalities observed in mouse mutants for the Hoxa1, kreisler and fgf3 genes and those reported in retinoic acid-deficient quail. Still, the nature of the relation between the neural tube and otic development remains unclear. Gene targeting experiments in the mouse have provided evidence for genes potentially involved in regional and cell-fate specification in the inner ear. The disruption of the mouse Brn3.1 gene identifies the first mutation affecting sensory hair-cell specification; mutants for the Pax2 and Nkx5.1 genes show that these two genes are required for the the development of specific regions of the otic vesicle. Several growth-factors contribute to the patterned cell proliferation of the otic vesicle. Among these, IGF-I and FGF-2 are expressed in the otic vesicle and may act in an autocrine manner. Little is known about early mechanisms involved in guiding ear innervation. However, targeted disruption of genes coding for neurotrophins and Trk receptors have shown that once synaptic contacts are established, they depend on specific trophic interactions that involve these two gene families. The accessibility of new cellular and molecular approaches are opening new perspectives in vertebrate developmental biology (Torres, 1998).

Targeted mutagenesis in mice demonstrates that the POU-domain gene Brn4/Pou3f4 plays a crucial role in the patterning of the mesenchymal compartment of the inner ear. Brn4 is expressed extensively throughout the condensing mesenchyme of the developing inner ear. Mutant animals display behavioral anomalies that result from functional deficits in both the auditory and vestibular systems, including vertical head bobbing, changes in gait, and hearing loss. Anatomical analyses of the temporal bone, which is derived in part from the otic mesenchyme, demonstrate several dysplastic features in the mutant animals, including enlargement of the internal auditory meatus. Many phenotypic features of the mutant animals result from the reduction or thinning of the bony compartment of the inner ear. Histological analyses demonstrated a hypoplasia of those regions of the cochlea derived from otic mesenchyme, including the spiral limbus, the scala tympani, and strial fibrocytes. A reduction in the coiling of the cochlea was observed, which suggests that Brn-4 plays a role in the epithelial-mesenchymal communication necessary for the cochlear anlage to develop correctly. Finally, the stapes demonstrates several malformations, including changes in the size and morphology of its footplate. Because the stapes anlage does not express the Brn4 gene, stapes malformations suggest that the Brn4 gene also plays a role in mesenchymal-mesenchymal signaling. On the basis of these data, it is suggested that Brn-4 enhances the survival of mesodermal cells during the mesenchymal remodeling, which forms the mature bony labyrinth and regulates inductive signaling mechanisms in the otic mesenchyme (Phippard, 1999).

Mutations in the POU domain gene Brn-3c cause hearing impairment in both the human and mouse as a result of inner ear hair cell loss. During murine embryogenesis, Brn-3c is expressed in postmitotic cells committed to hair cell phenotype but not in mitotic progenitors in the inner ear sensory epithelium. In developing auditory and vestibular sensory epithelia of Brn-3c-/- mice, hair cells are generated and undergo initial differentiation as indicated by their morphology, laminar position and expression of hair cell markers, including myosins VI and VIIa, calretinin and parvalbumin. However, a small number of hair cells are anomalously retained in the supporting cell layer in the vestibular sensory epithelia. Furthermore, the initially differentiated hair cells fail to form stereociliary bundles and degenerate by apoptosis in the Brn-3c-/- mice. These data indicate a crucial role for Brn-3c in maturation, survival and migration of hair cells, but not in proliferation or commitment of hair cell progenitors (Xiang, 1998b).

Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival

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. This study reports 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-x_L. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-x_L, 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 (Wiggins, 2004).

Brn3 POU-domain transcription factors in mouse retinal neurogenesis

In mammalian retinal differentiation, the POU domain factor Brn-3b is required for specific sets of retinal ganglion cells. Brn-3b is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer. Brn-3b knockouts show a reduction in the number of nuclei in each of the retinal layers: 30% in the ganglion cell layer, 15% in the inner nuclear layer, and 10% in the outer nuclear layer. The lack of cell death in the retinas of knockout mice suggests that in the absence of Brn-3b, the affected ganglion cell precursors fail to appear during development. It is thought that the modest reduction in cell number in the inner and outer nuclear layers of the mature retina may follow as a secondary effect of reduced ganglion cell number (Gan, 1996).

The selective spatial and temporal expression patterns exhibited in distinct sensorineural cells by Brn-3.1 and Brn-3.2, critically modulates terminal differentiation. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia (Erkman, 1996).

