Bearded

DEVELOPMENTAL BIOLOGY

Embryonic

Only a very low level of Brd mRNA is observed in preblastoderm embryos, including a small amount of presumably maternal transcript at 0-2 hours. In 4- to 6- and 6- to 8-hour embryos, the abundance of the transcript is sharply higher; interestingly, this is the period during which neuroblast segregation, a Notch-regulated process, takes place. By the next time interval (8-12 hours), Brd transcript levels have declined sharply, and remain low throughout the rest of embryogenesis. This finding indicates that, at least in the embryo, wild-type Brd mRNA is likely to be short-lived. It is concluded that embryonic levels of Brd transcript are subject to strong temporal modulation (Leviten, 1997).

Larval

Brd is expressed in a proneural cluster pattern in imaginal discs. Brd transcript accumulates specifically in a proneural cluster pattern in both wild-type and Brd mutant imaginal discs. Brd mRNA is significantly more abundant in the mutant discs, as reflected by a much stronger in situ hybridization signal. These observations provide further support for the interpretation that the gain-of-function alleles of Brd are hypermorphs (Leviten, 1996), since Brd is apparently overexpressed, and not spatially misexpressed, in these mutants. In addition, the finding that Brd is normally expressed in wild-type proneural clusters is consistent with the hypothesis that the gene has a wild-type function in adult sensory organ development. In the wing imaginal disc, Brd transcript is detectable at the positions of all developing adult external sensory organs, analogous to the expression of the proneural genes achaete and scute (Leviten, 1997).

To examine the spatial and temporal relationship of Brd expression to SOP specification, double-labeling experiments were carried out with the SOP-specific enhancer trap marker A101. Double-labeled third-instar wing discs demonstrate clearly that Brd transcript is localized to sites of SOP development These experiments also reveal that Brd expression, like ac/sc expression, precedes SOP specification as marked by A101. For example, in the anterior scutellar (aSC) macrochaete proneural cluster, uniform Brd expression can clearly be seen prior to detectable A101 staining. Subsequently, a single A101-positive cell is observed among the Brd-positive cells. In many clusters there is clearly a period in which the multiplied SOPs express both Brd and A101. This is most evident in regions in which a cross-sectional view of the disc epithelium can be seen, and the A101-positive nucleus of an SOP cell is clearly surrounded by cytoplasmic Brd transcript. Brd expression in A101-positive cells appears to become diminished at some positions in late third-instar and early pupal discs. For example, in the A101-positive SOP cells at the posterior scutellar (pSC) macrochaete position, Brd expression is much weaker than in the aSC region at the same time. Other positions, including the anterior postalar (aPA) macrochaete site, also exhibit undetectable levels of Brd transcript while expressing A101 strongly. The pSC and aPA are two of the first macrochaete SOPs to be determined, suggesting that the decreased Brd mRNA levels observed there represent the turnover of transcript and not simply a relatively lower level of Brd expression throughout the development of these particular clusters. In summary, there is a general pattern for the progression of Brd expression within imaginal disc proneural clusters. Initially, Brd transcript is present at roughly equal levels throughout the cluster, and this expression precedes that of the early SOP marker A101. Subsequently, Brd and A101 are coexpressed within the SOP cell (with Brd transcript remaining in the non-SOP cells as well), and then Brd transcript levels become diminished in both the SOPs and the surrounding cluster cells (Leviten, 1997).

Effects of mutation or deletion

A novel class of gain-of-function mutations that specifically affect the development of adult sensory organs in Drosophila was isolated at the Bearded locus. These Brd alleles cause bristle multiplication and bristle loss phenotypes resembling those described for the neurogenic genes Notch (N) and Delta (Dl). Supernumerary sensory organ precursor cells develop in the proneural clusters of Brd mutant imaginal discs; like normal SOPs, these are dependent on the function of the proneural genes achaete and scute, and express elevated levels of ac protein. At cuticular positions exhibiting the Brd bristle loss phenotype, the progeny of the multiplied SOPs develop aberrantly; neurons and thecogen (sheath) cells appear but not trichogen (shaft) and tormogen (socket) cells. This appears to represent a transformation of the pIIa secondary shaft and socket precursor cell within the SOP lineage to a pIIb secondary neuron and sheath precursor cell fate. These results suggest that Brd gain-of-function alleles interfere with Notch pathway-dependent cell-cell interactions at two distinct stages of adult sensory organ development. Brd null mutants are viable and exhibit no mutant phenotypes, suggesting that Brd may be a component of an overlapping function (Leviten, 1996).

