BIOLOGICAL OVERVIEW

Among neurogenic genes in Drosophila, big brain is an enigma. The term neurogenic creates its own confusion, as it refers to the mutant phenotype of a family of genes. The function of neurogenic genes is to reduce, rather than engender, the number of neural precursors; therefore, the so-called neurogenic genes may be thought of as functionally anti-neurogenic. big brain does not interact genetically with other neurogenic genes (such as Notch and Delta) suggesting that Bib does not act in the same pathway as the classic neurogenic genes. In addition, Bib structurally resembles aquaporins (water pores), the channel proteins that facilitate uptake of small molecules into cells and cell-cell communication. Compounding the confusion, Bib has no hydrophobic transmembrane sequences, suggesting that the protein, unlike other aquaporins, may be totally intracellular (Rao, 1990 and references).

What is the evidence that Bib is neurogenic? Ventral clusters of sensory neurons and their support cells are missing in bib mutants probably due to the overgrowth of the central nervous system. Lateral and dorsal clusters show an increase of sensory neurons and support cells. All three types of embryonic sensory neurons are increased by up to two fold in bib mutants: (1) external sensory neurons (sn), which innervate mechansosensory or chemosensory structures; (2) the chordotonal neurons (ch) that innervate an elongated internal sensory structure, and (3) multiple dendritic (md) organs. The duplication of sensory neurons, their support cells and the external sensory structures formed by the support cells suggests that the primary effect of bib mutation is an increase in the number of precursor cells that give rise to both neurons and the support cells. Examination of the division rate of precursors confirms that sensory neurons are more numerous in mutant embryos but that the division patterns of precursors are similar to those in wild-type embryos (Rao, 1992).

Drosophila adult sensory organs develop independently during the pupal stage. To determine whether bib also gives an adult neurogenic phenotype, clones of bib mutant cells were generated. bib mutant clones show an increased number of macrochaetae in all of the body parts examined including the notum (derived from the wing imaginal disc) the scutellum, the sternopleura, the head and wings. Supernumerary bristles are found in locations where bristles normally form (Rao, 1992).

Previous studies of big brain genetic interactions and expression suggest that bib acts as a channel protein in proneural cluster cells that adopt the epidermal cell fate and serves a necessary function in the response of these cells to the lateral inhibition signal. These genetic studies had not revealed any interaction between big brain and the other neurogenic genes. bib cannot rescue the phenotype in embryos mutant for other neurogenic genes. Ectopic bib expression does not rescue the cuticle-defect of embryos mutant for Dl, N, E(spl), mam or neu. In reciprocal experiments, ectopic Dl and neu do not rescue the neurogenic phenotype in bib mutant embryos. In contrast, ectopically activated N still has an antineurogenic effect in bib mutant embryos. These results indicate that bib, Dl and neu cannot functionally replace one another and that bib functions upstream of or parallel to activated N. Using mosaic analysis in the adult, it has been demonstrated that big brain activity is required autonomously in epidermal precursors to prevent neural development. Ectopically expressed big brain acts synergistically with ectopically expressed Delta and Notch, providing the first evidence that big brain may function by augmenting the activity of the Delta-Notch pathway (Doherty, 1997).

big brain mutants are different from other neurogenic phenotypes in two ways:

• bib is not required for the refinement of the entire neurogenic cluster. bib causes only a two fold increase in the number of sensory organ precursors as compared to the 8 to 12-fold increase in N or Dl null mutants. Not all cells within a proneural cluster become sensory organ precursors simultaneously. It is likely that bib is required for only a subset of proneural cells and not for all the cells of a proneural cluster.
  
  
• Notch functions both at the level of proneural precursors and at the level of sensory neurons and their support cells. bib functions only at the level of proneural precursors (Rao, 1992).

The enigma remains. Does Bib brain function have anything to do with its aquaporin structure? If Bib has an aquaporin function, is this reflected in the corresponding effect of Bib on cell size or cytoskeletal dynamics, and how is this translated into altered cell fate?