The Brn-3 subfamily of POU domain transcription factors consists of Brn-3a, -3b, and -3c, which are important regulators for sensorineural development. Despite the expression of all three factors in retinal ganglion cells, earlier studies have shown that Brn-3b is the only one among the three Brn-3 genes that is essential for development of approximately 70% of ganglion cells in the murine retina. Brn-3b displays a spatiotemporal expression pattern characteristic of the dynamic profile of ganglion cell genesis during murine retinal development. It is initially turned on in postmitotic ganglion cell precursors 2 days before the onset of Brn-3a and -3c expression in differentiated ganglion cells. During the entire period of retinal ganglion cell genesis, the postmitotic ganglion cell precursors that would normally become Brn-3b+ cells fail to properly differentiate in Brn-3b-/- mice, as evidenced by a twofold reduction in the optic nerve size and diminished expression of several ganglion cell markers. The undifferentiated ganglion cell precursors appear to be degenerated by apoptosis within the ganglion cell layer during the perinatal and early postnatal period. It is proposed that retinal ganglion cells develop following two separate differentiation pathways: Brn-3b dependent and Brn-3b independent. In the Brn-3b-dependent mechanism, Brn-3b may be required to initiate a particular differentiation program, thus enabling a large set of postmitotic ganglion precursors to properly differentiate into the 70%, Brn-3b-dependent retinal ganglion cells (Xiang, 1998a).

While the mammalian retina is well understood at the anatomical and physiological levels, little is known about the mechanisms that give rise to the retina's highly ordered pattern or its diverse neuronal cell types. Previous investigations have shown that gene disruption of the POU-IV class transcription factor Brn-3b (Brn-3.2) results in the loss of most retinal ganglion cells in retinas of postnatal mice. lacZ and human placental alkaline phosphatase genes knocked into the brn-3b locus were used to follow the fate of brn-3b-mutant cells in the developing retina. Brn-3b is not required for the initial commitment of retinal ganglion cell fate or for the migration of ganglion cells to the ganglion cell layer. However, Brn-3b is essential for the normal differentiation of retinal ganglion cells; without it, a subset of cells undergo enhanced apoptosis. Retinal ganglion cells lacking brn-3b extend processes at the appropriate time in development, but these processes are disorganized, resulting in a thinner optic nerve. Explanted retinas from brn-3b-null embryos also extend processes when cultured in vitro, but the processes are shorter and less bundled than in wild-type retinas. Ultrastructural and marker analyses show that the processes of mutant ganglion cells have dendritic rather than axonal features, suggesting that mutant cells form dendrites in place of axons. These results suggest that Brn-3b regulates the activity of genes whose products play essential roles in the formation of retinal ganglion cell axons (Gan, 1999).

math5 is a murine ortholog of atonal, a bHLH proneural gene essential for the formation of photoreceptors and chordotonal organs in Drosophila. The expression of math5 coincides with the onset of retinal ganglion cell (RGC) differentiation. Targeted deletion of math5 blocks the initial differentiation of 80% of RGCs and results in an increase in differentiated amacrine cells. Furthermore, the absence of math5 abolishes the retinal expression of brn-3b and the formation of virtually all brn-3b-expressing RGCs. These results imply that math5 is a proneural gene essential for RGC differentiation and that math5 acts upstream to activate brn-3b-dependent differentiation processes in RGCs (Wang, 2001).

The Class IV POU domain-containing brn-3b gene is required for the terminal differentiation of RGCs and null mutations in brn-3b result in axon growth defects and programmed cell death in ~70% of newly formed RGCs. The loss of RGCs observed in the math5-deficient mice resembles that of brn-3b-null mice. To test whether math5 and brn-3b are involved in the differentiation of the same population of RGCs, mice were generated with compound mutations in math5 (math5-GFP) and brn-3b (brn-3b-lacZ). Nuclear expression of lacZ in brn-3b-lacZ mice serves to effectively mark brn-3b-positive RGCs. Compared with normal retinas (brn-3b-lacZ/+, math5+/+), math5-null retinas (brn-3b-lacZ/+, math5-GFP/math5-GFP) show a loss of ~97% of brn-3b-lacZ-expressing cells, thus demonstrating that math5 is required for the differentiation of most brn-3b-expressing RGCs. The remaining RGCs in math5-deficient retina appear to be expressing brn-3b but are generated by a math5-independent pathway (Wang, 2001).

