Big brain (bib) is a neurogenic gene; when mutated, it causes defects in cell fate determination during Drosophila neurogenesis through an unknown mechanism. The protein Big Brain (Bib) has sequence identity with the major intrinsic protein family that includes the water- and ion-conducting aquaporin channels. Bib expressed heterologously in Xenopus oocytes provides a voltage-insensitive, nonselective cation channel function with permeability to K+ > Na+> tetraethylammonium. The conductance, activated in response to endogenous signaling pathways in BIB-expressing oocytes, is decreased after treatment with 20 µM insulin and is enhanced with 10 µM lavendustin A, a tyrosine kinase inhibitor. Western blot analysis confirms that Bib is tyrosine-phosphorylated. Both tyrosine phosphorylation and the potentiating effect of lavendustin A are removed by partial deletion of the C terminus (amino acids 317-700). Current activation is not observed in control oocytes or in oocytes expressing a nonfunctional mutant (BIB E71N) that appears to be expressed on the plasma membrane by confocal microscopy and Western blotting. These results indicate that Bib can participate in tyrosine kinase-regulated transmembrane signaling and may suggest a role for membrane depolarization in the neurogenic function of Bib in early development. The effect of insulin in Xenopus oocytes and neuronal cells can be mediated through cascades involving phospholipase C, phosphatidylinositol-3 kinase, and protein kinase C; however, the step that mediates the direct phosphorylation at the Bib channel itself is not identified in this work (Yanochko, 2002).

The method used here for activation of ion channel function in Bib-expressing oocytes by electrode insertion may be related to a well known phenomenon of parthenogenic oocyte activation that mimics fertilization. Sperm entry, as well as artificial stimulation by pricking, electric shock, and chemicals, triggers intracellular kinase signaling pathways in oocytes that mediate cell cycle progression in a process termed 'oocyte activation'. Although the oocytes used for heterologous expression are not as mature, it is reasonable to hypothesize that the kinase-sensitive activation of Bib-expressing oocytes by electrode penetration may occur by an analogous mechanism. Artificially stimulated oocytes show both activation and inactivation of different kinases, including the src-related p57 Xenopus tyrosine kinase, c-Jun N-terminal kinase, Eg2, cdc2K, and mitogen-activated protein kinase. The relevant signaling pathway for studies described here remains to be defined, but pharmacological assays indicate the likely candidates include tyrosine kinases (Yanochko, 2002).

Four lines of evidence demonstrate that the current observed in Bib-expressing oocytes is mediated by Bib channels and not by unidentified oocyte channels: (1) there is a lack of a conductance response from control oocytes; (2) mutation of Glu at position 71 to Asn (E71N) results in nonfunctional channels that appear to be expressed on the plasma membrane but do not recruit any ionic conductance response; (3) mechanisms of activation differ for the ionic conductance responses mediated by various channels in the MIP family, including AQP1 (activated by cGMP), AQP6 (activated by acidic pH), and Bib (regulated by tyrosine phosphorylation) when expressed in oocytes, and (4) partial truncation of the C-terminal tail domain interferes with tyrosine phosphorylation and pharmacological modulation of the current in Bib-expressing oocytes. In addition, preliminary results from cell-attached patch-clamp experiments of Bib-expressing oocytes have revealed a large, novel single-channel conductance not seen in control oocytes. Large single-channel conductances have been observed in other MIP family ion channels (Yanochko, 2002).

This study shows that the Bib protein is a target of tyrosine phosphorylation in oocytes and that the conductance associated with Bib expression is enhanced by lavendustin A, a tyrosine kinase inhibitor, and decreased by insulin, which acts on endogenously expressed insulin receptor and IGF-I tyrosine kinase receptors. These data support a model in which signals promoting dephosphorylation enhance Bib ion channel activation. Alternatively, Bib channels may associate with other proteins that are modulated by tyrosine phosphorylation and dephosphorylation. Several different mechanisms may regulate Bib ion channel activity, because the C-terminal tail contains many additional sites of potential regulatory interactions, including consensus sequences for phosphorylation by serine/threonine kinases, polyglutamine stretches, and three internal PDZ binding domains. Although insulin and IGF-I receptors signal through many pathways, including tyrosine phosphatases in oocytes, serine/threonine kinase pathways do not appear to be involved in the regulation of Bib in oocytes on the basis of the lack of effects of H7 and staurosporine (Yanochko, 2002).