Bearded was expressed under the control of the Hsp70 heat-shock promoter. Heat-shock pulses applied during the late larval and early pupal stages generate bristle multiplication and bristle loss phenotypes resembling those of Brd gain-of-function mutations. The bristle multiplication effects are not as severe as those observed for the Brd 1 and Brd 3 mutations, even in fly lines carrying four copies of the Hs-Brd construct. Pulses of late third-instar larvae affect the earlier-developing macrochaetes, causing bristle duplications at some macrochaete positions, and a more reliable and general macrochaete loss phenotype. Heat pulses applied during early pupal development (0, 6 and 16 hours APF) result in increases in microchaete number and density on the head and notum, comparable to those characteristic of weak Brd mutant genotypes. In addition, significant microchaete loss is observed, and in some positions sockets without shafts are found, along with microchaetes with severely reduced shafts. These phenotypes are all observed in Brd gain-of-function mutants. No general effects on epidermal cell or wing vein development were observed (Leviten, 1997).

The P[Hs-Brd] transformant lines were exposed to P transposase activity in order to mobilize the transgene construct and potentially reposition it adjacent to strong enhancers in the genome, which would confer more stable, high levels of expression on the Brd gene. Two lines of flies were generated that exhibit strong bristle defects. One of these, Brd HSJ1, displays strong dominant macrochaete and microchaete multiplication phenotypes similar to Brd 3 heterozygotes. Heterozygotes of this line exhibit microchaete tufting primarily in the anterior half of the notum, with a nearly wild-type microchaete pattern posteriorly. Homozygotes exhibit stronger and more uniform notum microchaete multiplication, strong head and notum macrochaete multiplication, and severely roughened eyes. The dominant bristle phenotypes of Brd HSJ1 are almost certainly due to overexpression of the Brd gene. The recessive eye roughening is potentially caused by a mutational effect of the transposon insertion on an endogenous gene, although gain-of-function alleles of Brd are capable of producing similar eye roughening defects. For comparison, an investigation was carried out of the ability of four copies of a Hs-Brd construct containing a wild-type Brd 3' UTR to phenocopy Brd gain-of-function phenotypes. This construct is incapable of conferring any dominant mutant phenotypes under a variety of heat-shock regimens. These results are consistent with the hypothesis that the blood transposon-mediated truncation of the mutant Brd transcript (and the consequent loss of two Brd boxes and one GY box) is important for its ability to interfere with cell fate decisions in the adult PNS (Leviten, 1997).

The 3' untranslated regions (UTRs) of many genes involved in Notch signaling, including Bearded (Brd) and the genes of the Enhancer of split complex (E(spl)-C), contain (often in multiple copies) two novel heptanucleotide sequence motifs: the Brd box (AGCTTTA) and the GY box (GTCTTCC). The molecular lesion associated with a strong gain-of-function Brd mutant suggests that the loss of these sequence elements from its 3' UTR might be responsible for the hyperactivity of the mutant gene. The wild-type Brd 3' UTR confers negative regulatory activity on heterologous transcripts in vivo; this activity requires its three Brd box elements and, to a lesser extent, its GY box. Brd box-mediated regulation decreases both transcript and protein levels, and the results suggest that deadenylation or inhibition of polyadenylation is a component of this regulation. Whereas bearded box mutation causes a modest increase in the amount of transcript (about 1.5 fold) and a greater increase (about 2.3 fold) in the relative amount of polyadelylated transcript, Brd box activity has an even greater effect (3 to 5 fold) on reporter protein accumulation. Though Brd and the E(spl)-C genes are expressed in spatially restricted patterns in both embryos and imaginal discs, the regulatory activity that functions through the Brd box is both temporally and spatially general. A Brd genomic DNA transgene with specific mutations in its Brd and GY boxes exhibits hypermorphic activity that results in characteristic defects in PNS development, demonstrating that Brd is normally regulated by these motifs. Ectopic bristles in mutant lines include both sockets and shafts, and are thus likely to represent complete sensory organs. All ectopic bristles are present as tufts in the normal postions of single sensory organs, indicating that the extra bristles arise from the normal complement of proneural clusters. Brd boxes and GY boxes in the (E(spl) region transcript m4) E(spl)m4 gene are specifically conserved between two distantly related Drosophila species, strongly suggesting that E(spl)-C genes are regulated by these elements as well (Lai, 1997).