The absence of 80% of RGCs in mature math5-deficient retinas could be the result of an insufficient number of RGC progenitors or a failure of RGC differentiation or survival. Because a similar number of math5-lacZ-expressing cells are detected in heterozygous and homologous math5-mutant retinas between E11 and E15.5, it is unlikely that there are an insufficient number of progenitor cells. TUNEL analysis also reveals no overt difference in apoptosis in retinas at E12.5-E19 in math5-deficient and wild-type embryos. A reasonable hypothesis is that math5-deficient retinas are unable to form RGCs. To test whether math5-null retinal progenitor cells fail to differentiate into RGCs, the presence of newly differentiated RGCs was examined by detecting the expression of the early RGC markers, brn-3b and p75, the latter of which encodes a receptor for nerve growth factor (NGF) and neurotrophins. Section in situ hybridization with a brn-3b antisense probe and expression of the brn-3b-lacZ allele at E13.5 shows that brn-3b is highly expressed in wild-type retinas but is virtually absent in homozygous math5-null retinas. Similarly, immunostaining of E13.5 retinas with anti-p75 showed that the expression of p75 in math5-deficient retina is severely reduced. The absence of most RGCs in mature math5-deficient retinas, and the lack of brn-3b and p75 expression in math5-deficient embryonic retinas, argues strongly that in math5-null retinas, retinal progenitor cells are unable to differentiate into RGCs. Because brn-3b expression is essentially absent in math5-null retinas, the results also demonstrate that math5 is genetically upstream of brn-3b (Wang, 2001).

In mice, Brn3 POU domain transcription factors play essential roles in the differentiation and survival of projection neurons within the retina, inner ear, dorsal root and trigeminal ganglia. During retinal ganglion cell differentiation, Brn3b is expressed first, followed by Brn3a and Brn3c. Targeted deletion of Brn3b, but not Brn3a or Brn3c, leads to a loss of most retinal ganglion cells before birth. However, as a few retinal ganglion cells are still present in Brn3b^-/- mice, Brn3a and Brn3c may partially compensate for the loss of Brn3b. To examine the role of Brn3c in retinal ganglion cell development, Brn3b/Brn3c double knockout mice were generated and their retinas and optic chiasms were examined. Retinal ganglion cell axons from double knockout mice are more severely affected than are those from Brn3b-deficient mice, indicating that Brn3c is required for retinal ganglion cell differentiation and can partially compensate for the loss of Brn3b. Moreover, Brn3c had functions in retinal ganglion cell differentiation separate from those of Brn3b. Ipsilateral and misrouted projections at the optic chiasm are overproduced in Brn3b^-/- mice but are missing entirely in optic chiasms of Brn3b/Brn3c double knockout mice, suggesting that Brn3c controls ipsilateral axon production. Forced expression of Brn3c in Brn3b^-/- retinal explants restores neurite outgrowth, demonstrating that Brn3c can promote axon outgrowth in the absence of Brn3b. These results reveal a complex genetic relationship between Brn3b and Brn3c in regulating the retinal ganglion cell axon outgrowth (Wang, 2002).

POU-domain transcription factors play essential roles in cell proliferation and differentiation. Previous studies have shown that targeted deletion of each of the three POU-domain Brn3 factors in mice leads to the developmental failure and apoptosis of a unique set of sensory neurons in retina, dorsal root ganglia, trigeminal ganglia and inner ear. The specific defects associated with the removal of each Brn3 gene closely reflect their characteristic spatiotemporal expression patterns. Nevertheless, it remains elusive whether Brn3 factors are functionally equivalent and act through a common molecular mechanism to regulate the development and survival of these sensory neurons. By knocking-in Brn3a (Brn3a^ki) into the Brn3b locus, it is shown that Brn3a^ki is expressed in a spatiotemporal manner identical to that of endogenous Brn3b. In addition, Brn3a^ki functionally restores the normal development and survival of retinal ganglion cells (RGCs) in the absence of Brn3b and fully reinstates the early developmental expression profiles of Brn3b downstream target genes in retina. These results indicate that Brn3 factors are functionally equal and that their unique roles in neurogenesis are determined by the distinctive Brn3 spatiotemporal expression patterns (Pan, 2005).