The Big brain aquaporin is required for endosome maturation and Notch receptor trafficking

Activity of the big brain (bib) gene influences Notch signaling during Drosophila nervous system development. This study demonstrates that Bib, which belongs to the aquaporin family of channel proteins, is required for endosome maturation in Drosophila epithelial cells. In Drosophila mutants for hrs, lethal(2) giant discs (lgd), and genes encoding ESCRT complex proteins, mislocalization of Notch in endosomes is associated with the accumulation of other receptors and ligands In the absence of Bib, early endosomes arrest and form abnormal clusters, and cells exhibit reduced acidification of endocytic trafficking organelles. Bib acts downstream of Hrs in early endosome morphogenesis and regulates biogenesis of endocytic compartments prior to the formation of Rab7-containing late endosomes. Abnormal endosome morphology caused by loss of Bib is accompanied by overaccumulation of Notch, Delta, and other signaling molecules as well as reduced intracellular trafficking of Notch to nuclei. Analysis of several endosomal trafficking mutants reveals a correlation between endosomal acidification and levels of Notch signaling. These findings reveal an unprecedented role for an aquaporin in endosome maturation, trafficking, and acidification (Kanwar, 2008).

Mutants in the big brain gene were among the first Drosophila mutants isolated with specific patterning defects in embryonic neurogenesis. Together with five other founding members of the 'neurogenic' gene family, Notch, Delta, mastermind, Enhancer of split, and neuralized, the bib locus prevents ectopic neuroblast specification and lethal hypertrophy of the embryonic nervous system. Subsequent molecular analyses have demonstrated that the five other genes encode core components of the Notch signaling pathway, but the relevance of Bib function to Notch activation has long remained unclear. Based on the current findings, it is proposed that the primary defect caused by loss of Bib function is a failure in endosome maturation, which indirectly impairs transmission of the activated Notch signal during its endosomal trafficking (Kanwar, 2008).

The results support a model in which the initial steps of Notch activation, including ligand binding, ectodomain removal, endocytosis, and biochemical cleavage of the Notch receptor by the γ-secretase complex, do not require Bib. However, these events are associated with the entry of ligand-activated Notch into endosome trafficking compartments (Vaccari, 2008), and efficient movement of Notch through these compartments depends upon Bib-promoted maturation of the endosomes. The morphological features of the bib endosome arrest phenotype could potentially account for the specificity of the bib mutant effects on Notch signaling as well as the partial preservation of Notch signaling in the absence of Bib function. Unlike other major developmental signaling pathways that involve signal amplification through an effector cascade, Notch signaling relies upon direct cleavage of Notch to produce stoichiometric amounts of NICD needed for transcriptional regulation of target genes. The tightly packed early endosomes in bib mutant cells might prevent proper intracellular trafficking of most but not all NICD, thus leading to reduced but not completely blocked Notch signaling. Arguing against this idea, however, is the finding that bib lgd double mutant cells exhibit a rather different phenotype of enlarged, possibly arrested MVB (multivesicular body)-like structures accompanied by suppression of the ectopic Notch activation seen in lgd single mutants. Hence, the specific early endosome arrest seen in bib mutants is not required per se for the observed negative effects on Notch signaling, and instead Bib seems capable of facilitating Notch activity at different steps along the endosome-lysosome pathway (Kanwar, 2008).

A clustering phenotype of early endosomes similar to that in bib mutant cells has been observed upon loss of KIF16B, a kinesin-3 microtubule motor that regulates plus-end motility of early endosomes, their intracellular distribution, and their ability to convert into late endosomes, maintaining the crucial balance between receptor recycling and degradation. Bib may regulate the assembly or activity of these key determinants, such that loss of Bib disrupts dynamic interactions of endosomes with the cytoskeleton and causes clustering through an arrest in motility (Kanwar, 2008).

How does the Bib aquaporin promote the endosome maturation? Mammalian aquaporins act as tetramers, each subunit of which forms a channel through which water, ions, or other small solutes are transported. The channel is formed by two NPA motif-containing loops that project into the lipid bilayer from opposite sides, and residues located near the narrowest channel point influence permeability characteristics through size restriction and electrostatic repulsion. Bib lacks a conserved histidine that is present in all water-transporting aquaporins, and Drosophila Bib expressed in Xenopus oocytes is permeable to cations but not water molecules (Yanochko, 2002; Kanwar, 2008 and references therein).

There are two primary models for Bib function that are suggested by these findings. In one model, Bib directly alters a biochemical property of endosomes by transporting ions across the limiting membrane. In the second model, the ion channel domain of Bib acts as a sensor for ion flux across the membrane, perhaps linking endosomes to different cytoplasmic factors in response to changes in endosome ion concentrations. With respect to the first model, endosomes undergo increasing acidification as they mature from early endosomes to lysosomes, and acidification plays a causal role in the formation of multivesicular/late-endosome-like membrane structures in vivo and in biochemical liposome reconstitution studies. The progression from early to late endosomes is also accompanied by a conversion from Rab5 to Rab7 GTPases. The absence of Rab7 in bib mutant endosomes along with their reduced acidification suggests that endosomal pH regulation by Bib may modulate aspects of intralumenal vesicle generation and endosome biogenesis. These processes could also influence interactions of endosomes with the cytoskeletal network, contributing to the bib endosome clustering phenotype as noted above (Kanwar, 2008).