Regulation of epithelial polarity by the E3 ubiquitin ligase Neuralized and the Bearded inhibitors in Drosophila

Understanding how epithelial polarity is established and regulated during tissue morphogenesis is a major issue. This study identified a regulatory mechanism important for mesoderm invagination, germ-band extension and transepithelial migration in the Drosophila embryo. This mechanism involves the inhibition of the conserved E3 ubiquitin ligase Neuralized by proteins of the Bearded family. First, Bearded mutant embryos exhibited a loss of epithelial polarity associated with an early loss of the apical domain. Bearded regulated epithelial polarity by antagonizing neuralized. Second, repression of Bearded gene expression by Snail was required for the Snail-dependent disassembly of adherens junctions in the mesoderm. Third, neuralized was strictly required to promote the downregulation of the apical domain in the midgut epithelium and to facilitate the transepithelial migration of primordial germ cells across this epithelium. This function of Neuralized was independent of its known role in Notch signalling. Thus, Neuralized has two distinct functions in epithelial cell polarity and Notch signalling (Chanet, 2012).

In this study, the Brd genes were identified as Sna molecular targets acting as key regulators of gastrulation. Second, it was shown that the Brd genes are required for the stabilization of the apical domain in the ectoderm at stages 6–8. Third, it was shown that Neur is the main target of Brd for the maintenance of epithelial polarity and that neur is required for the remodelling of the midgut epithelium at stage 10. Fourth, this function of Neur in the regulation of epithelial polarity is independent of its known activity in Notch signalling. Fifth, a model is proposed for the regulation of PGC transepithelial migration based on the regulation in space and time of the activity of Neur by its Brd inhibitors (Chanet, 2012).

The genetic analysis of gastrulation in Drosophila has indicated that Sna regulates both apical constriction and SAJ disassembly. How Sna regulates these two processes has remained unknown and the existence of a zygotic gene 'x' corresponding to a Sna target repressed in the mesoderm with the ability to block SAJ disassembly has been postulated. This study shows that the Brd genes have the proposed properties of this 'x'gene. Indeed, it was shown that Brd proteins inhibited junction disassembly in sna mutant embryos. Moreover, the early expression of the Tom and mαgenes was sufficient to inhibit apical constriction and delay mesoderm invagination. One possible interpretation is that Brd proteins regulate gastrulation by inhibiting Neur. As loss of zygotic neur did not perturb junction disassembly nor mesoderm invagination, it is suggested that maternal Neur may be sufficient for these processes. Alternatively, Brd proteins might regulate gastrulation through an unknown Neur-independent mechanism (Chanet, 2012).

One of the main findings of this study is the identification of the E3 ubiquitin ligase Neur as a regulator of epithelial polarity in the Drosophila embryo. This activity of Neur did not involve Dl–Notch signalling. How Neur stabilizes both the apical domain and adherens junctions is at present unclear; the loss of the apical domain may possibly result from increased apical endocytosis, as seen during apical constriction in Xenopus. Interestingly, increased apical endocytosis in Cdc42-compromised embryos correlated with adherens junction destabilization. Similarly, inhibition of Rab11 led to a loss of Crb preceding adherens junction. Whether increased Neur activity disrupts the balance between apical endocytosis and recycling remains to be investigated. Importantly, cross-regulatory interactions between apical polarity complexes, Rho/Rok signalling, actomyosin and adherens junctions make it difficult to predict the molecular targets of Neur on the sole basis of phenotypic analysis. Nevertheless, components and/or regulators of the Crb complex are possible candidates. Indeed, similarly to Brd proteins, Crb seems to stabilize epithelial polarity during cell–cell intercalation at germ-band extension. Moreover, high Neur levels correlate with low Crb in wild-type neuroblasts and neuroblast polarity did not depend on Brd activity. Finally, loss of Brd led to the premature apical relocalization of adherens junctions in the ectoderm, an effect that is consistent with the proposed role of Crb in excluding Baz and adherens junctions from the apical domain. Whether and how Neur counteracts the function of Crb will require the molecular identification of the Neur targets (Chanet, 2012).