Brn3b probably executes its roles in RGCs by regulating the expression of its downstream target genes. Many Brn3b downstream target genes have been identified by representational difference analysis, cDNA microarrays and in situ hybridization screening. They include the transcription factor genes Brn3a, Irx4 and Irx6 (homeodomain), Ablim (LIM-domain), Gfi1 and Gli1 (zinc-finger), Isl2 (LIM-homeodomain), Olf1 (bHLH), and Dlx1 and Dlx2 (homeodomain). Some of these factors have known or postulated roles in retinal development. Ablim and Irx4 have previously been reported to regulate RGC axon pathfinding. Shh is essential for the formation of the optic disc and the optic nerve. Gap43 and L1 NCAM are associated with RGC axon guidance (Pan, 2005).

To test whether Brn3a and Brn3b share the identical transcription activities, as well as to determine whether Brn3a^ki rescues the RGC defects by restoring the RGC expression of Brn3b downstream target genes, the expression of these genes was compared in E14.5 retinal sections of wild-type, Brn3b^lacZ/AP and Brn3b^3a/3a embryos. The control Brn3b probe confirmed the absence of Brn3b expression in Brn3b^lacZ/AP and Brn3b^3a/3a retinas. Brn3a ORF probe detected the reduced expression of endogenous Brn3a in Brn3b^lacZ/AP retina and Brn3b-like expression pattern of Brn3a^ki in Brn3b^3a/3a retina. Compared with wild-type controls, loss of Brn3b in Brn3b^lacZ/AP mice resulted in the downregulation of Brn3a, Irx2, Irx6, Ablim, Gfi1, Gli1, Isl2, Olf1, L1, Gap43, Shh and Hermes (Rbpms -- Mouse Genome Informatics) and the upregulation of Dlx1 and Dlx2 expression. When retinas from Brn3b^3a/3a knock-in mice were examined, the expression levels of all of above genes were restored to those observed in wild-type retinas. The expression analyses demonstrate the identical ability for Brn3a and Brn3b to activate or suppress in RGCs the expression of Brn3b downstream genes and support the notion that rescue of Brn3b-null phenotypes by Brn3a^ki is achieved by restoring the expression of Brn3b downstream genes. Furthermore, Brn3a^ki expression in Brn3b^3a/3a retina lead to the activation of endogenous Brn3a expression , implying the presence of positive feedback regulation of Brn3a expression in the developing retina (Pan, 2005).

Regulation of Brn3b by Dlx1 and Dlx2 is required for retinal ganglion cell differentiation in the vertebrate retina

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix loop helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. It was hypothesized that Dlx1/Dlx2 (see Drosophila Distalless) and Brn3b (see Drosophila Acj6) homeobox genes function in parallel intrinsic pathways to determine RGC fate, and Dlx1/Dlx2/Brn3b triple knockout mice were generated. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b null retinas than predicted by combining features of the Brn3b single and Dlx1/Dlx2 double knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, it was discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain-of-function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo. It is suggested that Atoh7 (see Drosophila Atonal) specifies RGC committed progenitors and that Dlx1/Dlx2 functions both downstream of Atoh7 and in parallel but cooperative pathways involving regulation of Brn3b expression to determine RGC fate (Zhang, 2017).

Brn3a and developing sensory ganglia

Brn-3a protein is first detected in a subset of migrating cranial and trunk neural crest cells as early as E9-E9.5. As the development of sensory ganglia progresses, the number of Brn-3a-immunoreactive cells increases and Brn-3a is observed in both proliferating cells and differentiating neurons. By stages E10.5 and E11.5, the trigeminal (V), vestibulococchlear (VIII) and dorsal root ganglia (DRG) show intense Brn-3a expression, whereas the geniculate (VII) and nodose-petrosal ganglia of the cranial ganglia IX-X complex show no evidence of Brn-3a expression. The superior-jugular ganglia of the IX-X complex, however, do contain a small number of Brn-3a- immunoreactive cells. The expression pattern of Brn-3a and the phenotype of mice lacking Brn-3a indicate an important role for Brn-3a in sensory ganglia development. Because Brn-3a is present in proliferating precursors, it seemed possible that the absence of Brn-3a might affect the precursors and consequently ganglion development prior to neurogenesis. To test this hypothesis, the number of BrdU-incorporating precursors in trigeminal ganglia was assessed at E10.5 and E11.5. No differences were found between wild-type and mutant ganglia in the number of proliferating precursors that incorporated BrdU during a 2 hour pulse. To assess the effect of the Brn-3a mutation on the neuronal population, the morphologies of wild-type and mutant ganglia at various stages were assessed using a Cresyl violet (Nissl) stain. Interestingly, although the morphologies of cells in the mutant ganglia do not appear to differ from those in wild-type ganglia at E11.5 and E12.5, the E11.5 mutant ganglia show fewer apoptotic profiles. Unlike the wild-type ganglia, the mutant ganglia show a sharp increase in apoptotic cell death at E15.5, which continues through E17.5 to P0 (Huang, 1999).

