One of the most widely studied phenomena in the establishment of neuronal identity is the determination of neurosecretory phenotype, in which cell-type-specific combinatorial codes direct distinct neurotransmitter or neuropeptide selection. However, neuronal types from divergent lineages may adopt the same neurosecretory phenotype, and it is unclear whether different classes of neurons use different or similar components to regulate shared features of neuronal identity. This question was addressed by analyzing how differentiation of the Drosophila larval leucokinergic system, which is comprised of only four types of neurons, is regulated by factors known to affect expression of the FMRFamide neuropeptide. All leucokinergic cells express the transcription factor Squeeze (Sqz). However, based on the effect on Leucokinin (LK) expression of loss- and gain-of-function mutations, three types of LK regulation are described. In the brain LHLK (lateral horn leukokinin) cells, both Sqz and Apterous (Ap) are required for LK expression, but surprisingly, high levels of either Sqz or Ap alone are sufficient to restore LK expression in these neurons. In the suboesophageal SELK cells, Sqz, but not Ap, is required for LK expression. In the abdominal ABLK neurons, inhibition of retrograde axonal transport reduces LK expression, and although sqz is dispensable for LK expression in these cells, it can induce ectopic leucokinergic ABLK-like cells when over-expressed. Thus, Sqz appears to be a regulatory factor for neuropeptidergic identity common to all leucokinergic cells, whose function in different cell types is regulated by cell-specific factors (Herrero, 2007).

It has been shown that Ap is required for LK expression only in LHLK cells. Ap is also necessary for proper transcription of the Fmrf gene in the thoracic Tv neurons. In attempts to understand the mechanisms underlying leucokinergic differentiation, it was asked whether other factors known to control expression of the FMRFamide neuropeptide, i.e., Sqz and the BMP signalling pathway, affected LK expression. Indeed, the number of LK-immunopositive cells is strongly reduced in sqz^lacZ mutant larvae. It has been proposed that Apterous is not necessary for the emergence and maintenance of LHLK cells. Earlier reports have established that the Tv cells are present in sqz mutants, although they do not express Fmrf. Are the LK cells present in sqz mutants? This question could not be directly addressed due to the lack of independent markers for following the fate of the LK cells, but the results presented in this study indicate that in sqz mutants, leucokinergic cells are born, but fail to express LK: (1) LK expression is restored by postmitotic expression of Sqz; (2) LK-immunoreactive cells are detected in sqz mutant brains during early larval stages, and later disappear; (3) the few LK-expressing cells detected in sqz mutants often show very faint immunostaining, which suggests that reduction of Sqz protein decreases Lk transcription but does not affect cell survival. The reduction in the number of leucokinergic cells in sqz mutants even in first instar larvae indicates that sqz is necessary for induction of LK expression, and the weaker phenotype observed in early larval stages may reflect perdurance of the wild type product supplied by the mother, or a requirement for sqz also for maintenance of LK expression (Herrero, 2007).

Although all leucokinergic neurons express sqz, they differ in how this transcription factor affects LK expression. In this study, at least three neuronal types have been identified based on the components that regulate LK expression: (1) in the LHLK neurons, LK expression is controlled by the transcription factors Ap and Sqz; (2) in the SELK neurons, Sqz, but not Ap, is necessary for wild type Lk transcription; (3) in the ABLK neurons, Sqz is dispensable for LK expression even though it can induce leucokinergic ABLK-like cells, and LK expression is affected by inhibition of retrograde axonal transport only in the ABLK cells. Regarding the ALK neurons, the number of this cell type is highly variable, and thus was not further analyzed in this study (Herrero, 2007).

By analyzing two different sqz alleles, it was shown that, besides differences in the components that control LK expression, the amount of Sqz protein required to achieve wild type LK expression also varies. RealTime PCR analysis indicates that the sqz^lacZ allele has undetectable levels of sqz transcripts, while the sqz^GAL4 allele has reduced, but measurable sqz transcription. Consistent with these results, sqz^lacZ mutant larvae show a large reduction in the number of LHLK and SELK cells detected by anti-LK immunostaining, while sqz^GAL4 mutants do not affect LK expression in the LHLK neuron, but do show a significant decrease in the number of LK-immunopositive SELK cells. Moreover, restoring neuronal Sqz protein with the elav^GAL4 driver can completely rescue LK expression in LHLK neurons in sqz^lacZ mutants, but only partially rescue LK in SELK neurons. The differential effect of these two sqz alleles suggests that there is a threshold of Sqz protein below which Lk transcription is prevented, and this threshold is higher in SELK cells than in LHLK cells (Herrero, 2007).