The lack of tyrosine phosphorylation of Delta317 HABib and the lack of effect of lavendustin A on these channels suggests that the C-terminal tail between amino acids 317 and 700 contributes to tyrosine kinase modulation of HABib in Xenopus oocytes. There are multiple regulatory domains in the C-terminal tail, including putative Src homology 3 (SH3) and PDZ binding domains and polyglutamines that may affect protein-protein interactions and mechanisms of regulation in addition to tyrosine phosphorylation. The C-terminal tail of Bib contains two elements important for regulation by tyrosine kinases: tyrosine residues and SH3 binding domains. Twenty-two tyrosine residues are located on intracellular portions of the Bib channel: 1 in the N terminus and 21 in the C terminus. Ongoing experiments are aimed at identifying the specific tyrosine residues involved in modulation of Bib channel activity; however, on the basis of optimal sequences for phosphorylation, 5 of the 21 tyrosines in the C-terminal tail of Bib are likely candidates for phosphorylation by tyrosine kinases such as src or Abl (Yanochko, 2002).

In addition to putative sites of tyrosine phosphorylation, the C-terminal tail contains four potential SH3 binding domains, proline-rich sequences that mediate protein interactions, notably between tyrosine kinases and their substrates. For example, for both hKv1.5 and connexin 43, intact SH3 binding domains are critical for regulatory interaction of the channels with src. SH3 binding domains present in the Bib C-terminal tail domain may mediate the interactions between tyrosine kinases and Bib channels (Yanochko, 2002).

The properties of Bib channels and the nature of their regulation during Drosophila neurogenesis clearly need to be investigated. The finding that Bib expression results in a regulated cationic channel function in Xenopus oocytes suggests that Bib activation in vivo would result in membrane depolarization. Results obtained from grasshopper embryos support this idea; the resting membrane potentials of differentiated neuroblasts were -60 to -80 mV, whereas those of surrounding non-neural cells were slightly depolarized, ranging from -40 to -60 mV (Yanochko, 2002 and references therein).

Both tyrosine kinases and phosphatases are involved in neuronal development in Drosophila. Although this study did not test for an interaction between insulin or IGF-I receptors and Bib in Drosophila, it is interesting that Drosophila embryos carrying mutations in the insulin receptor lacked populations of both neurons and glia, suggesting a role for the Drosophila insulin receptor in neurogenesis. In this sense, the Xenopus oocyte may have provided a better model system for evaluating Bib regulation than was expected. Further experiments are needed to determine the importance of the putative ion channel function of Bib in Drosophila neuronal development and the Notch signaling pathway, the role of membrane depolarization mediated by Bib in cell fate determination, and the relationship between the insulin receptor tyrosine kinase and Bib channel regulation in vivo (Yanochko, 2002).

The data support the hypothesis that Bib forms a nonselective cation channel when expressed in Xenopus oocytes. The novel finding that the Bib-mediated conductance is regulated by a mechanism involving tyrosine kinase pathways appears to fit logically with the role of Bib in neurogenesis, a process that is governed by growth factors and other environmental cues. The nature of the native regulation of Bib in Drosophila and the relationship between ion channel activity and the Notch signaling pathway remain to be determined. Further studies into the functional domains of the Bib protein should provide more clues to the unique involvement of bib in neurogenesis and further evidence of the diversity of function in the MIP family of channels (Yanochko, 2002).