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 the 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 in Brn-3a mutants survive to birth. Approximately 70% of these surviving neurons 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. These 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 (Huang, 1999).

In conclusion, this analysis of the Brn-3a mutant phenotype in the trigeminal ganglia indicates that Brn-3a is required for the induction of TrkC expression and for the maintenance of TrkA and TrkB expression. Because of this, loss of Brn-3a leads to two distinct phases of neuronal loss, one associated with the induction of TrcC expression (lack of TrcC expression leads to apoptotic cell death of neurons that would have been TrcC positive), and the second associated with the maintenance of TrkA and TrkB expression (the major wave of cell death in the trigeminal ganglia of Brn-3a mutants occurs at E15.5, after TrkA and TrkB receptors have been reduced below the critical level required to maintain neuronal survival). Absence of Brn-3a does not reduce the expression of p75 NTR, c-ret or parvalbumin, indicating that Brn-3a acts specifically to promote expression of the Trk receptors, but does not affect expression of several other differentiation markers (Huang, 1999).

Brn3a is a POU-domain transcription factor expressed in peripheral sensory neurons and in specific interneurons of the caudal CNS. Sensory expression of Brn3a is regulated by a specific upstream enhancer, the activity of which is greatly increased in Brn3a knockout mice, implying that Brn3a negatively regulates its own expression. Brn3a binds to highly conserved sites within this enhancer, and alteration of these sites abolishes Brn3a regulation of reporter transgenes. Furthermore, endogenous Brn3a expression levels in the sensory ganglia of Brn3a^+/+ and Brn3a^+/- mice are similar, demonstrating that autoregulation can compensate for the loss of one allele by increasing transcription of the remaining gene copy. Conversely, transgenic overexpression of Brn3a in the trigeminal ganglion suppresses the expression of the endogenous gene. These findings demonstrate that the Brn3a locus functions as a self-regulating unit to maintain a constant expression level of this key regulator of neural development (Trieu, 2003).

Negative autoregulation by Brn3a raises the possibility that the general function of this factor will be to repress the transcription of all of its direct targets, at least in the developing sensory system. In the developing spinal cord, where the molecular mechanisms of neural specification are best understood, several homeodomain proteins that characterize progenitor cell domains in the spinal neuroepithelium act as transcriptional repressors. These repressive activities prevent the inappropriate expression of transcription factors that characterize adjacent domains, and refine the boundaries between their domains. By contrast, examples of direct positive regulation of downstream target genes by neural transcription factors are relatively few, and it appears increasingly likely that neurons are transcriptionally defined to a large extent by the repression of inappropriate gene expression (Trieu, 2003).

The role of Brn3a as a negative regulator of its own expression in vivo is somewhat surprising given that it strongly activates transcription when co-transfected with reporter plasmids in cultured epithelial cells. This difference cannot be accounted for by the Brn3a recognition elements used or their immediate context, because in transfection assays Brn3a strongly activates transcription from reporters containing the same 200 base pair autoregulatory region that confers negative regulation in vivo. Although it is possible that this reversal of transcriptional effect depends on the broader context of the cis-acting sequences, it seems more likely that sensory neurons express a co-repressor that converts Brn3a from a positive to a negative regulator of transcription, or that dividing epithelial cells express an essential Brn3a co-activator (Trieu, 2003).

The TrkA/NGF receptor is essential for the survival and differentiation of sensory neurons. The molecular mechanisms regulating tissue and stage-specific expression of TrkA are largely unknown. The Brn3a POU-domain transcription factor has been implicated in the development of the PNS and proposed as a transcription regulator for TrkA. The molecular mechanisms underlying the regulation of TrkA by Brn3a is unclear. In this study, genetic, transgenic and biochemical evidence is provided that Brn3a binds to novel, specific sites in the 457 bp enhancer that regulates TrkA expression in embryonic sensory neurons. Bax-knockout mice, in which sensory neurons no longer require neurotrophins for survival, have been employed to uncouple TrkA-dependent cell death from downregulation of TrkA expression. In addition, when mutagenized, the novel Brn3a-binding sites identified fail to drive appropriate reporter transgene expression in sensory neurons. Thus, TrkA, a gene that is crucial for the differentiation and survival of sensory nociceptive neurons, requires Brn3a to maintain normal transcriptional activity (Ma, 2003).