The pH gradient across invaginating endosomal membranes is tightly regulated and could potentiate Notch signaling efficiency by additional mechanisms. First, the pH gradient might be needed for proper invagination or other membrane curvature events, involving dynamic changes in membrane composition that facilitate receptor/ligand dissociation or influence interactions of Notch with γ-secretase. Indeed, subtle changes in membrane lipid composition affect Notch and Egfr endosome sorting and signaling in Drosophila imaginal tissues. In addition, the catalytic activity of γ-secretase itself is optimal in a low pH environment. Although biochemical data indicate that Bib is unlikely to be an essential cofactor for γ-secretase, a caveat is that the cell lysis procedure might itself cause pH changes or other effects that bypass a normal γ-secretase requirement for Bib in intact cells (Kanwar, 2008).

Endosome-associated ion channels have been implicated in organelle acidification and biogenesis. Loss of endosome/lysosome-associated chloride ion channels leads to a bone resorption disorder in humans and defective receptor endocytosis and recycling in knockout mice. Cells lacking Bib exhibit a pronounced reduction in the acidification of endosomal organelles, even in combinations with other trafficking mutants that produce different abnormal endosome morphologies. Thus, the idea is favored that the ion channel activity of Bib is directly involved in the progressive acidification of the endosome compartments. Moreover, inhibition of vacuolar ATPase impairs endosomal acidification and protein trafficking to late endosomes, leading to the proposal that vacuolar ATPase is a component of an endosomal pH-sensing machinery that recruits cytosolic factors to endosomes. Bib might function in a similar manner, mediating pH-dependent interactions between endosomes and the cytoskeleton or recruiting cytoplasmic factors needed for endosome maturation and/or transport. Further studies will be required to elucidate the relationship of Bib to other ion channels and vacuolar ATPases implicated in endosome acidification, as well as to the cytoskeletal and trafficking machinery (Kanwar, 2008).

The involvement of Bib in endosome biogenesis illustrates the complexity of endocytosis-dependent membrane trafficking and its relationship to signal transduction. The striking defects in endosome maturation in cells lacking Bib reveal an unprecedented role for an aquaporin in organelle biogenesis. The findings also demonstrate that impairment of a specific step of endosome maturation can have profound yet relatively specific effects on a single developmental signaling pathway. Further analysis will be needed to understand the biophysical role of Bib in endosome maturation and acidification and to determine if mammalian aquaporins might also regulate endosomal processes and the membrane trafficking of internalized signaling molecules (Kanwar, 2008).

GENE STRUCTURE

Bases in 5' UTR - 302

Exons -

Bases in 3' UTR - 915

PROTEIN STRUCTURE

Amino Acids - 700

Structural Domains

The N-terminal half of Bib protein is highly hydrophobic, whereas the C-terminal portion is generally hydrophilic and glutamine-rich. There are six hydrophobic stretches of more than 21 amino acid residues that could potentially span the membrane. Since there is not signal peptide, it is thought that Bib is intracellular (Rao, 1990).

The Bib protein has significant sequence homology to the major intrinsic protein (MIP) of bovine lens fiber cell membrane; 40% of the 224 amino acids in the N-terminal half are identical to those in MIP. This region has sequence similarity to a soybean membrane protein, nodulin (with 27% amino-acid identity), and an E. coli protein, GlpF (with 23% identity). The Bib protein is about twice as large as the other proteins; the C-terminal half of Bib shows no significant homology to any known protein (Rao, 1990).

The Drosophila Big Brain (Bib) protein plays a critical role in the determination of neuroblasts in the embryonic ectoderm and many other cell types. Bib is a member of the MIP family of transmembrane channel proteins. The conserved channel domain of Bib is flanked by amino- and carboxy-terminal cytoplasmic domains of unique sequence, which comprise over two-thirds of the protein. To determine whether the cytoplasmic domains of Bib are important for Bib function, the bib gene of D. virilis has been cloned and sequenced and compared with that of D. melanogaster. The channel domain and both cytoplasmic domains are highly conserved between the two species. The conservation of the cytoplasmic domains indicates that they are critical to Bib function. bib transcripts are found in similar patterns in both species, indicating that the developmental function(s) of Bib has also been conserved (Burris, 1999).

date revised: 5 August 2021

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