This study has demonstrated that the reduced LK expression observed in the LHLK cells of sqz mutants is fully rescued when over-expressing the lost protein, indicating that this protein is indeed responsible for the observed phenotype. In addition, the data show that LK expression in these cells does not depend on the Gbb/Wit or Babo/Activin signalling cascades, or any extrinsic retrograde signal. Thus, neuropeptidergic identity of the LHLK neurons is controlled by Ap and Sqz, but not BMP. These cells also express the bHLH transcription factor Dimm, which has been shown to act post-tanscriptionally on the regulation of LK expression in these cells. This combination of factors (i.e., Ap, Sqz, Dimm, but not BMP signalling) is different from any of the known codes that regulate the neuropeptidergic differentiation of the peptidergic neurons in the thoracic ap cluster, which includes the Furin 1-expressing Ap-let cells that do not express Sqz, and the Fmrf-expressing Tv cell, which requires extrinsic signalling through Gbb/Wit (Herrero, 2007).

These results demonstrate that high levels of either Ap or Sqz protein alone are sufficient to induce LK expression in the LHLK cells. Thus, based on the cross-rescue experiments, it can be inferred that Sqz can form transcriptionally active complexes with proteins other than Ap to promote Lk expression. Similarly, Ap is able to promote Lk expression when it complexes with proteins other than Sqz. However, wild type levels of LK expression are only achieved when both proteins are present. It has been shown that Sqz and Ap can physically interact, and that both proteins can form separate complexes with Chip. Chip is involved in Lk regulation, because LK expression in LHLK cells is affected when dLMO, a cofactor that binds Chip and prevents Ap action, is expressed in ap-expressing cells. Thus, Ap and Sqz could weakly activate Lk transcription independently by interacting with Chip and/or other transcription factors, but strong activation would require a synergistic interaction mediated by complexes containing both proteins. A similar mechanism is exerted by Brn2 and Otp to stimulate transcription of the corticotrophin-releasing hormone gene in the neuroendocrine hypothalamic neurons, and by Cdx-2 and Brn-4 to activate expression of the proglucagon gene in pancreatic B-cell lines (Herrero, 2007).

It has been reported that Lk transcription in dimm mutants is downregulated in ABLK cells, while LHLK and SELK cells show slightly upregulated Lk transcription but reduced levels of LK peptide. It was found that the ABLK neurons also differ from LHLK and SELK neurons in that Sqz appears to be dispensable for LK expression in these cells. However, neuronal over-expression of Sqz can frequently induce ectopic LK expression in ABLK-like cells in an ap-independent fashion. Likewise, misexpression of the mammalian homeodomain transcription factor Phox2a in the sympathetic ganglion induces ectopic noradrenergic neurons, even though in the absence of Phox2a, sympathetic development is largely normal (Herrero, 2007).

Another peculiarity of the ABLK cells is that LK expression in these cells seems to depend on a retrograde signal, different from BMP, because the number of ABLK LK-expressing cells is reduced when axonal transport is inhibited. Although the degree of reduction is variable, it is indeed highly significant, because as few as two cells can be found when Glued^DN is pan-neurally expressed. This variability could be due to partial inhibition of retrograde transport, as suggested by the late pupal lethality of Glued^DN-expressing flies, instead of the earlier embryonic lethality of Glued amorphic mutations. In the thoracic ap-cluster, projection of the Tv cell outside the CNS is essential for its response to Gbb and subsequent Fmrf expression. Likewise, ABLK cells are known to project outside the CNS, and this may be essential for them to react to an extrinsic signal important for induction and/or maintenance of LK expression. Alternatively, extrinsic signalling may control guidance and/or targeting of ABLK axons, as suggested by the ectopic leucokinergic varicosities found in the abdominal neuropile of Glued^DN-expressing ganglia, and proper axonal targeting may be an important factor in the regulation of LK expression. Further experiments will be necessary to test these hypotheses, and rule out a possible deleterious effect of Glued^DN on ABLK viability (Herrero, 2007).

Ectopic leucokinergic cells can be induced by over-expressing Sqz, but not by Ap. Moreover, ectopic LK neurons can also be classified into three different groups: (1) the ectopic LHLK-like cells, which appear to depend on the relative amount of Sqz and Ap; (2) the ABLK-like cells, which require high levels of Sqz expression, but not ap expression, and (3) the ectopic brain cells, which are the only leucokinergic ectopic cells generated by ectopic expression of Sqz. These latter cells must have unidentified components present in other leucokinergic cells that enable them to activate Lk transcription when Sqz is present (Herrero, 2007).