Mice lacking the POU-domain transcription factor Brn3a exhibit marked defects in sensory axon growth and abnormal sensory apoptosis. The regulatory targets of Brn3a in the developing trigeminal ganglion have been determined using microarray analysis of Brn3a mutant mice. These results show that Brn3 mediates the coordinated expression of neurotransmitter systems, ion channels, structural components of axons and inter- and intra-cellular signaling systems. Loss of Brn3a also results in the ectopic expression of transcription factors normally detected in earlier developmental stages and in other areas of the nervous system. Target gene expression is normal in heterozygous mice, consistent with prior work showing that autoregulation by Brn3a results in gene dosage compensation. Detailed examination of the expression of several of these downstream genes reveals that the regulatory role of Brn3a in the trigeminal ganglion appears to be conserved in more posterior sensory ganglia but not in the CNS neurons that express this factor (Eng, 2004).

The array results clearly demonstrate that Brn3a has a major role in determining the neurotransmitter phenotype of the developing trigeminal ganglia. Expression of the neuropeptides PACAP and galanin and the NPY1 receptor are highly dependent on Brn3a, and the rate-limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, is also significantly reduced in Brn3a knockouts. In contrast, the expression of somatostatin and the 5HT3A receptor are markedly increased. Studies in the developing rat have shown that somatostatin is strongly expressed throughout the sensory ganglia soon after neurogenesis, but by mid-gestation its expression is restricted to a relatively small subset of sensory neurons. Thus the increased expression of somatostatin at E13.5 is very likely to represent a failure in the normal developmental attenuation of this gene, consistent with the idea that Brn3a knockout ganglia exhibit a pervasive maturation defect (Eng, 2004).

Also notable are changes in the expression of sodium channels, including Scn6 and Scn9, which are markedly decreased in Brn3a knockout ganglia, and Scn10, which is moderately decreased. Remarkably, these changes affect only those sodium channels that appear to have specific expression in the sensory nervous system, suggesting that Brn3a directly or indirectly coordinates expression of these channels. In contrast, expression levels of most neurotransmitter receptors, such as the GABA and glutamate receptors, and several classes of ion channels with wide expression in the CNS and PNS, are unchanged. Two other markedly changed genes, calretinin and the regulator of G-protein signaling RGS10, have putative roles in the modulation of neurotransmitter signals mediated by Ca²⁺-dependent and G-protein pathways, respectively. Altered expression of these genes may represent primary changes, or they may occur in an attempt to compensate homeostatically for other changes in neurotransmitter systems (Eng, 2004).

Mice lacking Brn3a have marked defects in sensory axon growth, including defasciculation of axon bundles and failure to innervate peripheral and central targets. The transcripts for several proteins known to be involved in axon growth and synaptogenesis were significantly decreased in Brn3a null mice. Among the proteins in this category is advillin (pervin), an actin-binding protein with specific expression in sensory and sympathetic ganglia, which increases neurite outgrowth in cultured dorsal root ganglia. Apolipoprotein E knockout mice exhibit anatomical and functional defects in unmyelinated nerve fibers. Although this has been attributed to loss of ApoE expressed in associated glia, these results suggest that the defect may be intrinsic to sensory neurons (Eng, 2004).

Also decreased in Brn3a knockout ganglia were transcripts for the functionally interrelated proteins insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 5 (IGFBP5). Mice lacking IGF1 have abnormalities in sensory neurons, and show defective cortical dendritic growth. IGFBP5 is a widely expressed protein whose role in vivo has not been clearly defined. However, it is highly expressed in the axon terminals of developing sensory neurons, where it is frequently co-localized with IGF1, suggesting that it also has a role in axon growth. Because these proteins are known to interact, relative deficiencies in their expression may have a synergistic effect (Eng, 2004).