Expression of big brain begins soon after the cell membrane starts to form around the nuclei in the syncytial blastoderm and is detectable in all the somatic cells that are forming in an early stage-5 embryo. The BIB mRNA soon disappears in a ventral region: the width of this region is about 10 cells at the stage of blastoderm formation when roughly one-fifth of the cell membrane is formed, increasing to about 17 cells when cellularization is complete. This region of about 17 cells includes all presumptive mesodermal cells and two rows of ectodermal cells. During gastrulation at stages 6 and 7 (see [Images]), the mesoderm invaginates, leaving one row of ectodermal cells without detectable bib expression on either side of the ventral furrow (Rao, 1990).

During germ-band elongation, BIB mRNA is detectable and is maintained in the ectoderm. As prospective neuroblasts delaminate, BIB mRNA is still found as nuclei move basally, but BIB transcript disappears from the neuroblasts after they have completely segregation from the epidermal layer. Toward the end of germ-band extension, mesodermal cells start to express bib. Just before germ-band shortening, bib expression begins to disapper first from the epidermis and then from the mesoderm (Rao, 1990).

Big brain is localized along the basolateral cell membranes of neuroectoderm cells. In addition to being concentrated apically in the region of the adherens junctions, it also appears to be concentrated basally, where the cells contact each other and the nascent neuroblast (Nb). Concentration of Bib and other proteins may be important for efficient signaling between proneural cells. In stage 10-11 embryos, Bib protein is expressed strongly in the epidermis and mesoderm and at a much lower level in the developing CNS, generating a 'two-stripe' pattern similar to that described for Dl expression. In stage 9 Notch mutant embryos, all the neuroectoderm cells become Nbs; Bib protein is only present in the mesoderm, confirming that the Nbs lose Bib expression. Using confocal microscopy, it is difficult to determine exactly when Bib protein disappears from the Nbs, because nascent Nbs are surrounded by the Bib-containing membranes of adjacent epidermal cells; however, in electron micrographs, there are far fewer grains associated with the membranes of segregating Nbs than with the membranes of adjacent neuroectoderm cells, indicating that Bib is lost from the Nb membrane early during Nb segregation. Thus it appears that the maintenance of the epidermal fate in the neuroectoderm cells surrounding the delaminating Nb does not require the presence of Bib protein in the Nb (Doherty, 1997).

bib expression is maintained in the epidermis and mesoderm until stage 12, consistent with the observed bib mutant phenotypes in somatic muscles, peripheral glia, oenocytes, optic lobs, stomatogastric nervous system, salivary glands. Malpighian tubules and dorsal vessel Bib protein expression is observed in all of these tissues or their precursors. Bib expression is also observed in the anterior and posterior midgut invaginations, ventral midline cells and tracheal pit cells. While the midgut, ventral midline cells and tracheae are not defective in bib mutant embryos, they are all disrupted by loss of other neurogenic gene function. Complementary expression of bib and Dl at the midgut/hindgut boundary and in rings of cells where the adult midgut precursors form in the proventriculus suggests that these genes play a role in formation of the adult midgut. Conspicuous Bib expression is observed in the adult muscle precursors and in subsets of the larval ventral nerve cord and brain lobes. The Bib antibody also results in strong signal in a subset of hemocytes. This signal must represent a cross-reacting antigen, because the signal is not present in bib - embryos; it is not competed away by bib peptide, and there is no BIB mRNA expression in these cells (Doherty, 1997).

Big brain and Delta proteins colocalize. In the prospective mesoderm just before gastrulation, Bib protein disappears from the plasma membrane and is present in punctate cytoplasmic structures basal to the nucleus. The Delta protein is expressed in a similar manner. To address whether Bib and Dl colocalize, embryos were simultaneously labeled with Bib and Dl antibodies. The Bib and Dl proteins did in fact colocalize in the plasma membrane and in the punctate cytoplasmic structures of prospective mesoderm cells, although the intensity of the two signals was not always similar. By immunoelectron microscopy, it was found that bib is associated with the plasma membrane and concentrated in apical adherens junctions as well as in small cytoplasmic vesicles (Doherty, 1997).