An ectopic LHLK-like cell is present in a small percentage of the sqz^GAL4 mutants. Appearance of this ectopic cell requires Ap, because ap is expressed in this cell and its appearance is prevented in the ap^UGO35 null allele. In the thoracic ganglion, sqz determines the number and identities of cells of the ap cluster, so that in szq null mutants an extra Dimm and Furin-expressing cell was generated in every ap cluster. Attempts to analyze changes in the number of ap and dimm expressing cells in sqz mutants was precluded by the large number of ap cells surrounding the LHLK cell, and the inconsistent expression of the dimm-GAL4 driver c929 in the LHLK cells, in both wild type and mutant brains. However, the results obtained when Sqz and/or Ap were over-expressed in a wild type background suggest a more complex scenario. First, a phenotype was obtained equivalent to that observed in sqz^GAL4 mutants, i.e., the appearance of ectopic LHLK-like cells, when Sqz was over-expressed with sqz^GAL4 and with the postmitotic ap- and elav- GAL4 drivers. Moreover, these ectopic LK cells disappeared if Ap was coexpressed with Sqz, even though over-expression of Ap alone had no effect whatsoever on LK expression. Based on these data, and on the different levels of Ap protein in cells surrounding the LHLK neuron, it is hypothesized that the relative dose of Ap and Sqz, rather than the absolute amount of Sqz, is essential for specifying the correct number of LHLK cells with detectable LK, and that this process is controlled postmitotically. According to this model, when Sqz is reduced, those cells with low Ap levels would reach an optimum stoichiometric Sqz/Ap ratio leading to ectopic LK expression, while when Sqz is increased, the correct ratio will be present in cells with high Ap levels. In this last case, further increasing Ap would drive the Sqz/Ap ratio away from the optimum for LK expression, and ectopic cells would not be produced (Herrero, 2007).

Two other situations lead to ectopic leucokinergic LHLK-like cells. (1) A small percentage of ectopic LHLK cells was detected in dac mutants. Because Ap has been shown to repress dac expression in the thoracic Tvb cell, it is possible that repression of dac is required to restrict LK expression to the LHLK neuron. (2) Over-expression of the transcription factor Dimm produces ectopic LHLK-like cells, albeit at much higher frequency. Ap also regulates dimm transcription in most cells. Thus, Dimm over-expression might overcome the requirement for a Sqz/Ap optimum ratio to induce LK expression, provided additional necessary factors are also present in the cell. More experiments will be needed to understand the role of Dac and Dimm, and their interaction with Ap and Sqz, in regulating LK expression in the LHLK cell (Herrero, 2007).

Deciphering how cells of different origin acquire the same neurosecretory identity is one of the major challenges in neuronal development. Much of the progress in this field has been achieved by studying the vertebrate catecholaminergic system, in which central and peripheral noradrenergic neurons use a similar combination of factors to specify their neurotransmitter phenotype, but they differ in the hierarchical relations between these factors, and recruit components specific to each neuronal type. It is tempting to speculate that analogous mechanisms may control Lk expression in leucokinergic cells in Drosophila. Consistent with this hypothesis, Sqz, and probably Dimm, appear to be regulatory factors for neuropeptidergic identity common to all leucokinergic cells. However, sqz affects LK expression in each cell subclass in very different ways, and cell-type-specific regulatory modes on Lk have also been described for Dimm. Moreover, additional cell-type-specific factors regulate LK expression in different leucokinergic cells, such as Ap in the LHLK cells, and an unidentified retrograde signal in the ABLK cells. Thus, the leucokinergic neurons comprise a simple, genetically amenable system for understanding how complex regulatory networks confer a similar neurosecretory identity on cells of different origins (Herrero, 2007).

GENE STRUCTURE

cDNA clone length - 3246 bp

Bases in 5' UTR - 866

Exons - 3

Bases in 3' UTR - 752

PROTEIN STRUCTURE

Amino Acids - 535

Structural Domains

Rotund, a zinc finger protein of the C₂H₂ Krüppel-type belongs to a conserved subfamily of zinc finger proteins together with Drosophila CG5557, C. elegans Lin-29, and rat CIZ. Squeeze is most closely related to Rotund, with identity greater than 90% throughout the zinc finger region; Squeeze is 78% identical to LIN-29 in the conserved zinc finger region. Both rotund and CG5557 are expressed in subsets of cells in the developing CNS. CG5557 has a larval lethal phase. Mutants eclose at a low frequency as immotile adults that die within 24 hr. Mutant larvae display a motility defect whereby the body wall musculature over-contract radially during the peristaltic wave typical of insect larval motility, apparent as a 'squeezing' of the intestine. Since this motility phenotype is fully penetrant, CG5557 was renamed squeeze (sqz) (Allan, 2003).

date revised: 1 September 2007

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.