Another group of Brn3a-regulated proteins likely to have a role in axon growth are those involved in cell signaling and intracellular signal transduction. Transcripts with significantly changed expression include N-chimaerin, downstream of tyrosine kinase 4 (Dok4), the low affinity neurotrophin receptor p75, the small GTPases RAP (Ras family) and WRCH1 (Rho family), and Dusp6/MKP3. The expression and potential role of some of these factors in the sensory nervous system has been described; in other cases, the function of related proteins suggest that they may have significant and synergistic effects on axon growth (Eng, 2004).

Loss of Brn3a results in profound changes in the expression of several transcriptional regulators of various types, suggesting a web of cross-regulation between genes involved in sensory neurogenesis. The expression of a few transcription factors expressed late in sensory development, such as Runx1, were decreased in the absence of Brn3a, but the majority of the changes were increases, suggesting that Brn3a functions as a repressor of transcription factors that would be temporally or spatially inappropriate in the maturing trigeminal ganglion (Eng, 2004).

The clearest example of the role of Brn3a in restricting the spatial expression of other transcription factors is the ectopic expression of GATA3, Irx1, Irx2, NeuroD1 and MyoR/musculin in Brn3a knockout mice. These factors are all expressed in the developing vestibulocochlear ganglion in control embryos, and in the absence of Brn3a are markedly increased in the trigeminal and IX/X ganglia, demonstrating an expansion of the expression domain of these genes in both directions of the rostrocaudal axis. It is likely that some of the downstream changes in gene expression in Brn3a knockout ganglia are mediated by these factors, but current knowledge of their role in neural development is not sufficient to predict the effect of their mis-expression in the trigeminal ganglion. GATA3 has a known role in the development of motor neurons in the inner ear and motor neurons originating in rhombomere 4, which innervate the inner ear. NeuroD1 is also required for normal development of the sensory neurons of the inner ear, and may have a cross-regulatory relationship with GATA3. Although the role of the Irx genes in sensory development has not been described in mice, the zebrafish protein iro7, a possible paralogue of Irx1, is required for trigeminal placode development in fish. The bHLH factor MyoR (musculin) is normally expressed in the developing facial muscles of the first branchial arch, which are innervated by trigeminal neurons, but not in the trigeminal ganglion itself. The observation that MyoR is expressed in the developing auditory system is the first report of the sensory expression of this gene, and its role in neurogenesis is unknown (Eng, 2004).

Although it was not detected in the vestibulocochlear ganglion at this stage, AP2ß shows a similar pattern of ectopic expression in the trigeminal and IX cranial ganglia in E13.5 Brn3a knockout embryos. AP2ß is normally expressed in the embryonic hindbrain and spinal cord, but little is known about its role in neural development. The nervous system of AP2ß mutant mice, which die from polycystic kidney disease, has no obvious abnormalities. However, mice lacking the related factor AP2alpha, which is highly expressed in migrating neural crest and in the developing sensory ganglia, exhibit extensive cranial abnormalities and dysgenesis of the cranial ganglia. There is some evidence that AP2ß is a weak transcriptional activator, and may oppose gene activation by AP2alpha. Thus the increased expression of AP2ß observed in this study may mimic some aspects of the loss of AP2alpha (Eng, 2004).

The increased expression of Math3 and NeuroD1 in Brn3a knockout trigeminal ganglia, together with decreased expression of the inhibitor of bHLH function Id1, suggest a marked increase in bHLH activity in the absence of Brn3a. Math3 and NeuroD1 have been characterized in the early development of the trigeminal ganglion (E9.0), where both factors appear to be downstream of the neurogenic HLH factor Ngn1. Thus the increased expression of bHLH factors in Brn3a knockout mice may reflect the abnormal persistence of genes normally down-regulated as sensory development progresses. Although the loss of NeuroD1 or Math3 alone does not have an obvious effect on neurogenesis in the trigeminal, the increased expression of multiple bHLH genes may have a synergistic effect in Brn3a knockout mice (Eng, 2004).

