Isabel Molina

Squeeze involvement in the specification of Drosophila leucokinergic neurons: Different regulatory mechanisms endow the same neuropeptide selection

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. We have addressed this question 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. We show that all leucokinergic cells express the transcription factor Squeeze (Sqz). However, based on the effect on LK expression of loss- and gain-of-function mutations, we can describe three types of Lk regulation. In the brain LHLK 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.

Ih Current Is Necessary to Maintain Normal Dopamine Fluctuations and Sleep Consolidation in Drosophila

HCN channels are becoming pharmacological targets mainly in cardiac diseases. But apart from their well-known role in heart pacemaking, these channels are widely expressed in the nervous system where they contribute to the neuron firing pattern. Consequently, abolishing Ih current might have detrimental consequences in a big repertoire of behavioral traits. Several studies in mammals have identified the Ih current as an important determinant of the firing activity of dopaminergic neurons, and recent evidences link alterations in this current to various dopamine-related disorders. We used the model organism Drosophila melanogaster to investigate how lack of Ih current affects dopamine levels and the behavioral consequences in the sleep∶activity pattern. Unlike mammals, in Drosophila there is only one gene encoding HCN channels. We generated a deficiency of the DmIh core gene region and measured, by HPLC, levels of dopamine. Our data demonstrate daily variations of dopamine in wild-type fly heads. Lack of Ih current dramatically alters dopamine pattern, but different mechanisms seem to operate during light and dark conditions. Behaviorally, DmIh mutant flies display alterations in the rest∶activity pattern, and altered circadian rhythms. Our data strongly suggest that Ih current is necessary to prevent dopamine overproduction at dark, while light input allows cycling of dopamine in an Ih current dependent manner. Moreover, lack of Ih current results in behavioral defects that are consistent with altered dopamine levels.

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, we have used a very highly specific marker that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. Our data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, we show that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, we have performed a targeted genetic screen to identify players involved in ABCA specification. We have found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, we show that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third suboesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification.

Lineage-unrelated neurons generated in different temporal windows and expressing different combinatorial codes can converge in the activation of the same terminal differentiation gene

It is becoming increasingly clear that the activation of specific terminal differentiation genes during neural development is critically dependent upon the establishment of unique combinatorial transcription factor codes within distinct neural cell subtypes. However, it is still unclear to which extent these codes are shared by lineage-unrelated neurons expressing the same terminal differentiation genes. Additionally, it is not known if the activation of a specific terminal differentiation gene is restricted to cells born at a particular developmental time point. Here, we utilize the terminal differentiation gene FMRFa which is expressed by the Ap4 and SE2 neurons in the Drosophila ventral nerve cord, to explore these issues in depth. We find that the Ap4 and SE2 neurons are generated by different neural progenitors and use different combinatorial codes to activate FMRFa expression. Additionally, we find that the Ap4 and SE2 neurons are generated in different temporal gene expression windows. Extending the investigation to include a second Drosophila terminal differentiation gene, Leucokinin, we find similar results, suggesting that neurons generated by different progenitors might commonly use different transcription factor codes to activate the same terminal differentiation gene. Furthermore, these results imply that the activation of a particular terminal differentiation gene in temporally unrestricted.

A new role of Klumpfuss in establishing cell fate during the GMC asymmetric cell division.

Studies in the Drosophila embryonic NB4-2 lineage have suggested that the transcription factor Klumpfuss (Klu) functions within embryonic neuroblast lineages to differentiate between the identities of two adjacent ganglion mother cells (GMCs). However, because of the limited lineage markers available, these observations have been made only for the NB4-2 lineage. Recent findings have placed this transcription factor in the vanguard of Drosophila neural stem cell biology by demonstrating that Klu is necessary for larval neuroblast growth and self-renewal. Here, we have studied the role of klu in an incipient model in order to address basic mechanisms of neural specification: the Va system. None of the previously reported roles of Klu satisfactorily explain our observations. Unexpectedly, in this lineage, klu is necessary for differentiating between the fates of the two neurons born from a unique GMC; klu mutants produce two B-type cells, rather than one B-type (Notch-OFF) and one A-type (Notch-ON) cell. Additionally, our results demonstrate that Klu operates in the GMC and/or in the newly born neuron, but not in the neuroblast. Unlike in larval neuroblasts in which Klu is an executor of Notch signaling, we have found that Klu does not lie downstream of the Notch pathway in this cell division context.

Klumpfuss controls FMRFamide expression by enabling BMP signaling within the NB5-6 lineage

A number of transcription factors that are expressed within most, if not all, embryonic neuroblast (NB) lineages participate in neural subtype specification. Some have been extensively studied in several NB lineages (e.g. components of the temporal gene cascade) whereas others only within specific NB lineages. To what extent they function in other lineages remains unknown. Klumpfuss (Klu), the Drosophila ortholog of the mammalian Wilms tumor 1 (WT1) protein, is one such transcription factor. Studies in the NB4-2 lineage have suggested that Klu functions to ensure that the two ganglion mother cells (GMCs) in this embryonic NB lineage acquire different fates. Owing to limited lineage marker availability, these observations were made only for the NB4-2 lineage. Recent findings reveal that Klu is necessary for larval neuroblast growth and self-renewal. We have extended the study of Klu to the well-known embryonic NB5-6T lineage and describe a novel role for Klu in the Drosophila embryonic CNS. Our results demonstrate that Klu is expressed specifically in the postmitotic Ap4/FMRFa neuron, promoting its differentiation through the initiation of BMP signaling. Our findings indicate a pleiotropic function of Klu in Ap cluster specification in general and particularly in Ap4 neuron differentiation, indicating that Klu is a multitasking transcription factor. Finally, our studies indicate that a transitory downregulation of klu is crucial for the specification of the Ap4/FMRFa neuron. Similar to WT1, klu seems to have either self-renewal or differentiation-promoting functions, depending on the developmental context.

Segmentally homologous neurons acquire two different terminal neuropeptidergic fates in the Drosophila nervous system

In this study, we identify the means by which segmentally homologous neurons acquire different neuropeptide fates in Drosophila. Ventral abdominal (Va)-neurons in the A1 segment of the ventral nerve cord express DH31 and AstA neuropeptides (neuropeptidergic fate I) by virtue of Ubx activity, whereas the A2-A4 Va-neurons express the Capa neuropeptide (neuropeptidergic fate II) under the influence of abdA. These different fates are attained through segment-specific programs of neural subtype specification undergone by segmentally homologous neurons. This is an attractive alternative by which Hox genes can shape Drosophila segmental neural architecture (more sophisticated than the previously identified binary “to live” or “not to live” mechanism). These data refine our knowledge of the mechanisms involved in diversifying neuronal identity within the central nervous system.