In third instar wing discs, Bib expression is found in the proneural clusters for wing margin bristles, sensory organs along the dorsal radius and SOs of the notum, where there is a known requirement for bib. In late third instar wing discs, Bib is also expressed in 3-6 rows of cells at the dorsal/ventral boundary of the wing pouch and in the cells that form the wing veins. Despite the fact that N and Dl are required for wing margin and wing vein formation, loss of bib function does not disrupt these tissues (data not shown). Bib is expressed at a high level in the cells that later form the flight muscles (Doherty, 1997).


No BIB mRNA is found in the ovary, consistent with the observation that bib is the only known neurogenic gene that has no detectable maternal contribution (Rao, 1990).

Effects of mutation or deletion

big brain (bib) is one of the six known zygotic neurogenic genes involved in the decision of an ectodermal cell to take on the neurogenic or the epidermogenic cell fate. Previous studies suggest that bib functions in a pathway separate from the one involving Notch and other known neurogenic genes, that is, bib shows no genetic interactions with other neurogenic genes. Loss of bib function approximately doubles the number of neuronal precursors and their progeny cells in the embryonic peripheral nervous system. Mosaic studies reveal a hypertrophy of sensory bristles in bib mutant patches in adult flies. bib appears to function in the specification of neuronal precursors in both the embryonic and adult sensory nervous system. This is in contrast to the function of Notch, which continues to be required at multiple stages of neural development subsequent to this initial determination event (Rao, 1992).

The neurogenic genes of Drosophila control mesoderm development. Embryonic cells that express the muscle-specific gene nautilus are overproduced in each of seven neurogenic mutants (Notch, Delta, Enhancer of split, big brain, mastermind, neuralized, and almondex), at the apparent expense of neighboring, nonexpressing mesodermal cells. The mesodermal defect does not appear to be a simple consequence of associated neural hypertrophy, suggesting that the neurogenic genes may function similarly and independently in establishing cell fates in both ectoderm and mesoderm. Altered patterns of beta 3-tubulin and myosin heavy chain gene expression in the mutants indicate a role for the neurogenic genes in development of most visceral and somatic muscles. It is proposed that the signal produced by the neurogenic genes is a general one, effective in both ectoderm and mesoderm (Corbin, 1991).

The complex embryonic phenotype of the six neurogenic mutations (Notch, mastermind, big brain, Delta, Enhancer of split and neuralized) was analyzed by using different antibodies and PlacZ markers that allowed labelling of most of the known embryonic tissues. All of the neurogenic mutants show abnormalities in many different organs derived from all three germ layers. Defects caused by the neurogenic mutations in ectodermally derived tissues fell into two categories:

Abnormalities in tissues derived from the mesoderm were observed in all six neurogenic mutations. Most importantly, somatic myoblasts do not fuse and/or form an aberrant muscle pattern. Cardioblasts (which form the embryonic heart) are increased in number and show differentiative abnormalities; other mesodermal cell types (fat body, pericardial cells) are significantly decreased. The development of the endoderm (midgut rudiments) is disrupted in most of the neurogenic mutations (Notch, Delta, Enhancer of split and neuralized) during at least two stages. Defects occur as early as during gastrulation when the invaginating midgut rudiments prematurely lose their epithelial characteristics. Later, the transition of the midgut rudiments to form the midgut epithelium does not occur. In addition, the number of adult midgut precursor cells that segregate from the midgut rudiments is strongly increased. It is proposed that, at least in the ectodermally and endodermally derived tissues, neurogenic gene function is primarily involved in interactions among cells that need to acquire or to maintain an epithelial phenotype (Hartenstein, 1992).


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big brain: Biological Overview | Evolutionary Homologs | Characterization of Channel Function | Developmental Biology | Effects of Mutation

date revised: 15 January 2011 

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