Brn3a target gene recognition in embryonic sensory neurons

Numerous transcription factors have been identified that have profound effects on developing neurons. A fundamental problem is to identify genes downstream of these factors and order them in developmental pathways. Eighty-five genes have been identified with changed expression in the trigeminal ganglia of mice lacking Brn3a, a transcription factor encoded by the Pou4f1 gene. This study used locus-wide chromatin immunoprecipitation in embryonic trigeminal neurons to show that Brn3a is a direct repressor of two of these downstream genes, NeuroD1 and NeuroD4, and also directly modulates its own expression. Comparison of Brn3a binding to the Pou4f1 locus in vitro and in vivo reveals that not all high affinity sites are occupied, and several Brn3a binding sites identified in the promoters of genes that are silent in sensory ganglia are also not occupied in vivo. Site occupancy by Brn3a can be correlated with evolutionary conservation of the genomic regions containing the recognition sites and also with histone modifications found in regions of chromatin active in transcription and gene regulation, suggesting that Brn3a binding is highly context dependent (Lanier, 2004).

Brn3 POU-domain transcription factors and axon pathfinding

The POU domain transcription factors Brn3a, Brn3b and Brn3c are required for the proper development of sensory ganglia, retinal ganglion cells, and inner ear hair cells, respectively. The roles of Brn3a in neuronal differentiation and target innervation in the facial-stato-acoustic ganglion have been investigated. Absence of Brn3a results in a substantial reduction in neuronal size, abnormal neuronal migration and downregulation of gene expression, including that of the neurotrophin receptor TrkC, parvalbumin and Brn3b. Selective loss of TrkC neurons in the spiral ganglion of Brn3a^-/- cochlea leads to an innervation defect similar to that of TrkC^-/- mice. Most remarkably, these results uncover a novel role for Brn3a in regulating axon pathfinding and target field innervation by spiral and vestibular ganglion neurons. Loss of Brn3a results in severe retardation in development of the axon projections to the cochlea and the posterior vertical canal as early as E13.5. In addition, efferent axons that use the afferent fibers as a scaffold during pathfinding also show severe misrouting. Interestingly, despite the well-established roles of ephrins and EphB receptors in axon pathfinding, expression of these molecules does not appear to be affected in Brn3a^-/- mice. Thus, Brn3a must control additional downstream genes that are required for axon pathfinding (Huang, 2001).

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. The murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, Brn-3.2 gene targets likely to act on axon guidance have been identified at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. Evidence is presented that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. These findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding (Erkman, 2000).

To understand the molecular mechanisms by which Brn-3.2 exerts its effects on RGC axon guidance, candidate target genes have been identified using a modification of representational difference analysis. Thus far, screening has yielded three potentially novel genes, and seven with matching sequences in mouse EST and human genomic and cDNA databases, the structure and function of which have not been reported. A number of genes were obtained that share a very low degree of homology with known genes and cannot be classified at this time. In addition, five candidate target genes have been identified that represent previously characterized molecules, including the transcription factors Irx6, EBF/Olf-1, EBF/Olf-2, and a mouse homolog of rat Neuritin. Irx6 is a homeodomain transcription factor with homology to the Drosophila genes of the iroquois complex, Olf-1 and Olf-2, belonging to the early B cell factor (EBF) family of HLH transcription factors, and Neuritin, a GPI-anchored neuronal cell surface protein, are all well characterized. One RDA product that did not present homology to published sequences was ultimately identified by analysis of cDNA clones as part of the 3'UTR of the mouse homolog of the human actin binding zinc finger protein abLIM. The amino acid sequence of the LIM domain containing all four LIM motifs specific for the retinal isoform is highly conserved and shows 97% identity to the human sequence. In situ hybridization analysis of m-abLIM in E15.5 mice shows, in addition to its expression in the inner layer of the retina, expression in other neuronal structures including peripheral sensory ganglia, spinal cord, SC, and nonneural tissues such as the thymus (Erkman, 2000).

If these genes are regulated by Brn-3.2, their mRNA levels should decrease in Brn-3.2^-/- retina, and their spatial and temporal patterns of expression should support such an assumption. Indeed, comparison of mRNA levels in E15.5 wild-type and Brn-3.2^-/- retinas reveals a dramatic decrease in the levels of Irx6, Olf-1, m-abLIM, and Neuritin, and a modest effect on Olf-2 mRNA levels, indicating that even relatively small differences can be detected by the modified RDA protocol. Temporal expression patterns of Brn-3.2, Irx6, Olf-2, and m-abLIM were determined using adjacent sections of retina at different developmental stages. Expression of Brn-3.2 mRNA, first detectable in the retina at E11.5, precedes initial detectable expression of Irx6 and Olf-2 around E12.5, and m-abLIM around E13.5. Thus, these genes may represent components of a molecular cascade regulated by Brn-3.2 (Erkman, 2000).

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