Another important finding is the identification of Neur and Brd as developmental regulators of the transepithelial migration of primordial germ cells (PGCs). Importantly, the migration of PGCs may be used as a model system to investigate the molecular basis of the process of transepithelial migration that is important for innate immune response in humans, as neutrophils migrate across the endothelium to reach sites of inflammation or infection. PGC migration was shown to involve a developmentally regulated remodelling of the midgut epithelium as suggested earlier and that Neur and Brd are essential regulators of this remodelling. Further analysis of the role of Neur in this morphogenetic regulation may shed light on conserved mechanisms involved in transepithelial migration (Chanet, 2012).

Finally, the finding that Neur functions both in Notch signalling and cell polarity raises the exciting possibility that these two processes are coupled. Indeed, Neur-dependent endocytosis of Dl occurs along the basolateral membrane of signal-sending cells. Thus, epithelial remodelling by Neur might facilitate Dl activity along the basolateral membrane. It is speculated that Neur may mechanistically link Dl signalling and epithelial polarity remodelling to ensure efficient signalling. As Neur plays an essential role in Notch signalling in Drosophila but not in mammals, it is possible that the evolutionarily conserved function of Neur is not the regulation of Notch activity but rather the remodelling of epithelial cells. Accordingly, Neur would have been co-opted for the regulation of Dl signalling in insects. It will thus be of interest to examine the potential role of Neur in epithelial polarity in mammals (Chanet, 2012).

Bardin, A. J. and Schweisguth, F. (2006). Bearded family members inhibit Neuralized-mediated endocytosis and signaling activity of Delta in Drosophila. Dev. Cell 10(2): 245-55. 16459303

Chanet, S. and Schweisguth, F. (2012). Regulation of epithelial polarity by the E3 ubiquitin ligase Neuralized and the Bearded inhibitors in Drosophila. Nat. Cell Biol. 14(5): 467-76. PubMed Citation: 22504274

De Renzis, S., Yu, J., Zinzen, R. and Wieschaus, E. (2006). Dorsal-ventral pattern of Delta trafficking is established by a Snail-Tom-Neuralized pathway. Dev. Cell 10(2): 257-64. 16459304

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Lai, E. C. and Posakony, J. W. (1997). The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression. Development 124(23): 4847-4856. PubMed Citation: 9428421

Lai, E. C. and Posakony, J. W. (1998). Regulation of Drosophila neurogenesis byRNA:RNA duplexes? Cell 93: 1103-1104. PubMed Citation: 9657143

Lai, E. C., et al. (2000a). Antagonism of Notch signaling activity by members of a novel protein family encoded by the Bearded and Enhancer of split gene complexes. Development 127: 291-306. PubMed Citation: 10603347.

Lai, E. C., Bodner, R. and Posakony, J. W. (2000b). The Enhancer of split Complex of Drosophila includes four Notch-regulated members of the Bearded gene family. Development 127: 3441-3455. PubMed Citation: 10903170

Lai, E. C. (2002). Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat. Genet. 30(4): 363-4. 11896390

Lai, E. C., Tam, B. and Rubin, G. M. (2005). Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs. Genes Dev. 19: 1067-1080. 15833912

Lee, Y. S., Nakahara, K., Pham, J. W. Kim, K., He, Z., Sontheimer, E. J. and Carthew, R. W. (2004). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117: 69-81. 15066283

Leviten, M. W. and Posakony, J. W. (1996). Gain-of-function alleles of interfere with alternative cell fate decisions in Drosophila adult sensory organ development. Dev. Biol. 176(2): 264-283, PubMed Citation: 8660866.

Leviten, M. W., Lai, E. C. and Posakony, J. W. (1997). The Drosophila gene Bearded encodes a novel small protein and shares 3' UTR sequence motifs with multiple Enhancer of split Complex genes. Development 124: 4039-4051. PubMed Citation: 9374401

Morel, B., Le Borgne, R. and Schweisguth, F. (2003). Snail is required for Delta endocytosis and Notch-dependent activation of single-minded expression. Dev. Genes Evol. 213: 65-72. 12632175

Singson, A., et al. (1994). Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling. Genes Dev. 8(17): 2058-2071. PubMed Citation: 7958878

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date revised: 30 September 2012

 

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